Dhanasekaran, Dharumadurai PanneErselvam, A. Thajuddin, Nooruddin Antimicrobials Synthetic and Natural Compounds

ANTIMICROBIALS
Synthetic and Natural Compounds
Edited by
Dharumadurai Dhanasekaran
Nooruddin Thajuddin
Annamalai Panneerselvam
Contents
Preface...............................................................................................................................................ix
Editors...............................................................................................................................................xi
Contributors.................................................................................................................................. xiii
Chapter 1 Antibiotics: From discovery to journey................................................................1

Deene Manikprabhu and Wen-Jun Li
Section I: Broad spectrum antimicrobial compounds from microorganisms
Chapter 2 Antimicrobial potential of marine actinobacteria: A review........................ 17

Panchanathan Manivasagan and Se-Kwon Kim
Chapter 3 Antimicrobial compounds from microorganisms: Production,

characterization, and applications......................................................................29
W.M. Muiru
Chapter 4 Animal fecal actinomycetes: A new source for the discovery

of drug leads............................................................................................................53
Yi Jiang, Li Han, Xueshi Huang, and Chenglin Jiang
Chapter 5 Potentially novel Actinobacteria-derived antibiotics from unique

microenvironments................................................................................................83
Diana R. Cundell and Michael P. Piechoski
Chapter 6 Antimicrobial agents from actinomycetes: Chemistry and applications....99

Chapter 7 Actinobacteria: A predominant source of antimicrobial compounds....... 117

Ramasamy Vijayakumar, Govindaraj Vaijayanthi, Annamalai Panneerselvam,
and Nooruddin Thajuddin
Chapter 8 Novel antimicrobial and anticancer drugs from bacteria............................ 143

Ranjith N. Kumavath
Chapter 9 Bacteriocin: A natural alternative to synthetic antibacterial antibiotics...... 155

S. Latha and Dharumadurai Dhanasekaran
v
vi Contents
Chapter 10 Protease inhibitors from marine organisms................................................... 175

L. Karthik and A. Vishnu Kirthi
Chapter 11 Ganoderma: A bioresource of antimicrobials.................................................189

K. Rajesh and Dharumadurai Dhanasekaran
Chapter 12 Marine cyanobacteria: A prolific source of

antimicrobial natural products..........................................................................203
Natesan Sivakumar and Gopal Selvakumar
Chapter 13 Antimicrobial and natural compounds from edible mushrooms..............233

Annamalai Panneerselvam, V. Ambikapathy, and M. Nithya
Chapter 14 Aspergillosis and its resistance: Marine natural products as future
treatment.................................................................................................................255
Kumar Saurav, Subhasish Saha, Manoj Singh,
Soumik Sarkar, Dharumadurai Dhanasekaran, and K. Kannabiran
Section II: Broad spectrum antimicrobial compounds from animals
Chapter 15 Secondary metabolites from microorganisms isolated from

marine sponges from 2000 to 2012��279
Mohammad F. Mehbub, Christopher M.M. Franco, and Wei Zhang
Section III: Broad spectrum antimicrobial compounds from

plants and rhizosphere microorganisms
Chapter 16 Antimicrobial compounds and their chemical entities on therapeutic

herbals for agricultural and medical applications��319
P. Rajesh, K. Natarajan, and V. Rajesh Kannan
Chapter 17 Role of antimicrobial compounds from Trichoderma spp. in plant

disease management��359
Someshwar Bhagat, O.P. Sharma, Ajanta Birah, Natarajan Amaresan,
Israr Ahmad, Nasim Ahmad, and C. Chattopadhyay
Chapter 18 Antimicrobial compounds from rhizosphere bacteria and their role

in plant disease management��371
Natarajan Amaresan, Nallanchakravarthula Srivathsa, Velusamy Jayakumar,
Someshwar Bhagat, and Nooruddin Thajuddin
Section IV: Synthetic chemical compounds as broad spectrum antimicrobials
Chapter 19 Microbe-mediated synthesis of silver nanoparticles: A new drug of

choice against pathogenic microorganisms.....................................................389
Contents vii
Chapter 20 Nanomaterials: Source of antimicrobial products.........................................403

Atanu Bhattacharyya, P.M. Gopinath, A. Ranjani, and Dharumadurai Dhanasekaran
Chapter 21 Platinum-based anticancer therapeutics and their mechanistic

aspects: An overview...........................................................................................419
Bhaskar Biswas
Section V: Narrow-spectrum antimicrobials
Chapter 22 Marine actinobacteria as potential drug storehouses: A future

perspective on antituberculosis compounds..................................................435
N. Tamilselvan, Ernest David, Dharumadurai Dhanasekaran, and Kumar Saurav
Chapter 23 Antiprotozoal agents derived from natural soil and aquatic
actinobacteria: Fighting one microbe with another......................................457
Chapter 24 Bioactive compounds from actinomycetes and their antiviral

properties: Present trends and future prospectives......................................479
Avilala Janardhan, Arthala Praveen Kumar, and Golla Narasimha
Chapter 25 Novel antidermatophytic drug candidates from nature...............................487

Didem Deliorman Orhan and Nilüfer Orhan
Preface
Antimicrobials are secondary metabolites found in microorganisms, plants, and animals.
Herbal plants and microorganisms such as bacteria, actinobacteria, cyanobacteria, fungi,
and algae attracted more attention in research that led to the discovery of natural antimi-
crobial compounds. The exploration of natural and synthetic antimicrobial compounds
subsequently led to the development of drugs for the treatment of human microbial dis-
eases. Microbial diseases are one of the main causes of morbidity and mortality worldwide.
Today, many of such diseases are often caused by multidrug-resistant microorganisms
and are very difficult to treat by using conventional antibiotics, and, consequently, lead to
substantial increases in health-care costs.
Research in natural and synthetic compounds has attained significant progress
with the discovery of novel compounds with antimicrobial potentials. In fact, nature is a
wealthy resource of antimicrobial compounds with the potential to treat diseases in plants
and animals, including tuberculosis, dermatomycosis, aspergillosis, cancer, and viral and
protozoan diseases. Among the known sources of natural and synthetic antimicrobials,
we have highlighted plants, sponges, and marine and terrestrial microorganisms, includ-
ing bacteria and fungi. Nevertheless, there is still a vast fauna and flora that, when system-
atically explored, can provide additional antimicrobial leads and new drugs.
The book consists of an introductory overview of antimicrobials, which are clas-
sified into two main sections: broad spectrum and narrow spectrum antimicrobial
compounds. Broad spectrum antimicrobials are further divided into five categories:
antimicrobial compounds from microorganisms, animals, plants, rhizosphere micro-
organisms, and synthetic chemical compounds as antimicrobials. This book provides
a comprehensive account of microorganisms and sponge- and plant-derived natural
compounds with antibacterial, antifungal, antituberculosis, anticancer, antiviral, anti-
dermatophytic, and antiprotozoan properties. It also discusses synthetic chemical com-
pounds and biogenic nanoparticles. The 25 chapters have been contributed by authors
around the world, including Australia, China, Korea, Kenya, the United States, India,
Israel, Russia, and Turkey.
The book is considered as necessary reading for microbiologists, biotechnologists,
biochemists, pharmacologists, botanists, marine biologists, and those who are doing
research in natural and synthetic antimicrobial compounds. It should also be useful to
MSc students, MPhil and PhD scholars, scientists, and faculty members of various science
disciplines.
We are thankful to all the contributors for the submission of their valuable work. Our
special thanks to Hayley Ruggieri for her encouragement in bringing out this book. We
also thank Dr. Leong Li-Ming and Dr. Gagandeep Singh for their valuable suggestions
ix
x Preface
during the review of the chapters. We deeply appreciate the technical support from
P.M. Gopinath, S. Latha, A. Ranjani, and G. Vinothini, research scholars, Department of
Microbiology, Bharathidasan University. We are also grateful to CRC Press for their con-
cern, efforts, and encouragement in the task of publishing this book.
Dharumadurai Dhanasekaran
Nooruddin Thajuddin
Annamalai Panneerselvam
Editors
Dr. Dharumadurai Dhanasekaran, PhD, is an assistant
professor, Department of Microbiology, School of Life
Sciences, Bharathidasan University, Tiruchirappalli,
Tamil Nadu, India. He has experience in the fields of actino-
bacteriology and mycology. His current research focus is on
actinobacteria, microalgae, fungi, and mushroom for ani-
mal and human health improvement. He has deposited
around 54 nucleotide sequences in GenBank; published 77
research and review articles and one book, Fungicides for
Plant and Animal Diseases; guided four PhD candidates; and
organized several national-level symposia, conferences, and
workshops. Dr. Dhanasekaran has received major research grants from the Department of
Biotechnology (DBT), the University Grants Commission (UGC), the Indian Council of
Medical Research (ICMR) and the International Foundation for Science (IFS), Sweden. He
is a life member of the Mycological Society of India and the National Academy of Biological
Sciences, an editorial board member of national and international journals, and a doctoral
committee member and board of study member in microbiology. As per the Indian Journal
of Experimental Biology (Vol. 51, 2013), Dr. Dhanasekaran was ranked in second position in
top five institutions in the field of actinobacterial research in India.
Prof. Nooruddin Thajuddin, PhD, is the dean, Faculty of

Science, Engineering and Technology, head, Department
of Microbiology, School of Life Sciences, Bharathidasan
University, Tiruchirappalli, Tamil Nadu, India. He has vast
experience in microbial taxonomy, isolation, cultivation,
harvesting, and extraction of valuable products. He is an
expert in employing molecular tools in the identification
and phylogeny of various microorganisms and bioremedi-
ation of effluents and bioenergy from microalgae and cya-
nobacteria. He underwent one-year postdoctoral training
in molecular taxonomy and phylogeny of cyanobacteria in
the Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, through
the Department of Biotechnology (Government of India) overseas fellowship. Professor
Thajuddin has deposited around 500 nucleotide sequences in GenBank; published 227
research and review articles and 3 books on microbiology-related topics; developed
germplasm of cyanobacteria, microalgae, bacteria, actinobacteria, and fungi in his labo-
ratory; guided 27 PhD candidates; and organized several national-level symposia,
xi
xii Editors
workshops, refresher courses, and DST-INSPIRE (Innovation in Science Pursuit for

Inspired Research) programs. He has received major research grants from the Department
of Biotechnology (DBT), the Department of Science and Technology (DST), the University
Grants Commission (UGC) and the Ministry of Earth Sciences (MoES) amounting to
$44 million. He is a life member of various academic bodies and an editorial board mem-
ber of national and international journals. Professor Thajuddin has visited the United
States of America, the United Kingdom, Germany, Honk Kong, Malaysia, Kingdom of
Saudi Arabia, Republic of Korea and Singapore to disseminate his expertise and to keep
abreast of the advanced techniques in the field of microbiology. Recently, the Department
of Biotechnology (Gov’t of India) sanctioned a major grant to Professor Thajuddin for the
establishment of national repository for freshwater microalgae and cyanobacteria.
He received the 2014 Dr. G. S. Venkataraman Memorial NABS-Best Scientist Award from
the National Academy of Biological Sciences.
Prof. Annamalai Panneerselvam, PhD, is an associate

professor and head, Department of Botany and
Microbiology, A.V.V.M. Sri Pushpam College (Autonomous)
Poondi, Thanjavur District, Tamil Nadu, India. He has
more than 32 years of experience in teaching and research.
His areas of specialization are mycology, plant pathology,
and actinobacteriology. He has visited the National
University of Singapore, Nanyang Technical University,
TEMSEK Laboratory Singapore, Malaysian Agricultural
University, Kuala Lumpur, Malaysia, Bangkok, and also
national-level institutes in India. He has received the fol-
lowing awards: INSA visiting fellowship, New Delhi; Member of Research Board of
Advisors; Excellent Faculty in Botany; and Man of Year Commemorative model. Professor
Panneerselvam has deposited more than 85 nucleotide sequences in GenBank and main-
tains more than 500 microbial cultures in his laboratory. He is a reviewer for reputed
journals and a doctoral committee member for more than 100 research scholars working
in Bharathidasan University. He has published more than 300 research and review arti-
cles, 23 popular articles, and chapters in more than 12 books. He has guided 30 PhD
scholars and organized several symposia, seminars, workshops, DST-INSPIRE programs
for plus one students, YSSP program, etc. Professor Panneerselvam has conducted more
than 100 training programs on edible mushroom cultivation technology for the welfare
of women under a self-help group scheme, Dissemination of Innovative Technology
(DIT). He received grants from various funding agencies like the Department of Science
and Technology (DST), the University Grants Commission (UGC), the Council of
Scientific and Industrial Research (CSIR), the Tamil Nadu State Council for Science and
Technology (TNSCST), the National Medicinal Plants Board (NMPB) and the District
Rural Development Agency (DRDA) amounting to $5 million. He is a life member of
various academic bodies and an editorial board member of reputed journals.
Contributors
Israr Ahmad Bhaskar Biswas
Central Institute of Subtropical Department of Chemistry
Horticulture Raghunathpur College
Indian Council of Agricultural Research West Bengal, India
Uttar Pradesh, India
C. Chattopadhyay
Nasim Ahmad
National Research Centre for Integrated
National Research Centre for Integrated
Pest Management
Pest Management
Indian Council of Agricultural Research
Indian Council of Agricultural Research
New Delhi, India
New Delhi, India
Natarajan Amaresan Diana R. Cundell

C.G. Bhakta Institute of Biotechnology College of Science, Health and
Uka Tarsadia University Liberal Arts
Gujarat, India Philadelphia University
Philadelphia, Pennsylvania
V. Ambikapathy
Department of Botany and Microbiology
Ernest David
A.V.V.M. Sri Pushpam College
Department of Biotechnology
(Autonomous)
Thiruvalluvar University
Tamil Nadu, India
Tamil Nadu, India
Someshwar Bhagat
National Research Centre for Integrated Dharumadurai Dhanasekaran
Pest Management Department of Microbiology
Indian Council of Agricultural Research Bharathidasan University
New Delhi, India Tamil Nadu, India
Atanu Bhattacharyya
Christopher M.M. Franco
Department of Biomedical Engineering
School of Medicine
Rajiv Gandhi Institute of Technology
Flinders University
Karnataka, India
Adelaide, South Australia, Australia
Ajanta Birah
National Research Centre for Integrated P.M. Gopinath
Pest Management Department of Microbiology
Indian Council of Agricultural Research Bharathidasan University
New Delhi, India Tamil Nadu, India
xiii
xiv Contributors
Li Han S. Latha
College of Life and Health Science Department of Microbiology
Northeastern University Bharathidasan University
Shenyang, People’s Republic of China Tamil Nadu, India
Xueshi Huang Wen-Jun Li

College of Life and Health Science Ministry of Education
Northeastern University and
Shenyang, People’s Republic of China Yunnan Institute of Microbiology
Yunnan University
Avilala Janardhan Kunming, People’s Republic of China
Department of Virology
Sri Venkateswara University Deene Manikprabhu
Andhra Pradesh, India Ministry of Education
and
Velusamy Jayakumar Yunnan Institute of Microbiology
Division of Crop Protection Yunnan University
Sugarcane Breeding Institute Kunming, People’s Republic of China
Tamil Nadu, India
Panchanathan Manivasagan
Chenglin Jiang Department of Marine-Bio Convergence
Yunnan Institute of Microbiology Science
Yunnan University Pukyong National University
Kunming, People’s Republic of China Busan, Republic of Korea
Yi Jiang Mohammad F. Mehbub

Yunnan Institute of Microbiology School of Medicine
Yunnan University Flinders University
Kunming, People’s Republic of China Adelaide, South Australia, Australia
K. Kannabiran W.M. Muiru

School of Biosciences and Technology Department of Plant Science and Crop
VIT University Protection
Tamil Nadu, India University of Nairobi
Nairobi, Kenya
L. Karthik
School of Life Sciences and Biotechnology Golla Narasimha
Shanghai Jiao Tong University Department of Virology
Shanghai, People’s Republic of China Sri Venkateswara University
Andhra Pradesh, India
Se-Kwon Kim
Department of Marine-Bio Convergence K. Natarajan
Science Department of Microbiology
Pukyong National University Bharathidasan University
Busan, Republic of Korea Tamil Nadu, India
Ranjith N. Kumavath M. Nithya

Department of Genomic Science Department of Botany and Microbiology
Central University of Kerala A.V.V.M. Sri Pushpam College (Autonomous)
Kerala, India Tamil Nadu, India
Contributors xv
Didem Deliorman Orhan Soumik Sarkar

Department of Pharmacognosy Department of Microbiology and
Gazi University Biotechnology
Ankara, Turkey Al-Ameen College of Arts, Science and
Commerce
Nilüfer Orhan Karnataka, India
Department of Pharmacognosy
Gazi University Kumar Saurav
Ankara, Turkey Department of Marine Biology
University of Haifa
Annamalai Panneerselvam Haifa, Israel
Department of Botany and Microbiology
A.V.V.M. Sri Pushpam College Gopal Selvakumar
(Autonomous) Department of Microbiology
Tamil Nadu, India Alagappa University
Tamil Nadu, India
Michael P. Piechoski
Arts Academy at Benjamin Rush O.P. Sharma
Philadelphia, Pennsylvania National Research Centre for Integrated
Pest Management
Arthala Praveen Kumar Indian Council of Agricultural
Department of Virology Research
Sri Venkateswara University New Delhi, India
Andhra Pradesh, India
Manoj Singh
K. Rajesh Laboratory of Biomedical
Department of Microbiology Nanomaterials
Bharathidasan University National University of Science and
Tamil Nadu, India Technology
Moscow, Russia
P. Rajesh
Department of Biochemistry Natesan Sivakumar
K.S. Rangasamy College of Arts and School of Biotechnology
Science Madurai Kamaraj University
Tamil Nadu, India Tamil Nadu, India
V. Rajesh Kannan Nallanchakravarthula Srivathsa

Department of Microbiology C.G. Bhakta Institute of Biotechnology
Bharathidasan University Uka Tarsadia University
Tamil Nadu, India Gujarat, India
A. Ranjani N. Tamilselvan
Department of Microbiology Department of Zoology
Bharathidasan University Voorhees College
Tamil Nadu, India Tamil Nadu, India
Subhasish Saha Nooruddin Thajuddin

South China Sea Institute of Oceanology Department of Microbiology
Chinese Academy of Sciences Bharathidasan University
Guangzhou, People’s Republic of China Tamil Nadu, India
xvi Contributors
Govindaraj Vaijayanthi A. Vishnu Kirthi

Department of Microbiology Department of Biotechnology
Bharathidasan University Constituent C. Abdul Hakeem College
College, Kurumbalur, Perambalur Tamil Nadu, India
Tamil Nadu, India
Ramasamy Vijayakumar Wei Zhang

Department of Microbiology Centre for Marine Bioproducts
Bharathidasan University Constituent Development/Medical Biotechnology
College, Kurumbalur, Perambalur Flinders University
Tamil Nadu, India Adelaide, South Australia, Australia
chapter one
Antibiotics
From discovery to journey
Contents
1.1 Introduction............................................................................................................................. 1
1.2 Classification of antibiotics.................................................................................................... 2
1.2.1 Aminoglycosides......................................................................................................... 2
1.2.1.1 Aminoglycoside mode of action................................................................4
1.2.2 Penicillin...................................................................................................................... 5
1.2.2.1 Penicillin mode of action............................................................................ 6
1.2.3 Cephalosporin............................................................................................................. 6
1.2.3.1 Cephalosporin mode of action...................................................................7
1.2.4 Carbapenems...............................................................................................................8
1.2.4.1 Carbapenem mode of action....................................................................... 9
1.2.5 Glycopeptides.............................................................................................................. 9
1.2.5.1 Glycopeptide mode of action.................................................................... 10
1.2.6 Lincosamides............................................................................................................. 10
1.2.6.1 Lincosamide mode of action.................................................................... 11
1.2.7 Macrolides.................................................................................................................. 11
1.2.7.1 Macrolide mode of action......................................................................... 11
Acknowledgments......................................................................................................................... 12
References........................................................................................................................................ 12
1.1 Introduction
An antibiotic is defined as the agent that is capable of killing or inhibiting the growth of a
microorganism. The action of an antibiotic is selective in nature: it affects some organisms
completely, some to a limited degree, while some not at all (Waksman, 1947).
Though antibiosis was first described by Louis Pasteur and Robert Koch (Landsberg,
1949), the use of the word “antibiosis” dates from the concept first expressed, in 1889, by
Vuillemin in the following terms: “one creature destroying the life of another in order
to sustain its own” (Vuillemin, 1889). The era of chemotherapy started with Paul Ehrlich
who found a toxic dye molecule, a “magic bullet,” that specifically binds to pathogens and
destroys them without affecting human cells. He further found that the dye, trypan red,
was active against the trypanosome that causes African sleeping sickness. Subsequently,
Ehrlich and a young Japanese scientist, Sahachiro Hata, discovered that arsphenamine
was active against syphilis (Foster and Raoult, 1974).
1
2 Antimicrobials: Synthetic and natural compounds
The story of antibiotics came into existence after the discovery of penicillin by Alexander
Fleming. Fleming after returning from a holiday noticed a petri dish containing Staphylococcus
plate culture was mistakenly left open; the plate was contaminated by blue-green mold
Penicillium, which formed a visible growth. Fleming noticed that there was a halo of inhib-
ited bacterial growth around the mold. Looking at the plate, Fleming concluded that the mold
might release a substance that suppressed the growth or killed the bacteria. He then began his
efforts to characterize what he called penicillin. He found that broth from a Penicillium culture
contained penicillin and that the antibiotic could destroy several pathogenic bacteria (Fleming,
1929). Unfortunately, Fleming’s next experiments convinced him that penicillin would not
remain active in the body for a long enough time to kill the pathogens. Similarly, another
professor, Howard Florey, was testing the bactericidal activity of numerous substances. After
reading Fleming’s paper, one of Florey’s coworkers, Chain, obtained Penicillum culture from
Fleming, and they both started to obtain pure compound from the Penicillum; both were suc-
cessful in purifying penicillin. When the purified penicillin was injected into the mice infected
with Staphylococcus, practically all the mice survived. Florey and Chain also had successful
trials on humans. The penicillin obtained saved many lives during the world war. For their
remarkable contribution, Fleming, Florey, and Chain received the Nobel Prize in 1945 for the
discovery and production of penicillin (Lansing et al., 2005).
The great success of penicillin boosted Waksman to conduct experiments for the
production of antibiotics. Within a few months of the discovery of penicillin, Waksman dis-
covered streptomycin in 1944 (Schatz et al., 1944; Kingston, 2008). Mayo Clinic found that
streptomycin surpassed the activity of inhibiting tuberculosis more than what penicillin did
(Kiple, 1993). The journey of antibiotics began then; at present, various antibiotics are avail-
able, which are obtained through either a natural or synthetic route. In this chapter, we will
discuss a broad classification of antibiotics and some major classes of antibiotics.
1.2 Classification of antibiotics

Antibiotics are classified mainly by the following bases:
1. Mechanism of action: An antibiotic may be bactericidal or bacteriostatic. The bacteri-

cidal antibiotics are the ones that kill the harmful microorganism, while the bacterio-
static antibiotics stop or slow down bacterial growth.
The bacterial damage is done by either interrupting cell wall synthesis, protein
synthesis, DNA synthesis, RNA synthesis, mycolic acid synthesis, or folic acid

synthesis.
2. Spectrum: Antibiotics are either narrow or wide spectrum.
3. Route of administration of the drug.
4. Class: This includes aminoglycosides, ansamycins, carbacephem (discontinued), car-
bapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides,
monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quinolones/
fluoroquinolones, sulfonamides, tetracyclines, and drugs against mycobacteria
(Sunil and Kirti, 2013).
1.2.1 Aminoglycosides
The aminoglycosides are a large, diverse, and key antibiotic, most popular during the
1970s and 1980s (Bosso et al., 2013). The aminoglycoside antibiotics are either derived
from Streptomyces spp. (streptomycin, neomycin, and tobramycin) or Micromonospora spp.
Chapter one: Antibiotics 3
(gentamicin) or synthesized in vitro (netilmicin, amikacin, arbekacin, and isepamicin)

(Durante et al., 2009). Aminoglycosides derived from bacteria of the Streptomyces genus
are named with the suffix mycin, whereas those that are derived from Micromonospora are
named with the suffix micin (Dewick, 2002; Kroppenstedt et al., 2005).
The first discovered aminoglycoside was streptomycin (Figure 1.1) by Waksman in
1944, which was obtained from Streptomyces griseus (Comroe, 1978). Streptomycin became
an even more famous antibiotic during that era due to its activity that surpassed that of
penicillin in inhibiting tuberculosis (Kiple, 1993). Soon after, another aminoglycoside,
neomycin, was discovered by Waksman, which was obtained from Streptomyces fradiae
in 1949. Neomycin contains two or more amino sugars connected by glycosidic bonds
typically used as a topical preparation, such as neosporin. For this extraordinary contribu-
tion toward the field of physiology and medicine, Waksman was awarded with the Nobel
Prize in 1951 (Schatz et al., 1944; Kingston, 2008). Similarly, kanamycin from Streptomyces
kanamyceticus, paromomycin from Streptomyces krestomuceticus (Garrod et al., 1981), gen-
tamicin from Micromonospora purpurea (Weinstein et al., 1963), and sisomicin (Figure 1.2)
from Micromonospora were obtained (Weinstein et al., 1970).
OH
H2N
HO O OH
NH2
HO HO N
O OH
HN
O
H3C O N
OHC NH2
OH CH3
NH2
Figure 1.1 Structure of streptomycin.
OH
OH
OH CH3
HO O HO OH H3C O H2N
H2N O HN HO O
HO
HO O NH2 H3C HOO NH2
O O
H2N NH2 H2N NH2
(a) (c)
OH
HO O
HO
H2N
NH2
HO OO NH2
OH
O OH
H3C O H2N
HN O
H2N NH2 O OH HO
H3C OH
HO O O NH2
HO O H2N NH2
(b) (d)
Figure 1.2 Structure of (a) kanamycin, (b) paromomycin, (c) gentamicin, and (d) sisomicin.
Structurally, aminoglycosides contain two or more amino sugars linked by glycosidic

bonds to an amino cyclitol component. The cyclitol is 2-deoxystreptamine in most cases,
one exception being streptomycin, which has a streptidine moiety streptomycin, which
has a streptidine moiety (David, 2000).
The antimicrobial spectrum among aminoglycosides varies, for example, tobramy-
cin is effective against the species of Pseudomonas, while kanamycin is effective against
Escherichia coli, Proteus spp., Serratia marcescens, and Klebsiella pneumoniae. Paromomycin is
used in treating intestinal infections such as cryptosporidiosis, amebiasis, and leishmani-
asis (Sundar et al., 2007). Gentamicin is more active than tobramycin against Serratia spp.,
while tobramycin has superior activity against Pseudomonas aeruginosa. Isepamicin exhib-
ited twofold to fourfold higher in vitro activities than amikacin against Enterobacteriaceae
(Cheng et al., 2005).
Aminoglycosides exhibit several characteristics that make them useful as antimicro-
bial agents:
1.
They show concentration-dependent bactericidal activity: The killing potential of amino-
glycosides increases with increasing concentrations of the antibiotic (Begg et al., 1992).
2.
They show the postantibiotic effect: They continue to kill bacteria, even after the amino-
glycoside has been removed, following a short incubation with the microorganism
(Hessen et al., 1989).
3.
Synergism with other antibiotics: They show enhanced bactericidal activity in com-
bination with antimicrobial agents. Synergism presumably arises as the result of
enhanced intracellular uptake of aminoglycosides caused by the increased perme-
ability of bacteria after incubation with cell wall synthesis inhibitors (Moellering and
Weinberg, 1971).
The recent emergence of infections due to gram-negative bacterial strains with advanced
patterns of antimicrobial resistance has prompted physicians to reevaluate the use of
these antibacterial agents. Current evidence shows that aminoglycosides do retain activity
against the majority of gram-negative clinical bacterial isolates in many parts of the world.
Still, the relatively frequent occurrence of nephrotoxicity and ototoxicity during aminogly-
coside treatment makes physicians reluctant to use these compounds in everyday practice.
Recent advances in the understanding of the effect of various dosage schedules of ami-
noglycosides on toxicity have provided a partial solution to this problem, although more
research still needs to be done in order to overcome this problem entirely (Maurin and
Raoult, 2001).
1.2.1.1 Aminoglycoside mode of action

The mode of aminoglycoside action is through inhibition of protein synthesis via bind-
ing to the bacterial ribosome. Aminoglycosides first cross the bacterial cell walls. A bac-
terial ribosome complex structure usually comprises three RNA molecules and more
than 50 proteins. This complex catalyzes protein synthesis with the assistance of several
GTP-hydrolyzing protein factors. The bacterial ribosome consists of two subunits with
relative sedimentation rates of 50S and 30S. The large subunit comprises two RNA mol-
ecules, referred to as the 5S and 23S RNAs, and 33 proteins, while the small subunit is
made up of a single 16S RNA and 20–21 proteins. The ribosome has three functionally
important tRNA binding sites, designated A (for aminoacyl), P (for peptidyl), and E (for
exit). During protein synthesis, the ribosome decodes information stored in the mRNA
and catalyzes sequential incorporation of amino acids into a growing polypeptide chain.
Aminoglycoside antibiotics bind to the 30S ribosomal subunit (some work by binding to
the 50S subunit), inhibiting the translocation of the peptidyl-tRNA from the A-site to the
P-site and also causing misreading of mRNA, leaving the bacterium unable to synthesize
proteins vital to its growth, leading to the death of bacteria (Sergei and Shahriar, 2003).
1.2.2 Penicillin
Penicillin (Figure 1.3) was the first β-lactam antibiotic that was discovered and the most
important antibiotic of this group. Although penicillin was announced over eight decades
ago, it is of greater use today than it has ever been (Ozcengiz and Demain, 2013).
The parent structure of all penicillins is composed of a β-lactam ring condensed to
a thiazoline ring. Connected to this common backbone structure are characteristically
different amide-bonded side chains present in every member of this class (Weltzien and
Padovan, 1998). Penicillins include natural penicillins, penicillinase-resistant penicillins,
aminopenicillins, and beta-lactam–beta-lactamase inhibitor combinations.
Natural penicillin consists of penicillin V, while penicillinase-resistant p
enicillins include
methicillin, oxacillin, and nafcillin (Figure 1.4). Penicillinase-resistant penicillins were devel-
oped because of the increasing resistance of Staphylococci to natural penicillins. Penicillinase-
resistant penicillins are modified penicillins, which have a side chain that inhibits the action
of penicillinase.
The aminopenicillins includes ampicillin and amoxicillin (Figure 1.5); these amino
penicillins were the first penicillins discovered to be active against gram-negative rods
such as E. coli and Haemophilus influenzae. Beta-lactam–beta-lactamase inhibitor com-
binations include clavulanate, sulbactam, and tazobactam; this combination drug pro-
vides increased antimicrobial coverage of beta-lactamase–producing strains (Wright and
Wilkowske, 1987).
H H
R N
S
CH3
O N CH3
O
COOH
Figure 1.3 Structure of penicillin where “R” is the variable group.
O N O
H H O
N H H H H
S N N
O S S
O O O
N N N
O O O
O OH OH
HO O O
(a) (b) (c)
Figure 1.4 Structure of (a) methicillin, (b) oxacillin, and (c) nafcillin.
NH2
NH2
H H H H H
N N
S CH3 HO S
H O
O N CH3 N
O H O
OH OH
O O
(a) (b)
Figure 1.5 Structure of (a) ampicillin and (b) amoxicillin.
1.2.2.1 Penicillin mode of action

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that cata-
lyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. Penicillin
inhibits the formation of peptidoglycan cross-links in the bacterial cell wall that is achieved
through the binding of the four-membered β-lactam ring of penicillin to the enzyme
DD-transpeptidase. As a result, the DD-transpeptidase cannot catalyze formation of these
cross-links and an imbalance between cell wall productions. In addition, the buildup of
peptidoglycan precursors triggers the activation of bacterial cell wall hydrolases and auto-
lysins, which further digest the bacteria’s existing peptidoglycan causing the cell to die
rapidly (Yocum et al., 1980).
1.2.3 Cephalosporin
The basic structure of both penicillins and cephalosporins consists of a four-member
β-lactam ring. In penicillins, this ring is condensed with a five-member sulfur ring (the
thiazolidine ring) and in cephalosporins with a six-member ring (the dihydrothiazine
ring). Cephalosporins (Figure 1.6) can be classified according to different criteria, such
as their metabolism and stability to the action of beta-lactamases, the substitution of the
R2 side chain, their pharmacokinetic properties, or their microbial properties, mainly
related to their antibacterial spectrum.
Group I cephalosporins include cefadroxil, cefacetrile, and cephalexin (Figure 1.7);
these antibiotics have greater activity against gram-positive bacteria. Group II cephalo-
sporins include cefaclor, cefonicid, cefprozil, and cefuroxime (Figure 1.8). Unlike Group
I cephalosporins, these antibiotics also have greater activity against gram-negative bac-
teria. Group III cephalosporin includes cefcapene, cefdaloxime, and cefdinir (Figure 1.9).
These cephalosporins are active against P. aeruginosa. Group IV includes cefepime, which
is active against anaerobic bacteria.
From the clinical viewpoint, the most useful classification divides cephalosporins
according to their historical development plus their common microbiologic and struc-
tural characteristics. Within this classification, according to different generations of
R2 H H
N S
O
N
R1
O
O OH
Figure 1.6 Core structure of cephalosporins.

HO O
HO O
O O
N O
O O N
OH O
S N
H H S N N
H2N H H
(a) (b)
NH2
H H
N S
O
N
O
O OH
(c)
Figure 1.7 Group I cephalosporins: (a) cefadroxil, (b) cefacetrile, and (c) cephalexin.
NH2
HO O
H H N N
N S N O
O N S N O
N O
O CI O S S N
HO H H
O OH OH
(a) (b)
O OH
O N O
N
O O H
OH H
S N S
H N
H O
H2N N O NH2
O
O
(c) (d) O OH
Figure 1.8 Group II cephalosporins: (a) cefaclor, (b) cefonicid, (c) cefprozil, and (d) cefuroxime.
cephalosporins, besides the well-known first-, second-, and third-generation cephalospo-

rins, we now include the fourth generation for which newly developed molecules are con-
tinually being added (Perez Inestrosa et al., 2005).
1.2.3.1 Cephalosporin mode of action

Cephalosporins, like other β-lactams, bind to the bacterial penicillin-binding proteins
(PBPs). These correspond to the d-ala-d-ala trans-, carboxy-, and endopeptidases responsible
for catalyzing the cross-linking of newly formed peptidoglycan. Resistance arises when
the PBPs and particularly the transpeptidases are modified or when they are protected by
β-lactamases or permeability barriers. Target-mediated cephalosporin resistance can involve
either the reduced affinity of existing PBPs component or the acquisition of a supplemen-
tary β-lactam-insensitive PBP (Livermore, 1987).
N OH
S H H S H H
N S N S
N N
H2N O H 2N O
N O NH2 N O
O O
O
(a) O OH (b) O OH
OH
N
H H H
N N S
H2N
S O N
O
(c) O OH
Figure 1.9 Group III cephalosporins: (a) cefcapene, (b) cefdaloxime, and (c) cefdinir.
1.2.4 Carbapenems
Carbapenems (Figure 1.10) are unique among the β-lactam antibiotics because of their
extremely broad spectrum of antibacterial activity that encompasses aerobic and anaero-
bic gram-positive and gram-negative bacteria (Neu, 1985). They have a structure that ren-
ders them highly resistant to most β-lactamases (Livermore and Woodford, 2000).
The carbapenems differ from the penicillins in having an unsaturated bond between
C2 and C3 and a carbon atom replacing sulfur at position 1 of the thiazolidine ring.
Various carbapenems differ primarily in the configuration of the side chains at C2 and
C6. Carbapenems include thienamycins, olivanic acids, carpetimycins, asparenomycins,
pluracidomycins, and other natural and semisynthetic compounds (Moellering et al., 1989).
Thienamycin was the first carbapenem from Streptomyces cattleya obtained and
would eventually serve as the parent or model compound for all carbapenems (Krisztina
et al., 2011). However, thienamycin could not be marketed due to chemical and biological
instabilities. Many carbapenem compounds have long been considered unstable even in
neutral conditions, much less in gastric juice and/or intestinal juice. To overcome these
problems, many groups started to develop stable compounds and ways of synthesizing
thienamycin. Under these circumstances, imipenem (2) containing cilastatin as an inhibi-
tor of renal dehydropeptidase I was marketed by Merck & Co., Inc. Next, the Sankyo
group marketed panipenem containing betamipron for the alleviation of nephrotoxicity
(Kumagai et al., 2002).
R2
R1 H
R3
N
O
COOH
Figure 1.10 Structure of the carbapenem backbone.

1.2.4.1 Carbapenem mode of action

Carbapenems enter gram-negative bacteria through outer membrane proteins, also known
as porins. After transversing into the periplasmic space, carbapenems permanently acyl-
ate the PBPs. PBPs are enzymes (i.e., transglycosylases, transpeptidases, and carboxypep-
tidases) that catalyze the formation of peptidoglycan in the cell wall of bacteria. Current
insights into this process suggest that the glycan backbone forms a right-handed helix
with a periodicity of three per turn of the helix. Carbapenems act as mechanism-based
inhibitors of the peptidase domain of PBPs and can inhibit peptide cross-linking as well
as other peptidase reactions. A key factor of the efficacy of carbapenems is their ability to
bind to multiple different PBPs. Since cell wall formation is a dynamic 3D process with
the formation and autolysis occurring at the same time, when PBPs are inhibited, autolysis
continues. Eventually, the peptidoglycan weakens, and the cell bursts due to osmotic pres-
sure (Krisztina et al., 2011).
1.2.5 Glycopeptides
Glycopeptide antibiotics represent a very important group of natural products with
regard to medicinal applications as antibacterial and anticancer agents. Glycopeptides
include vancomycin, teicoplanin, ramoplanin, bleomycin, mannopeptimycin, and
salmochelin (Falko et al., 2007). The first glycopeptide discovered was vancomycin
(Figure 1.11) isolated from a strain of Amycolatopsis orientalis found in a soil sample
collected in Borneo. Similarly, teicoplanin A2 (lipoglycopeptide antibiotic) obtained
from Actinoplanes teichomyceticus was also introduced for clinical use in the mid-1980s
(Grace et al., 2014).
Structurally, glycopeptides are complex molecules of unique structure that has a cen-
tral, relatively conserved heptapeptide domain in which five of the seven amino acid
residues are common to all glycopeptides but differ in the amino acids at positions 1 and
3 and in the substituents of the aromatic amino acid residues. Some of the carbons of the
OH
HO
OH
O OH
NH2 HN
NH O O O
H H H O
N N N
N N
H H
O O O
HO OH
O O
CI CI
O O
HO
HO O
OH
O
H2N
OH
Figure 1.11 Structure of vancomycin.

aromatic residue carry chlorine, hydroxyl, or methyl groups, and some of the hydroxyl
groups are substituted with sugar or amino sugar. The presence of phenolic residue per-
mits the formation of two- and three-ring structures in all glycopeptides. Such interac-
tion results in a large group of molecule with very similar rigid 3D structures. Those
compounds that have rings 1 and 3 joined in addition to 2, 4 and 6, and 5 and 7 have been
shown to adopt a bracelet-like configuration with a substantial cleft in the molecule into
which the bacterial site binds with exquisite position. Some glycopeptides, like teicopla-
nins, have the amino group of amino sugar substituted with a fatty acid chain containing
9–11 carbon atoms (Reynolds, 1989).
1.2.5.1 Glycopeptide mode of action

Biochemical studies indicate that glycopeptides inhibit the late stages of peptidoglycan
synthesis (Reynolds, 1989). During cell wall synthesis, muramylpentapeptide forms in the
cell cytoplasm, and an N-acetylglucosamine unit is added, together with any amino acids
needed for the interpeptide bridge of gram-positive organisms. It is then passed to a lipid
carrier molecule, which transfers the whole unit across the cell membrane to be added to
the growing end of the peptidoglycan macromolecule. The addition of the new building
block is prevented by the glycopeptides vancomycin and teicoplanin, which bind to the
acyl-d-alanyl-d-alanine tail of the muramylpentapeptide (Finch et al., 2011). For the rigid
polymer that protects bacterial cells from osmotic lysis, by binding to this terminal dipep-
tide, glycopeptide antibiotics interfere with the proper cell wall formation, which results
in eventual death of the cell (Marshall et al., 1998).
1.2.6 Lincosamides
Lincosamides are a class of antibiotics that include clindamycin, lincomycin, and pirli-
mycin (Figure 1.12). Among them, lincomycin was the first antibiotic to be produced by
Streptomyces lincolnensis isolated from a soil sample from Lincoln, NE (Chang et al., 1966).
Lincomycin proved to be a member of a new class of antibiotics characterized by an alkyl
6-amino-6,8-dideoxy-1-thio-d-erythro-α-d-galacto-octopyranoside joined with a proline
moiety by an amide linkage.
Lincomycin has gained clinical acceptance as a major antibiotic for the treatment of
diseases caused by gram-positive microbes including pathogenic Streptococci, Staphylococci,
and Mycoplasma (Spizek and Rezanka, 2004). While clindamycin is used to treat infec-
tions with anaerobic bacteria, it can also be used to treat some protozoal diseases, such
as malaria. It is a common topical treatment for acne and can be useful against some
CH3
S CI HO
O O OH CI
HO CH3 H H
N OH O
HO H
HO HN O N
N O S
H N
O
H
N CH3 OH
SCH3 HO OH
OH
CH3
(a) (b) (c)
Figure 1.12 Structure of (a) clindamycin, (b) lincomycin, and (c) pirlimycin.
methicillin-resistant Staphylococcus aureus infections (Daum, 2007). Pirlimycin is active

against gram-positive bacteria, specifically Staphylococcus aureus and coagulase negative
species of Staphylococcus and Streptococcus.
1.2.6.1 Lincosamide mode of action

Lincosamides prevent bacteria replication by interfering with the synthesis of proteins.
They bind to the 23S portion of the 50S subunit of bacterial ribosomes and cause prema-
ture dissociation of the peptidyl-tRNA from the ribosome (Tanel et al., 2003).
1.2.7 Macrolides
Macrolides, which are primarily antibiotics, belong to the polyketide group of natural
products. They derive their name from their characteristic structural features, a macro-
cyclic lactone ring to which various deoxy sugars, most commonly cladinose and deso-
samine, are attached (Steel et al., 2012). Macrolides are composed of a 12- to 16-member
macrolactone ring decorated with various amino sugars that includes azithromycin, clar-
ithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, midecamycin, mioc-
amycin, oleandomycin, rokitamycin, roxithromycin, spiramycin, troleandomycin, tylosin,
ketolides, telithromycin, cethromycin, and solithromycin (Alexander, 2008).
Erythromycin was the first macrolide antibiotic discovered from Streptomyces erythreus
isolated from soil samples (Zuckerman, 2004) and used to treat bacteria responsible
for causing infections of the skin and upper respiratory tract, including Streptococcus,
Staphylococcus, and Haemophilus genera (Roland, 2002). Similarly, clarithromycin was
invented by researchers at the Japanese drug company Taisho Pharmaceutical in the 1970s
(Zuckerman, 2004). Animal studies have shown that clarithromycin can induce fetal loss
in rabbits and monkeys when used in very low dosages and high dosages, respectively.
One observational study concerning pregnant women showed a doubling of the num-
ber of miscarriages in women exposed to clarithromycin in early pregnancy compared
to a match control group. There is limited knowledge concerning the risk of congenital
malformations among women exposed to clarithromycin during pregnancy (Andersen
et al., 2013).
Azithromycin is another macrolide antibiotic derived from erythromycin, which was
discovered by a team of researchers at the Croatian pharmaceutical company PLIVA (1980).
This product has a high bacteriostatic action in front of a wide spectrum of pathogenic
bacteria and is used mainly for the treatment of respiratory and dermatological infections
(Banic, 2011; Timoumi et al., 2014).
Bacteria resist macrolide antibiotics in three ways: (1) through target-site modification
by methylation or mutation that prevents the binding of the antibiotic to its ribosomal
target, (2) through efflux of the antibiotic, and (3) by drug inactivation. These mechanisms
have been found in the macrolide producers, which often combine several approaches to
protect themselves against the antimicrobial that they produce. In pathogenic microorgan-
isms, the impact of the three mechanisms is unequal in terms of incidence and of clinical
implications (Roland, 2002).
1.2.7.1 Macrolide mode of action

Macrolides reversibly bind to domain V of 23S ribosomal RNA of the 50S subunit of the bac-
terial ribosome inhibiting RNA-dependent protein synthesis (Douthwaite and Champney,
2001).
Acknowledgments
This research was supported by the Key Project of International Cooperation of Ministry
of Science and Technology (No. 2013DFA31980) and the Key Project of Yunnan Provincial
Natural Science Foundation (2013FA004). W.-J. Li was also supported by the Hundred Talents
Program of the Chinese Academy of Sciences and the Pearl River Scholar Funded Scheme
of the Higher Vocational Colleges & Schools of Guangdong Province (2014).
References
Alexander, S.M. 2008. Macrolide myths. Curr. Opin. Microbiol., 11, 414–421.
Andersen, J.T., Petersen, M., Jimenez-Solem, E., Broedbaek, K., Andersen, N.L., Torp-Pedersen,
C., Keiding, N., and Poulsen, H.E. 2013. Clarithromycin in early pregnancy and the risk
of miscarriage and malformation: A register based nationwide cohort study. PLoS ONE, 8,
e53327, 1–6.
Banic, T.Z. 2011. The story of azithromycin. Kemija u Industriji, 60, 603–617.
Begg, E.J., Peddie, B.A., Chambers S.T., and Boswell, D.R. 1992. Comparison of gentamicin dosing
regimens using an in-vitro model. J. Antimicrob. Chemother., 29, 427–433.
Bosso, J.A., Haines, L.H., and Gomez, J. 2013. Stable susceptibility to aminoglycosides in an age of
low level, institutional use. Infect. Dis. Ther., 2, 209–215.
Chang, F.N., Sih, C.J., and Weisblum, B. 1966. Lincomycin, an inhibitor of aminoacyl sRNA binding
to ribosomes. Proc. Natl. Acad. Sci. USA, 55, 431–438.
Cheng, N.C., Hsueh, P.R., Liu, Y.C., Shyr, J.M., Huang, W.K., Teng, L.J., and Liu, C.Y. 2005. In vitro
activities of tigecycline, ertapenem, isepamicin, and other antimicrobial agents against clini-
cally isolated organisms in Taiwan. Microb. Drug Resist., 11, 330–341.
Comroe J.H. Jr. 1978. Pay dirt: The story of streptomycin. Part I: From Waksman to Waksman. Am.
Rev. Respir. Dis., 117, 773–781.
Daum, R.S. 2007. Clinical practice, skin and soft-tissue infections caused by methicillin-resistant
Staphylococcus aureus. N. Engl. J. Med., 357, 380–390.
David, A.S. 2000. Current methodologies for the analysis of aminoglycosides. J. Chromatogr. B, 747,
69–93.
Dewick, P.M. 2002. Medicinal Natural Products: A Biosynthetic Approach. England, U.K.: John Wiley &
Sons.
Douthwaite, S. and Champney, W.S. 2001. Structures of ketolides and macrolides determine their
mode of interaction with the ribosomal target site. J. Antimicrob. Chemother., 48, Topic T1, 1–8.
Durante, M.E., Grammatikos, A., Utili, R., and Falagas, M.E. 2009. Do we still need the aminoglyco-
sides? Int. J. Antimicrob. Agents., 33, 201–205.
Falko, W, Sebastian, S., and Roderich, D.S. 2007. Synopsis of structural, biosynthetic, and chemical
aspects of glycopeptide antibiotics. Top. Curr. Chem., 267, 143–185.
Finch, R.G., Greenwood, D., Whitley, R.J., and Norrby, S.R. 2011. Antibiotic and Chemotherapy: Anti-
Infective Agents and Their Use in Therapy, 9th edn. Edinburgh, U.K.: Churchill-Livingstone,
Elsevier Health Sciences.
Fleming, A. 1929. On the antibacterial action of cultures of a Penicillium, with special reference to
their use in the isolation of B. influenza. Br. J. Exp. Pathol., 3, 226–236.
Foster, W. and Raoult, A. 1974. Early descriptions of antibiosis. J. Roy. Coll. Gen. Pract., 24, 889–894.
Garrod, L.P., Lambert, H.P., and O’Grady, F. 1981. Antibiotic and Chemotherapy, 5th edn. Edinburgh,
U.K.: Churchill-Livingstone, Elsevier Health Sciences.
Grace, Y., Maulik, N.T., Kalinka, K., and Gerard, W. 2014. Glycopeptide antibiotic biosynthesis.
J. Antibiot., 67, 31–41.
Hessen, M.T., Pitsakis P.G., and Levison, M.E. 1989. Postantibiotic effect of penicillin plus gentamicin
versus Enterococcus faecalis in vitro and in vivo. Antimicrob. Agents. Chemother., 33, 608–611.
Kingston, W. 2008. Irish contributions to the origins of antibiotics. Ir. J. Med Sci., 177, 87–92.
Kiple, K.F. 1993. Cambridge World History of Human Disease. Cambridge, U.K.: Cambridge University
Press.
Krisztina, M.P.W., Andrea, E., Magdalena, A.T., and Robert, A.B. 2011. Carbapenems: Past, present,
and future. Antimicrob. Agents. Chemother., 55(11), 4943–4960.
Kroppenstedt, R.M., Mayilraj, S., and Wink, J.M. 2005. Eight new species of the genus Micromonospora,
Micromonospora citrea sp. nov., Micromonospora echinaurantiaca sp. nov., Micromonospora
echinofusca sp. nov. Micromonospora fulviviridis sp. nov., Micromonospora inyonensis sp. nov.,
Micromonospora peucetia sp. nov., Micromonospora sagamiensis sp. nov., and Micromonospora
viridifaciens sp. nov. Syst. Appl. Microbiol., 28, 328–339.
Kumagai, T., Tamai, S., Abe, T., and Hikda, M. 2002. Current status of oral carbapenem development.
Curr. Med. Chem. Anti-Infect. Agents, 1, 1–14.
Landsberg, H. 1949. Prelude to the discovery of penicillin. Isis, 40, 225–257.
Lansing, M.P., John, P.H., and Donald, A. K. 2005. Microbiology. New York: McGraw-Hill.
Livermore, D.M. 1987. Mechanisms of resistance to cephalosporin antibiotics. Drugs, 34(Suppl. 2), 64–88.
Livermore, D.M. and Woodford, N. 2000. Carbapenemases: A problem in waiting? Curr. Opin.
Microbiol., 3, 489–495.
Marshall, C.G., Lessard, I.A., Park, I., and Wright, G.D. 1998. Glycopeptide antibiotic resistance genes
in glycopeptide-producing organisms. Antimicrob. Agents. Chemother. 42, 2215–2220.
Maurin, M. and Raoult, D. 2001. Use of aminoglycosides in treatment of infections due to intracel-
lular bacteria. Antimicrob. Agents. Chemother., 45, 2977–2986.
Moellering, R.C. Jr., Eliopoulos, G.M., and Sentochnik, D.E. 1989. The carbapenems: New broad spec-
trum beta-lactam antibiotics. J. Antimicrob. Chemother., 24(Suppl. A), 1–7.
Moellering, R.C. Jr. and Weinberg, A.N. 1971. Studies on antibiotic synergism against enterococci.
II. Effect of various antibiotics on the uptake of 14C-labeled streptomycin by enterococci. J. Clin.
Investig., 50, 2580–2584.
Neu, H.C. 1985. Carbapenems: Special properties contributing to their activity. Am. J. Med., 78, 33–40.
Ozcengiz, G. and Demain, A.L. 2013. Recent advances in the biosynthesis of penicillins, cephalospo-
rins and clavams and its regulation. Biotechnol. Adv., 31, 287–311.
Perez Inestrosa, E., Suau, R,. Montanez, M.I., Rodriguez, R., Mayorga, C., Torres, M.J., and Blanca,
M. 2005. Cephalosporin chemical reactivity and its immunological implications. Curr. Opin.
Allergy Clin. Immunol., 5, 323–330.
Reynolds, P.E. 1989. Structure, biochemistry and mechanism of action of glycopeptides antibiotics.
Eur. J. Clin. Microbiol. Infect. Dis., 8, 943–950.
Roland, L. 2002. Mechanisms of resistance to macrolides and lincosamides: Nature of the resistance
elements and their clinical implications. Clin. Infect. Dis., 34(4), 482–492.
Schatz, A., Bugie, E., and Waksman, S.E. 1944. Streptomycin, a substance exhibiting antibiotic activ-
ity against gram-positive and gram negative bacteria. Proc. Soc. Exp. Biol. Med., 55, 65–69.
Sergei, B.V. and Shahriar, M. 2003. Versatility of aminoglycosides and prospects for their future.
Clin. Microbiol. Rev., 16, 430–450.
Spizek, J. and Rezanka, T. 2004. Lincomycin, cultivation of producing strains and biosynthesis. Appl.
Microbiol. Biotechnol., 63, 510–519.
Steel, H.C., Theron, A.J., Cockeran, R., Anderson, R., and Feldman, C., 2012. Pathogen- and
host-directed anti-inflammatory activities of macrolide antibiotics. Mediat. Inflamm., Article
ID 584262.
Sundar, S., Jha, T.K., Thakur, C.P., Sinha, P.K., and Bhattacharya, S.K. 2007. Injectable paromomycin
for visceral leishmaniasis in India. N. Engl. J. Med., 356, 2571–2581.
Sunil, T.P. and Kirti, M.J. 2013. Antibiotics—A Manifold Cover Story, 1st edn. Toronto, Ontario, Canada:
Canadian Academic Publishing.
Tanel, T., Martin, L., and Mans, E. 2003. The mechanism of action of macrolides, lincosamides
and streptogramin B reveals the nascent peptide exit path in the ribosome. J. Mol. Biol., 330,
1005–1014.
Timoumi, S., Mangin, D., Peczalski, R., Zagrouba, F., and Andrieu, J. 2014. Stability and thermo-
physical properties of azithromycin dehydrate. Arabian J. Chem., 7(2), 189–195.
Vuillemin, P. 1889. Antibiose et symbiose, Assoc. franc. pour l’Avanc. des Sciences. Paris 2: 525–542.
Waksman, S.A. 1947. What is an antibiotic or an antibiotic substance? Mycologia, 39, 565–569.
Weltzien, H.U. and Padovan, E. 1998. Molecular features of penicillin allergy. J. Invest. Dermatol., 110,
203–206.
Weinstein, M.J., Luedemann, G.M., Oden, E.M., Wagman, G.H., Rosselet, J.P., Marquez, J.A., and
Black, J. 1963. Gentamicin, a new antimicrobial complex from Micromonospora. J. Med. Chem.,
6, 463–464.
Weinstein, M.J., Marquez, J.A., Testa, R.T., Wagman, G.H., Oden, E.M., and Waitz, J.A. 1970. Antibiotic
6640, a new Micromonospora-produced aminoglycoside antibiotic. J. Antibiot., 23, 551–554.
Wright, A.J. and Wilkowske, C.J. 1987. The penicillins. Mayo. Clin. Proc., 62, 806–820.
Yocum, R.R., Rasmussen, J.R., and Strominger, J.L. 1980. The mechanism of action of penicillin,
penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.
J. Biol. Chem., 255, 3977–3986.
Zuckerman, J.M. 2004. Macrolides and ketolides: Azithromycin, clarithromycin, telithromycin.
Infect. Dis. Clin. N. Am., 18, 621–649.
section one
Broad spectrum antimicrobial

compounds from microorganisms
chapter two
Antimicrobial potential
of marine actinobacteria
A review
Panchanathan Manivasagan and Se-Kwon Kim
Contents
2.1 Introduction........................................................................................................................... 17
2.2 Marine microorganisms...................................................................................................... 19
2.3 Antimicrobial activity.......................................................................................................... 19
2.3.1 Antibacterial activity................................................................................................ 19
2.3.2 Antifungal activity................................................................................................... 23
2.4 Conclusion............................................................................................................................. 24
Acknowledgment........................................................................................................................... 24
References........................................................................................................................................ 24
2.1 Introduction
Marine actinobacteria are the most economically and biotechnologically valuable prokary-
otes. They are able to produce a wide range of bioactive secondary metabolites such as
antibiotics, antitumor agents, immunosuppressive agents, and enzymes. These metabolites
are known to possess antibacterial, antifungal, neuritogenic, anticancer, antialgal, antima-
larial, and anti-inflammatory activities (Ravikumar et al., 2011). Actinobacteria have the
capacity to synthesize many different biologically active secondary metabolites such as
cosmetics, vitamins, nutritional materials, herbicides, antibiotics, pesticides, antiparasitic
agents, and enzymes used in waste treatment like cellulase and xylanase (Ogunmwonyi
et al., 2010). They are free-living, saprophytic bacteria and a major source for the produc-
tion of antibiotics (Atta et al., 2009).
Natural products are a boundless source for important novel compounds having
antagonistic activity against pathogenic organisms. The marine environment covers
almost 70% of the earth surface (Valli et al., 2012). Organisms present in these environ-
ments are extremely rich sources of bioactive compounds (Solanki et al., 2008; Hong et al.,
2009). The ocean remains an unexploited source for many drugs and pharmacologically
active substances (Sivasubramanian et al., 2011). Actinobacteria are gram-positive, fila-
mentous bacteria that are supreme secondary metabolite producers (Olano et al., 2009).
Among the members of the Actinomycetes genus, Streptomyces sp. are a dynamic producer
of f unctional, bioeffective metabolites with a broad pharmaceutical range having antimi-
crobial, antihelminthic, antitumor, and antiviral properties (Ravikumar et al., 2011; Reddy
et al., 2011).
17
Multidrug resistance in microorganisms is an emerging serious problem in the health-

care sector. The improper usage of antibiotics plays a major role in drug resistance in pathogenic
microbes. Microorganisms acquire resistance toward common antibiotics by altering their
metabolism and genetic structure (Raghunath, 2008; Rao, 2012). There is an incessant need
to find novel efficient drug molecules against multidrug-resistant microbes. Actinobacteria
from terrestrial origin produce hundreds of antibiotics that are widely used at present. Some
differences could be expected among organisms existing in marine and terrestrial environ-
ments due to variation in physical, chemical, and biological factors (Saurav and Kannabiran,
2010). It is apparent that the marine environment is a potent source for finding new actino
bacteria and new antibiotics or biologically active substances (Gärtner et al., 2011). In this
chapter, we focus on the antimicrobial potential of marine actinobacteria and classify them in
terms of their unique chemical structures (Table 2.1), covering the literature to date.
Table 2.1 Antimicrobial compounds produced by marine actinobacteria
Other biological
Compound Species activity References
Antibacterial activity
Abyssomicins Verrucosispora sp. — Riedlinger et al. (2004)
Bonactin Streptomyces sp. Antifungal Schumacher et al. (2003)
Chloro-dihydroquinones Streptomyces sp. Anticancer Soria-Mercado et al.
(2005)
Diazepinomicin Micromonospora sp. Anticancer; Charan et al. (2004)
anti-inflammatory
Frigocyclinone Streptomyces griseus — Bruntner et al. (2005)
Essramycin Streptomyces sp. — El-Gendy et al. (2008)
Lynamicins Marinispora sp. — McArthur et al. (2008)
Marinopyrroles Streptomyces sp. Cytotoxic Hughes et al. (2008)
Caboxamycin Streptomyces sp. Cytotoxic Hohmann et al. (2009)
Himalomycins Streptomyces sp. — Maskey et al. (2003b)
Marinomycins Marinispora Antifungal; anticancer Kwon et al. (2006)
Glyciapyrroles Streptomyces sp. — Macherla et al. (2005)
Tirandamycin Streptomyces sp. — Carlson et al. (2009)
Bisanthraquinone Streptomyces sp. — Socha et al. (2006)
Gutingimycin Streptomyces sp. — Maskey et al. (2004)
Helquinoline Janibacter limosus — Asolkar et al. (2004)
Lajollamycin Streptomyces nodosus — Manam et al. (2005)
TP-1161 Nocardiopsis sp. — Engelhardt et al. (2010)
Lincomycin Streptomyces — Peschke et al. (2006)
lincolnensis
Tirandamycins Streptomyces sp. — Carlson et al. (2009)
1,4-Dihydroxy-2-(3- Streptomyces sp. — Ravikumar et al. (2012)
hydroxybutyl)-9,
10-anthraquinone 9,
10-anthrac
Antifungal activity
Chandrananimycin Actinomadura sp. Antialgal; antibacterial; Maskey et al. (2003b)
anticancer
N-(2-hydroxyphenyl)-2- Nocardia dassonvillei Anticancer Gao et al. (2012)
phenazinamine (NHP)
Chapter two: Antimicrobial potential of marine actinobacteria 19
2.2 Marine microorganisms

Marine microorganisms are increasingly becoming a potential source in the search for
industrially important biomolecules. Today both academic and industrial interests in
marine microorganisms are on the rise because unique and bioactive metabolites have
been reported from marine organisms (Jensen and Fenical, 1994; Imada, 2004; Zhang et al.,
2005; Manivasagan et al., 2013, 2014b). Actinobacteria are present in various ecological hab-
itats such as soil, freshwater, backwater, lake, compost, sewage, and marine environments
(Goodfellow and Williams, 1983; Manivasaganet al., 2014c). They are considered highly
valuable as they produce various antibiotics and other therapeutically useful compounds
with diverse biological activities. The vast majority of these metabolites (70%) have been
isolated from actinobacteria with the remaining 20% from fungi, 7% from Bacillus sp., and
1%–2% from Pseudomonas sp. Hence, it is known that the actinobacteria are perhaps the
most important group of organisms studied extensively for the discovery of drugs and
other bioactive metabolites program (Prabavathy et al., 2006).
The marine environment contains a wide range of distinct microorganisms that are
not present in the terrestrial environment. Though some reports are available on antibi-
otic and enzyme production by marine actinobacteria, the marine environment is still a
potential source for new actinobacteria, which can yield novel bioactive compounds and
industrially important enzymes (Sharma and Pant, 2001). Since the late 1980s, the num-
ber of novel compounds isolated from terrestrial microorganisms has steadily decreased.
To cope up with the demand for new pharmaceutical compounds and to combat the
antibiotic-resistant pathogens, researchers have been forced to look for novel microorgan-
isms in unusual environments (Ramesh and Mathivanan, 2009).
2.3 Antimicrobial activity

Marine actinobacteria are important sources of new bioactive compounds such as antibi-
otics and enzymes (Bredholt et al., 2008; Aghamirian and Ghiasian, 2009; Manivasagan
et al., 2014a), which have diverse clinical effects and are active against many pathogenic
organisms. Actinomycetes and their bioactive compound show antibacterial and antifun-
gal effects against various multidrug-resistant pathogens such as vancomycin-resistant
Enterococci, methicillin-resistant Staphylococcus aureus, Shigella dysenteriae, Klebsiella sp., and
Pseudomonas aeruginosa (Saadoun et al., 1998; Bhatnagar and Kim, 2010). The need for new,
safe, and effective antimicrobial agent is the major challenge to the pharmaceutical indus-
try today, especially with the obvious increase in opportunistic infections in the immune-
compromised host via and multidrug-resistant strains (Bredholt et al., 2008).
2.3.1 Antibacterial activity

An antibacterial substance is an agent that inhibits bacterial growth or kills bacteria.
Infectious diseases remain one of the main causes of death due to antibiotic-resistant
microorganisms. The frequency of resistance in microbial pathogens continues to grow at
an alarming rate throughout the world (Schmitz et al., 1999). Decreased efficacy and resis-
tance of pathogens to antibiotics have necessitated the development of new alternatives
(Ravikumar et al., 2010). To overcome this problem, development of effective newer drugs
without any side effects is an urgent need.
Caboxamycin (1) (Figure 2.1) is a new benzoxazole antibiotic and was detected by
HPLC-diode array screening in extracts of Streptomyces sp. NTK 937, another strain that was
OH O
CI
H
HO O
O H
HO
O
HO
N
CI
O H
1. Caboxamycin 2. Chlorinated dihydroquinones
O
O HN N O
H O
O N N
H3C N N
H
O
3. Bisanthraquinone 4. Essramycin
Figure 2.1 Chemical structures of caboxamycin, chlorinated dihydroquinones, bisanthraquinone,

and essramycin.
isolated from the sediments collected from the Canary Basin. The compound was named
after the first letters of the collection site from where the organism was isolated and from the
letters drawn from its chemical structure. Caboxamycin showed inhibitory activity against
both gram-positive bacteria and the tumor cell lines gastric adenocarcinoma (AGS), hepato-
cellular carcinoma (Hep G2), and breast carcinoma cells (MCF7). The antibiotic also showed
an inhibitory activity against the enzyme phosphodiesterase (Hohmann et al., 2009).
Chlorinated dihydroquinones (2) (Figure 2.1) are novel antibiotics produced by a new
marine Streptomyces sp. (Soria-Mercado et al., 2005). The compounds formally possess
new carbon skeletons but are related to several previously reported metabolites of the
napyradiomycin class. Structures of the new molecules possess significant antibacterial
and cancer cell cytotoxicities. In general, marine actinobacteria are extensively studied for
antibacterial activity. Bisanthraquinone (3) (Figure 2.1) is a new antibiotic isolated from
Streptomyces sp. Biological activities were measured against clinically derived isolates of
vancomycin-resistant Enterococcus faecium (VRE) and methicillin-susceptible, methicillin-
resistant, and tetracycline-resistant Staphylococcus aureus (MSSA, MRSA, and TRSA, respec-
tively). The most potent antibiotic displayed MIC50 values of 0.11, 0.23, and 0.90 µM against
a panel (n = 25 each) of clinical MSSA, MRSA, and VRE, respectively, was determined to be
bactericidal by time–kill analysis (Socha et al., 2006). Essramycin (4) (Figure 2.1) is a novel
triazolopyrimidine antibiotic isolated from Streptomyces sp. The compound is antibacteri-
ally active with an minimum inhibitory concentration (MIC) of 2–8 µg/mL against gram-
positive and gram-negative bacteria (El-Gendy et al., 2008).
Diazepinomicin (5) (Figure 2.2) is a unique farnesylated dibenzodiazepinone produced
by a Micromonospora strain (Charan et al., 2004). It possesses antibacterial, anti-inflammatory,
and antitumor activities. It has a broad spectrum of in vitro cytotoxicity and has demon-
strated in vivo activity against glioma, breast cancer, and prostate cancer in mouse models.
Abyssomicin C (6) (Figure 2.2) is a novel polycyclic polyketide antibiotic produced by a marine
Verrucosispora strain (Riedlinger et al., 2004). It is a potent inhibitor of para-aminobenzoic acid
biosynthesis and, therefore, inhibits the folic acid biosynthesis at an earlier stage than the
CH3
H3C
O O O
O
N
HO O O
N CH3
H
OH HO HO
5. Diazepinomicin 6. Abyssomicin C
O
O
OH
OH O
O O
OH
O O O OH O
N
7. Bonactin 8. Frigocyclinone
Figure 2.2 Chemical structures of diazepinomicin, abyssomicin C, bonactin, and frigocyclinone.
well-known synthetic sulfa drugs (Bister et al., 2004). Abyssomicin C possesses potent activity
against gram-positive bacteria, including clinical isolates of multidrug-resistant and vanco-
mycin-resistant Staphylococcus aureus. Abyssomicin C or its analog (Rath et al., 2005) has the
potential to be developed as an antibacterial agent against drug-resistant pathogens.
A new compound, assigned the trivial name bonactin (7) (Figure 2.2), has been iso-
lated from the liquid culture of the Streptomyces sp. BD21-2, obtained from a shallow-water
sediment sample collected at Kailua Beach, Oahu, HI. Bonactin displayed antimicrobial
activity against both gram-positive and gram-negative bacteria as well as antifungal
activity (Schumacher et al., 2003). Frigocyclinone (8) (Figure 2.2) is a new angucyclinone
antibiotic isolated from Streptomyces griseus strain NTK 97, consisting of a tetrangomy-
cin moiety attached through a C-glycosidic linkage with the aminodeoxysugar ossamine.
Frigocyclinone showed antibacterial activities against gram-positive bacteria.
Lynamicins (9) (Figure 2.3) are chlorinated bisindole pyrroles, isolated from
Marinispora sp. The antimicrobial spectrum of these compounds was evaluated against a
panel of 11 pathogens, which demonstrated that these substances possess broad-spectrum
activity against both gram-positive and gram-negative organisms. Significantly, compounds
were active against drug-resistant pathogens such as methicillin-resistant Staphylococcus
aureus and vancomycin-resistant Enterococcus faecium (McArthur et al., 2008). Tirandamycin
C (10) (Figure 2.3) is a novel dienoyl tetramic acid isolated from Streptomyces sp. 307–9.
Tirandamycin C showed inhibitory activity against vancomycin-resistant Enterococcus
faecalis (Carlson et al., 2009). Marinopyrroles (11) (Figure 2.3) are densely halogenated and
axially chiral metabolites that contain an uncommon bis-pyrrole structure isolated from
Streptomyces sp. The marinopyrroles possess potent antibiotic activities against methicillin-
resistant Staphylococcus aureus (Hughes et al., 2008).
Himalomycins A (12) and B (13) (Figure 2.4) are anthracycline antibiotics. They were
obtained from Streptomyces sp. 6921, isolated from the marine sediments of Mauritius,
H
N
CI
H
HN
NH O OH O
O O
O
HN
CI O
9. Lynamicins 10. Tirandamycin C
HN CI
CI
HO
O O
N OH
CI
CI X
11. Marinopyrroles
Figure 2.3 Chemical structures of lynamicins, tirandamycin C, and marinopyrroles.
CH3
O O OH
O
H3C OH
O
O O
H3C O O
OH O O
O H3C
12. Himalomycin A
O
HO
CH3
O OH
HO H3C O
O H
O HO O
H 3C H3C O O
OH O O
O H 3C
13. Himalomycin B
O
HO
CH3
Figure 2.4 Chemical structures of himalomycins A and B.

CH3 H 3C CH3 CH3 CH3

O OH HO CH3
CH3 CH3
OH OH
O O
NH NH
14. Glyciapyrroles A 15. Glyciapyrroles B
CH3 CH3 CH3
CH3
O
O
NH
16. Glyciapyrroles C
Figure 2.5 Chemical structures of glyciapyrroles A, B, and C.
and exhibited strong antibacterial activity (Maskey et al., 2003a). Glyciapyrroles A (14),
B (15), and C (16) (Figure 2.5) are new pyrrolosesquiterpene antibiotics isolated from the
Streptomyces sp. NPS008187 (Macherla et al., 2005).
2.3.2 Antifungal activity

Numerous antibiotics have been isolated from a variety of microorganisms; however,
studies are still being conducted to identify novel antibiotics effective against pathogenic
fungi (Atlas and Bartha, 1986). Marine actinobacteria are useful biological tools for the
production of antifungal substances against fungi (Okami and Hotta, 1988). In general,
Streptomyces species are s aprophytic and are commonly associated with soils, where they
contribute significantly to the turnover of complex biopolymers and antibiotics (Wanner,
2009). The marine Streptomyces sp. DA11 isolated from South China, found to be associated
with sponge Craniella australiensis, produced the enzyme chitinase and showed antifungal
activities against Aspergillus niger and Candida albicans (Han et al., 2009). Chitin, a linear
β-1,4-linked homopolymer of N-acetylglucosamine, is one of the three most abundant
polysaccharides in nature besides cellulose and starch. The antifungal activity and highly
biocompatible quality make chitinase and its derivatives particularly useful for biomedi-
cal applications, such as wound healing, cartilage tissue engineering, drug delivery, and
nerve generation (Shi et al., 2006; Yan et al., 2006). The biodegradable and antifungal prop-
erties of chitinase are also useful for environmental and agricultural uses, food technology,
and cosmetics (Rabea et al., 2003; Lin and Lin, 2005).
N-(2-hydroxyphenyl)-2-phenazinamine (NHP) (17) (Figure 2.6) is a new antibiotic isolated
from Nocardia dassonvillei. The new compound showed significant antifungal activity against
Candida albicans, with an MIC of 64 µg/mL and high cancer cell cytotoxicity against HepG2,
A549, HCT-116, and COC1 cells (Gao et al., 2012). Chandrananimycin A (18) (Figure 2.6) is a
novel antibiotic isolated from Actinomadura sp. Chandrananimycin A possesses potent anti-
fungal activity against Mucor miehei. It also exhibits antialgal activity against the microalgae
Chlorella vulgaris and C. sorokiniana and antibacterial activity against Staphylococcus aureus
and Bacillus subtilis, along with anticancer activity (Maskey et al., 2003b).
H3C O
OH
N HO HN N
N N
H O O
17. N-(2-hydroxyphenyl)-2-phenazinamine (NHP) 18. Chandrananimycin A
Figure 2.6 Chemical structures of N-(2-hydroxyphenyl)-2-phenazinamine (NHP) and chandranani-

mycin A.
2.4 Conclusion
Marine actinobacteria have a tremendous potential to provide therapeutic leads with dis-
tinct chemical structures and biological activities. Actinobacteria, and in particular the
genus Streptomyces, have the ability to produce a wide variety of secondary metabolites
as bioactive compounds, including antibacterial, antifungal, anticancer, antitumor, anti-
inflammatory, antimalarial, antiviral, and anti-angiogenesis drugs.
Because of the immense biological diversity in the sea as a whole, it is increasingly
recognized that a large number of novel chemical entities exist in the oceans. As marine
microorganisms, particularly actinobacteria, have evolved the greatest genomic and met-
abolic diversity, efforts should be directed toward exploring marine actinobacteria as a
potential source for the discovery of novel secondary metabolites and for the development
of new antimicrobial drugs.
Acknowledgment
This research was supported by a grant from the Marine Bioprocess Research Center of the
Marine Biotechnology Program, funded by the Ministry of Oceans and Fisheries, R and
D/2004-6002, Republic of Korea.
References
Aghamirian, M.R. and Ghiasian, S.A. 2009. Isolation and characterization of medically important
aerobic actinomycetes in soil of Iran (2006–2007). Open Microbiology Journal. 3:53–57.
Asolkar, R.N., Dirk Schroeder, R.H., Siegmund Lang, I.W.D., and Hartmut, L. 2004. Helquinoline, a
new tetrahydroquinoline antibiotic from Janibacter limosus Hel 1. Journal of Antibiotics (Tokyo).
57:17–23.
Atlas, R.M. and Bartha, R. 1986. Microbial Ecology: Fundamentals and Applications, 4th edn., New York:
Benjamin-Cummings Publishing Company, pp. 174–217.
Atta, H.M., Dabour, S.M., and Desoukey, S.G. 2009. Sparsomycin antibiotic production by Streptomyces sp.
AZ-NIOFD1: Taxonomy, fermentation, purification and biological activities. American-Eurasian
Journal of Agricultural and Environmental Science 5(3):368–377.
Bhatnagar, I. and Kim, S.-K. 2010. Immense essence of excellence: Marine microbial bioactive com-
pounds. Marine Drugs. 8(10):2673–2701.
Bister, B., Bischoff, D., Stroebele, M., Riedlinger, J., Reicke, A., Wolter, F., and Suessmuth, R.D. 2004.
Abyssomicin C—A Polycyclic antibiotic from a marine Verrucosispora strain as an inhibi-
tor of the p-aminobenzoic acid/tetrahydrofolate biosynthesis pathway. Angewandte Chemie
International Edition 43(19):2574–2576.
Bredholt, H., Fjærvik, E., Johnsen, G., and Zotchev, S.B. 2008. Actinomycetes from sediments in the
Trondheim fjord, Norway: Diversity and biological activity. Marine Drugs. 6(1):12–24.
Bruntner, C., Binder, T., Pathom-aree, W., Goodfellow, M., Bull, A.T., Potterat, O., and Fiedler, H.P.
2005. Frigocyclinone, a novel angucyclinone antibiotic produced by a Streptomyces griseus
strain from Antarctica. The Journal of Antibiotics. 58(5):346–349.
Carlson, J.C., Li, S., Burr, D.A., and Sherman, D.H. 2009. Isolation and characterization of tirandamy-
cins from a marine-derived Streptomyces sp. Journal of Natural Products. 72(11):2076–2079.
Charan, R.D., Schlingmann, G., Janso, J., Bernan, V., Feng, X., and Carter, G.T. 2004. Diazepinomicin,
a new antimicrobial alkaloid from a marine Micromonospora sp. Journal of Natural Products.
67(8):1431–1433.
El-Gendy, M.M., Shaaban, M., Shaaban, K.A., El-Bondkly, A.M., and Laatsch, H. 2008. Essramycin:
A first triazolopyrimidine antibiotic isolated from nature. Journal of Antibiotics. 61(3):149–157.
Engelhardt, K., Degnes, K.F., Kemmler, M., Bredholt, H., Fjaervik, E., Klinkenberg, G., and Zotchev,
S.B. 2010. Production of a new thiopeptide antibiotic, TP-1161, by a marine Nocardiopsis species.
Applied and Environmental Microbiology. 76(15):4969–4976.
Gao, X., Lu, Y., Xing, Y., Ma, Y., Lu, J., Bao, W., and Xi, T. 2012. A novel anticancer and antifungus
phenazine derivative from a marine actinomycete BM-17. Microbiological Research. 167:616–622.
Gärtner, A., Ohlendorf, B., Schulz, D., Zinecker, H., Wiese, J., and Imhoff, J.F. 2011. Levantilides A
and B, 20-membered macrolides from a Micromonospora strain isolated from the Mediterranean
deep sea sediment. Marine Drugs. 9(1):98–108.
Goodfellow, M. and Williams, S.T. 1983. Ecology of actinomycetes. Annual Reviews in Microbiology.
37(1):189–216.
Han, Y., Yang, B., Zhang, F., Miao, X., and Li, Z. 2009. Characterization of antifungal chitinase from
marine Streptomyces sp. DA11 associated with South China Sea sponge Craniella australiensis.
Marine Biotechnology. 11(1):132–140.
Hohmann, C., Schneider, K., Bruntner, C., Irran, E., Nicholson, G., Bull, A.T., and Fiedler, H.P. 2009.
Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain
Streptomyces sp. NTK 937*. Journal of Antibiotics. 62(2):99–104.
Hong, K., Gao, A.H., Xie, Q.Y., Gao, H.G., Zhuang, L., Lin, H.P., and Ruan, J.S. 2009. Actinomycetes
for marine drug discovery isolated from mangrove soils and plants in China. Marine Drugs.
7(1):24–44.
Hughes, C.C., Prieto-Davo, A., Jensen, P.R., and Fenical, W. 2008. The marinopyrroles, antibiotics of
an unprecedented structure class from a marine Streptomyces sp. Organic Letters. 10(4):629–631.
Imada, C. 2004. Enzyme inhibitors of marine microbial origin with pharmaceutical importance.
Marine Biotechnology. 6(3):193–198.
Jensen, P.R. and Fenical, W. 1994. Strategies for the discovery of secondary metabolites from marine
bacteria: Ecological perspectives. Annual Reviews in Microbiology. 48(1):559–584.
Kwon, H.C., Christopher, A.K., Paul, R.J., and William, F. 2006. Marinomycins AD, antitumor-
antibiotics of a new structure class from a marine actinomycete of the recently discovered
genus “Marinispora.” Journal of the American Chemical Society. 128(5):1622–1632.
Lin, C.C. and Lin, H.-L. 2005. Remediation of soil contaminated with the heavy metal (Cd2+). Journal
of Hazardous Materials. 122(1):7–15.
Macherla, V.R., Jehnan, L., Christopher, B. et al. 2005. Glaciapyrroles A, B, and C, pyrrolosesquiter-
penes from a Streptomyces sp. isolated from an Alaskan marine sediment. Journal of Natural
Products. 68(5):780–783.
Manam, R.R., Teisan, S., White, D.J., Nishino, J.G., Neuteboom, S.T.C., Lam, K.S., Mosca, D.A., Lloyd,
G.K., and Potts, B.C.M. 2005. Lajollamycin, a nitro-tetraene spiro-β-lactone-γ-lactam antibiotic
from the marine actinomycete Streptomyces nodosus. Journal of Natural Products. 68:240–243.
Manivasagan, P., Kang, K.H., Sivakumar, K., Li-Chan, E.C., Oh, H.M., and Kim, S.K. 2014a. Marine
actinobacteria: An important source of bioactive natural products. Environmental Toxicology
and Pharmacology. 38(1):172–188.
Manivasagan, P., Venkatesan, J., Sivakumar, K., and Kim, S.K. 2013. Marine actinobacterial metabo-
lites: Current status and future perspectives. Microbiological Research. 168(6):311–332.
Manivasagan, P., Venkatesan, J., Sivakumar, K., and Kim, S.K. 2014b. Actinobacterial enzyme
inhibitors—A review. Critical Reviews in Microbiology. 41(2):261–272.
Manivasagan, P., Venkatesan, J., Sivakumar, K., and Kim, S.K. 2014c. Pharmaceutically active sec-
ondary metabolites of marine actinobacteria. Microbiological Research. 169:262–278.
Maskey, R.P., Helmke, E., and Laatsch, H. 2003a. Himalomycin A and B: Isolation and structure
elucidation of new fridamycin type antibiotics from a marine Streptomyces isolate. Journal of
Antibiotics. 56(11):942–949.
Maskey, R.P., Li, F.C., Qin, S., Fiebig, H.H., and Laatsch, H. 2003b. Chandrananimycins A approxi-
mately C: Production of novel anticancer antibiotics from a marine Actinomadura sp. isolate
M048 by variation of medium composition and growth conditions. Journal of Antibiotics.
56(7):622–634.
Maskey, R.P., Sevvana, M., Usón, I., Helmke, E., and Laatsch, H. 2004. Gutingimycin: A highly
complex metabolite from a marine streptomycete. Angewandte Chemie International Edition.
43(10):1281–1283.
McArthur, K.A., Scott, S.M., and Ginger, T. 2008. Lynamicins A−E, chlorinated bisindole pyrrole
antibiotics from a novel marine actinomycete. Journal of Natural Products. 71(10):1732–1737.
Ogunmwonyi, I.H., Ntsikelelo, M., and Leonard, M. 2010. Studies on the culturable marine actino-
mycetes isolated from the Nahoon beach in the eastern Cape Province of South Africa. African
Journal of Microbiology Research. 4(21):2223–2230.
Okami, Y. and Hotta, K. 1988. Search and discovery of new antibiotics. In: Goodfellow, M., Williams,
S.T., and Mordarski, M. (eds), Actinomycetes in Biotechnology. San Diego, CA: Academic Press,
Inc., pp. 33–67.
Olano, C., Carmen, M., and José, A.S. 2009. Antitumor compounds from marine actinomycetes.
Marine Drugs. 7(2):210–248.
Peschke, U., Heike, S., Hui-Zhan, Z., and Wolfgang, P. 2006. Molecular characterization of the lin-
comycin-production gene cluster of Streptomyces lincolnensis 78–11. Molecular Microbiology.
16(6):1137–1156.
Prabavathy, V.R., Narayanasamy, M., and Kandasamy, M. 2006. Control of blast and sheath blight
diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5. Biological
Control. 39(3):313–319.
Rabea, E.I., Mohamed, E.-T.B., Christian, V.S., Guy, S., and Walter, S. 2003. Chitosan as antimicrobial
agent: Applications and mode of action. Biomacromolecules. 4(6):1457–1465.
Raghunath, D. 2008. Emerging antibiotic resistance in bacteria with special reference to India.
Journal of Biosciences. 33(4):593–603.
Ramesh, S. and Mathivanan, N. 2009. Screening of marine actinomycetes isolated from the Bay of
Bengal, India for antimicrobial activity and industrial enzymes. World Journal of Microbiology
and Biotechnology. 25(12):2103–2111.
Rao, K.V.B. 2012. In-vitro antimicrobial activity of marine actinobacteria against multidrug resis-
tance Staphylococcus aureus. Asian Pacific Journal of Tropical Biomedicine. 2(10):787–792.
Rath, J.P., Stephan, K., and Martin, E.M. 2005. Synthesis of the fully functionalized core structure of
the antibiotic abyssomicin C. Organic Letters. 7(14):3089–3092.
Ravikumar, S., Gnanadesigan, M., Saravanan, A., Monisha, N., Brindha, V., and Muthumari, S.
2012. Antagonistic properties of seagrass associated Streptomyces sp. RAUACT-1: A source for
anthraquinone rich compound. Asian Pacific Journal of Tropical Medicine. 5(11):887–890.
Ravikumar, S., Jacob Inbaneson, S., Uthiraselvam, M., Rajini Priya, S., Ramu, A., and Banerjee, M.B.
2011. Diversity of endophytic actinomycetes from Karangkadu mangrove ecosystem and its
antibacterial potential against bacterial pathogens. Journal of Pharmacy Research. 4(1):294–296.
Ravikumar, S., Thajuddin, N., Suganthi, P., Jacob Inbaneson, S., and Vinodkumar, T. 2010. Bioactive
potential of seagrass bacteria against human bacterial pathogens. Journal of Environmental
Biology. 31(3):387–389.
Reddy, N.G., Ramakrishna, D.P.N., and Raja Gopal, S.V. 2011. A morphological, physiological and
biochemical studies of marine Streptomyces rochei (MTCC 10109) showing antagonistic activ-
ity against selective human pathogenic microorganisms. Asian Journal of Biological Sciences.
4(1):1–14.
Riedlinger, J., Reicke, A., Zähner, H.A.N.S., Krismer, B., Bull, A.T., Maldonado, L.A., and Fiedler,
H.P. 2004. Abyssomicins, inhibitors of the para-aminobenzoic acid pathway produced by the
marine Verrucosispora strain AB-18-032. Journal of Antibiotics. 57(4):271–279.
Saadoun, I., Hameed, K.M., and Moussauui, A. 1998. Characterization and analysis of antibiotic
activity of some aquatic actinomycetes. Microbios. 99(394):173–179.
Saurav, K. and Kannabiran, K. 2010. Diversity and optimization of process parameters for the
growth of Streptomyces VITSVK9 spp isoled from Bay of Bengal, İndia. Journal of Natural and
Environmental Sciences. 1(2):56–65.
Schmitz, F.J., Verhoef, J., and Fluit, A.C. 1999. Prevalence of resistance to MLS antibiotics in
20 European university hospitals participating in the European SENTRY surveillance pro-
gramme. Journal of Antimicrobial Chemotherapy 43(6):783–792.
Schumacher, R.W., Talmage, S.C., Miller, S.A., Sarris, K.E., Davidson, B.S., and Goldberg, A. 2003.
Isolation and structure determination of an antimicrobial ester from a marine sediment-
derived bacterium. Journal of Natural Products. 66(9):1291–1293.
Sharma, S.L. and Pant, A. 2001. Crude oil degradation by a marine actinomycete Rhodococcus sp.
Indian Journal of Marine Sciences. 30(3):146–150.
Shi, C., Zhu, Y., Ran, X., Wang, M., Su, Y., and Cheng, T. 2006. Therapeutic potential of chitosan and
its derivatives in regenerative medicine. Journal of Surgical Research. 133(2):185–192.
Sivasubramanian, K., Ravichandran, S., and Vijayapriya, M. 2011. Antagonistic activity of marine
bacteria Pseudoalteromonas tunicata against microbial pathogens. African Journal of Microbiology
Research. 5(5):562–567.
Socha, A.M., LaPlante, K.L., and Rowley, D.C. 2006. New bisanthraquinone antibiotics and semi-
synthetic derivatives with potent activity against clinical Staphylococcus aureus and Enterococcus
faecium isolates. Bioorganic and Medicinal Chemistry. 14(24):8446–8454.
Solanki, R., Khanna, M., and Lal, R. 2008. Bioactive compounds from marine actinomycetes. Indian
Journal of Microbiology. 48(4):410–431.
Soria-Mercado, I.E., Prieto-Davo, A., Jensen, P.R., and Fenical, W. 2005. Antibiotic terpenoid chloro-
dihydroquinones from a new marine actinomycete. Journal of Natural Products. 68(6):904–910.
Valli, S., Suvathi, S.S., Aysha, O.S., Nirmala, P., Vinoth, K.P., and Reena, A. 2012. Antimicrobial poten-
tial of Actinomycetes species isolated from marine environment. Asian Pacific Journal of Tropical
Biomedicine. 2(6):469–473.
Wanner, L.A. 2009. A patchwork of Streptomyces species isolated from potato common scab lesions
in North America. American Journal of Potato Research. 86(4):247–264.
Yan, J., Li, X., Liu, L., Wang, F., Zhu, T.W., and Zhang, Q. 2006. Potential use of collagen-chitosan-
hyaluronan tri-copolymer scaffold for cartilage tissue engineering. Artificial Cells, Blood
Substitutes and Biotechnology. 34(1):27–39.
Zhang, L., An, R., Wang, J., Sun, N., Zhang, S., Hu, J., and Kuai, J. 2005. Exploring novel bioactive
compounds from marine microbes. Current Opinion in Microbiology. 8(3):276–281.
chapter three
Antimicrobial compounds
from microorganisms
Production, characterization, and applications
W.M. Muiru
Contents
3.1 Introduction........................................................................................................................... 30
3.2 Sources of antimicrobial metabolites................................................................................. 30
3.3 Functions of antimicrobial compounds in nature........................................................... 31
3.4 Production of antimicrobial compounds.......................................................................... 31
3.4.1 Bacteria as producers of antimicrobial compounds............................................ 32
3.4.1.1 Endophytic bacteria................................................................................... 32
3.4.1.2 Bacillus.......................................................................................................... 32
3.4.1.3 Pseudomonas................................................................................................34
3.4.1.4 Lactic acid bacteria..................................................................................... 35
3.4.2 Actinomycetes as producers of antimicrobial compounds................................ 35
3.4.2.1 Streptomyces................................................................................................. 36
3.4.2.2 Micromonosporaceae..................................................................................... 37
3.4.2.3 Pseudonocardia............................................................................................. 37
3.4.2.4 Actinomadura............................................................................................... 37
3.4.3 Fungi as producers of antimicrobial compounds................................................ 38
3.4.3.1 Fungal endophytes..................................................................................... 38
3.4.3.2 Trichoderma.................................................................................................. 38
3.4.3.3 Diaporthe...................................................................................................... 39
3.4.3.4 Aspergillus.................................................................................................... 39
3.4.3.5 Penicillium.................................................................................................... 39
3.4.3.6 Nematophagous fungi............................................................................... 40
3.4.3.7 Ascomycetous fungi.................................................................................. 40
3.4.3.8 Marine fungi............................................................................................... 40
3.5 Detection/assay of microorganisms producing antagonistic metabolites................... 40
3.6 Production and detection of antimicrobial metabolites in shaken liquid media........ 41
3.7 Factors affecting the bioassay of antibiotic substances................................................... 41
3.7.1 Ingredients of the assay medium...........................................................................42
3.7.2 Choice of the medium and agar thickness............................................................42
3.7.3 Effect of pH................................................................................................................42
3.7.4 Temperature of incubation......................................................................................42
3.7.5 Volume of the sample...............................................................................................42
3.7.6 Nature of inocula propagules and amount of inoculum....................................43
29
3.8 Secondary screening for antimicrobial compounds.......................................................43

3.9 Identification and characterization of antimicrobial metabolites..................................43
3.9.1 Thin-layer chromatography....................................................................................44
3.9.2 High-performance liquid chromatography (HPLC)............................................44
3.9.3 Sephadex columns.................................................................................................... 45
3.9.4 Other methods of identifying antimicrobial metabolites................................... 45
3.9.5 Minimum inhibitory concentration test................................................................ 45
3.10 Utilization of antimicrobial compounds........................................................................... 46
3.10.1 Use in pharmaceutical industries........................................................................... 46
3.10.2 Food industry............................................................................................................ 46
3.10.3 Crop protection......................................................................................................... 46
3.10.4 Other applications.................................................................................................... 47
3.11 Challenges in the utilization of antimicrobial metabolites and future prospects...... 47
References........................................................................................................................................ 48
3.1 Introduction
Antimicrobials are substances that act against microorganisms by inhibiting their growth
or killing them. Antimicrobial metabolites produced by microorganisms are secondary
metabolites and are grouped according to the microorganisms they act against, for instance,
antibiotics are used against bacteria and antifungals are used for controlling fungal organ-
isms. Antibiotics are the most important antimicrobial metabolites (Basilio et al., 2003). The
use of antimicrobial products against microorganisms, either by killing them or inhibit-
ing their growth, has been in place for the past 2000 years (Liras and Martin, 2005). Molds
and plant extracts were used by ancient Egyptians and Greeks to treat various infections.
Alexander Fleming discovered penicillin, a natural antimicrobial product from Penicillium
rubens, and this has been successfully used to treat many infections in humans caused by
Streptococcus bacteria such as pneumonia and gonorrhea. Microbial secondary metabolites
have a great variety of chemical structures and are normally formed by microorganisms
after the growth phase is complete. Apart from the activity against other microbes, second-
ary metabolites have different biological activities such as enhancing plant growth and
acting as enzyme inhibitors or antitumor agents (Liras and Martin, 2005).
3.2 Sources of antimicrobial metabolites

Many microorganisms have been reported to produce secondary metabolites. Fungi pro-
duce a wide range of antimicrobial metabolites, and the production is observed when
mycelial growth stops due to some nutrients being limiting while there are still ample car-
bon sources. Actinomycetes are the most prolific and fruitful group of organisms produc-
ing antimicrobial metabolites, and as many as three-quarters of actinomycetes produce
antibiotics. Many species of bacteria also produce secondary antagonistic metabolites.
Although several hundreds of compounds with antibiotic properties have been isolated
and identified, only a few of them have therapeutic applications.
Some of the microorganisms are known to produce several metabolites with differ-
ent structures and biological activities, and a single microbial strain may produce more
than one secondary metabolite. The compounds produced by one particular strain may
have similar chemical structures or completely different structures. Microorganisms
contain cluster of genes for the formation of each secondary metabolite. Each cluster has
genes encoding structural biosynthetic enzymes and regulatory proteins for the control of
Chapter three: Antimicrobial compounds from microorganisms 31
metabolite formation. The microorganisms producing the metabolites also contain genes
for making the producer organism resistant to the metabolite for those cases where the
metabolites have lethal or deleterious biological activity (Liras and Martin, 2005).
3.3 Functions of antimicrobial compounds in nature

The role of secondary metabolites in the organisms that produce them is not very clear, but
they are reported to offer ecological advantage to the organism producing it in its natural
habitat. The microbial metabolites produced by many microorganisms possess specific or
broad-spectrum activities against coexisting microorganisms (Berleman and Kirby, 2009).
Some of these metabolites such as antibiotics confer advantage to the producing organ-
isms through direct suppression of other organisms in highly competitive and resource-
limited environments (Davies, 1990).
Sethi et al. (2013) postulated that many of these metabolites may act as chemical
defense mechanisms in competition for substrates by the various microbes. The same
observation has been reported by other workers. Microorganisms that thrive in unusual
and extreme habitats have exhibited capabilities of producing unique metabolites. When
antibiotics levels are produced at subinhibitory concentrations, they serve other purposes
such as in the acquisition of substrates or initiation of changes that enhance survival under
stressful conditions (Romero et al., 2011). Antibiotics have also been reported to play a
role in virulence on host plants, and they also influence multitrophic interactions that are
necessary for adapting to various environments.
3.4 Production of antimicrobial compounds

Different microorganisms vary in the type and quantity of antimicrobial metabolites they
produce. The ability of microbes to produce antimicrobial metabolites is greatly influenced
by factors such as conditions of nutrition and cultivation (temperature, pH) (Mangamuri
et al., 2011). The composition of the media and the intrinsic capacity of the microbe in pro-
ducing antimicrobial metabolites greatly influence the synthesis of the bioactive metab-
olites. The medium composition together with the metabolic capacity of the producing
microorganism greatly affects synthesis of bioactive metabolites. The environmental fac-
tors that influence the production of bioactive metabolites are pH, temperature, incubation
media, and duration of incubation (Mangamuri et al., 2011).
Sources of nitrogen and carbon play a critical role in the production of antimicro-
bial metabolites (Kumar et al., 2012). Carbon is required for growth and production of
antimicrobial metabolites, and the choice of carbon sources greatly influences secondary
metabolism and, therefore, antibiotic production. Carbon sources such as dextrose, lactose,
sucrose, fructose, starch, and glycerol are suitable for production of secondary metabolites
for various microorganisms. Glucose may allow the cell to achieve maximum cell growth
rates, but it inhibits the production of many secondary metabolites due to intermediates
generated from the rapid catabolism of glucose, which interferes with enzymes in the sec-
ondary metabolism process. In addition, the duration of fermentation plays a significant
role in the growth of culture, and production of antibiotics takes place in the late growth
phase of the producing organism. Although the production of antimicrobial metabolites is
genetically controlled, it is greatly influenced by environmental manipulations. Variation
in fermentation environment results in changes in the yields and compositions or profiles
of antimicrobial substances. Limiting the supply of essential nutrients not only restricts
the growth but has specific metabolic and regulatory effects (Ripa et al., 2009).
3.4.1 Bacteria as producers of antimicrobial compounds

3.4.1.1 Endophytic bacteria
Endophytes are microbes that colonize living, internal tissues of plants without caus-
ing any immediate or overt negative effects. Common genera of bacteria that serve as
endophytes are Serratia, Pseudomonas, Bacillus, Azospirillum, Burkholderia, Azoarcus, and
Streptomyces. Endophytic bacteria are known to have such effects as enhancing seed ger-
mination, promoting plant growth, and protecting plants from insect pests and pathogens.
Currently, only a few secondary metabolites have been isolated from endophytic bacteria
since only a few plant species have been studied in depth and their metabolites tested for
activity against plant, animal, or human pathogens. There is increasing interest in endo-
phytic bacteria as they are likely to possess new genes for novel bioactive compounds. The
bioactive compounds from endophytic bacteria can be used in pharmaceutical industries
in the production of drugs, in agriculture as biocontrol agents, and in food industry as
food preservatives (Adhikari et al., 2001; Gunatilaka, 2006).
The following antibiotics have been reported from bacterial endophytes: ecomycins
from Pseudomonas viridiflava and munumbicins from Streptomyces. They exhibit antibacte-
rial activities and also have activity against the malarial parasite Plasmodium falciparum
and the plant-pathogenic oomycete Pythium ultimum. Other antibiotics produced by endo-
phytic bacteria are fusaricidins A–D from Paenibacillus polymyxa and oocydin, which is a
chlorinated macrocyclic lactone produced by Serratia marcescens and has been reported to
contribute to the natural protection against oomycete pathogens (Menpara and Chanda,
2013). Table 3.1 shows some of the endophytes of host plants and their reported activity
against test pathogens.
3.4.1.2 Bacillus
Bacillus produces a wide range of antimicrobial metabolites, mainly polypeptide antibi-
otics, which have many applications in various fields such as in crop protection, phar-
maceutical, and food industry (Sethi et al., 2013). Up to 167 antibiotics are produced by
Bacillus with B. subtilis alone producing 66 antibiotics accounting for more than a third of
all the antibiotics produced by the genus. The main antibiotic producers of this genus are
B. subtilis, which produces polymyxin, mycobacillin, bacitracin, difficidin, and subtilin;
B. cereus, which produces cerexin and zwittermicin; B. brevis, which produces gramicidin
and tyrothricin; B. laterosporus, which produces laterosporin; B. circulans, which produces
circulin; B. polymyxa, which produces polymyxin and colistin; B. licheniformis, which pro-
duces bacitracin; and B. pumilus, which produces pumulin (Awais et al., 2010).
Most of the peptide antibiotics produced by Bacillus are active against gram-positive
bacteria; however, some have activity against gram-negative bacteria, whereas others such
as bacillomycin, mycobacillin, and fungistatin are effective against molds and yeasts. Two
compounds isolated from Bacillus and identified as iturin A were shown to have a suppres-
sive effect on common scab disease in a pot assay, decreasing the infection rate from 75%
to 35%. This strain also suppressed Fusarium oxysporum, the pathogen causing potato dry
rot disease.
Bacillus is reported to produce metabolites that suppress Meloidogyne incognita,
Radopholus similis, Ditylenchus dipsaci, and Heterodera glycines through inhibition of egg
development and root infection. Bacillus is the most numerous producer of peptide anti
biotics, producing gramicidins, tyrocidines, and bacitracins.
Table 3.1 Some of the reported bacterial endophytes and their associated
microbial activity to the test organisms
Host plant Potent endophytes Activity shown Tested organisms
Panax ginseng Paenibacillus polymyxa Antifungal Phytopathogenic fungi
GS01, Bacillus sp.
GS07, and
Pseudomonas poae
JA01
T. grandiflora, Streptomyces Antifungal Phytopathogenic fungi
Polyalthia sp., and fulvoviolaceus,
Mapania sp. Streptomyces
coelicolor, and
Streptomyces caelestis
Scutellaria baicalensis Bacillus Antibacterial and Phytopathogenic, food-
Georgi amyloliquefaciens antifungal borne pathogenic, and
spoilage bacteria and fungi
Panax notoginseng Bacillus Antifungal Phytopathogenic fungi and
amyloliquefaciens nematode
subsp. plantarum and
Bacillus
methylotrophicus
Azadirachta indica A. Streptomyces sp. and Antibacterial and Phytopathogenic fungi,
Juss. Nocardia sp. antifungal human pathogenic
bacteria, and fungus
Plectranthus Bacillus sp. and Antibacterial and Human pathogenic bacteria
tenuiflorus Pseudomonas sp. antifungal and fungus
Wheat Bacillus subtilis Antifungal Phytopathogenic fungi
Anthurium B. amyloliquefaciens Antibacterial Phytopathogenic bacteria
Platycodon Bacillus licheniformis, Antibacterial and Phytopathogenic fungi and
grandiflorum Bacillus pumilus, and antifungal anti-human food-borne
Bacillus sp. pathogenic organisms
Artemisia annua Streptomyces Antibacterial and Pathogenic bacteria, yeast,
antifungal and fungal
phytopathogens
Centella asiatica Bacillus subtilis and Antifungal Phytopathogenic fungi
P. fluorescens
Panicum virgatum L. Bacillus subtilis, Antifungal Phytopathogenic fungi
C. flaccumfaciens,
P. fluorescens, and
P. ananatis
Raphanus sativus L. Enterobacter sp. and B. Antibacterial and Phytopathogenic fungi and
subtilis antifungal human pathogenic
bacteria
Memecylon edule, Bacillus Antibacterial and Human pathogenic bacteria
Tinospora cordifolia, amyloliquefaciens antifungal and fungus
Phyllodium
pulchellum, and
Dipterocarpus
tuberculatus
(Continued)
Table 3.1 (Continued) Some of the reported bacterial endophytes and their associated
microbial activity to the test organisms
Host plant Potent endophytes Activity shown Tested organisms
S. lavandulifolia, Bacillus sp. Antibacterial and Human pathogenic bacteria
H. scabrum, and antifungal and saprophytic fungi
R. pulcher
Aloe chinensis Paenibacillus species Antibacterial and Pathogenic bacteria and
antifungal fungi
Epimedium Phyllobacterium Antibacterial and Phytopathogenic fungi and
brevicornum Maxim myrsinacearum antifungal phytopathogenic
bacterium
11 mangrove Bacillus thuringiensis Antibacterial Shrimp pathogens
halophytic plants and Bacillus pumilus
Kandelia candel Streptomyces sp. Antibacterial Several pathogenic bacteria
Codonopsis lanceolata Bacillus pumilus, Antifungal Phytopathogenic fungi
B. subtilis, and
B. licheniformis
Polygonum cuspidatum Streptomyces sp. Antifungal Pathogenic fungi
Manihot esculenta Paenibacillus sp. Antifungal Phytopathogenic fungus
Bruguiera gymnorrhiza, Bacillus Antibacterial and Phytopathogenic fungi and
Rhizophora stylosa, amyloliquefaciens antifungal phytopathogenic bacteria
and Kandelia candel
Monstera sp. Streptomyces sp. Antifungal and Pythiaceous fungi and the
antimalarial human fungal pathogen,
and malarial parasite
Piper nigrum L. P. aeruginosa, P. putida, Antifungal Phytopathogenic fungus
and B. megaterium
Huperzia serrata Burkholderia sp. Antifungal Phytopathogenic fungi
300 plants from upper Streptomyces sp., Antibacterial and Range of potential fungal
Amazonian Micromonospora sp., antifungal and bacterial pathogens
rainforests and Amycolatopsis sp.
Lycopersicon Streptomyces sp., Antibacterial and Phytopathogenic fungi and
esculentum Microbispora sp., antifungal phytopathogenic bacteria
Micromonospora sp.,
and Nocardia sp.
Source: Sharma, M., Int. J. Curr. Microbiol. Appl. Sci., 3(2), 801, 2014. Table courtesy of Prof. S. Chanda, Department
of Biosciences, Saurashtra University, Rajkot, Gujarat, India.
3.4.1.3 Pseudomonas
Pseudomonas spp. are widely distributed soil inhabitants belonging to the gamma subclass
of Proteobacteria, and many of them live in a commensal relationship with plants (Paulsen
et al., 2005). Consequently, some of the Pseudomonas such as Pseudomonas fluorescens are
used as plant growth–promoting rhizobacteria. It has the ability to colonize the rhizo-
sphere of host plants and produce a wide range of compounds inhibitory to a number of
economically important plant pathogens (Haas and Keel, 2003). Pseudomonads have an
exceptional capacity to produce a wide variety of metabolites, including antibiotics that
are toxic to plant pathogens.
P. fluorescens have been reported to produce the following antibiotic compounds:
phenazines, pyrrole derivatives, indole derivatives, 2,4-diacetylphloroglucinol (DAPG)
(Ahil et al., 2014; Nowak-Thompson et al., 1994), pyrrolnitrin, and pyoluteorin. Additionally,
it produces DAPG, which is toxic to plant juveniles of parasitic nematodes (Globodera
rostochiensis). A strain of P. fluorescens has been observed to be antagonistic to Meloidogyne
javanica (Raaijmakers et al., 2002). Apart from producing secondary metabolites that
suppress plant disease and signal gene expression to neighboring cells inhabiting the
rhizosphere, Pseudomonas also use siderophores such as pyochelin and pyoverdine from
other microorganisms to obtain iron, which increases their survival in iron-limited envi-
ronments (Paulsen et al., 2005). Production of hydrogen cyanide formed by oxidation of
glycine and the siderophores enables the P. fluorescens to suppress target pathogens in the
rhizosphere through iron competition (Buysens et al., 1996).
3.4.1.4 Lactic acid bacteria

Lactic acid bacteria are a physiologically diverse group of gram-positive, non–spore form-
ing cocci or rods that produce lactic acid as the major end product during carbohydrate fer-
mentation. Lactic acid bacteria (LAB) are able to produce antimicrobial compounds against
competing flora, including food-borne spoilage and pathogenic bacteria. Under unfavorable
environmental conditions, many species of LAB also produce exopolysaccharides (EPSs),
which protect themselves against desiccation, bacteriophage, and protozoan attack (Yang,
2000). LAB are involved in fermentation of a wide range of food products such as vegetable
foods, milk, meat, and cereals and have traditionally been used to improve flavor develop-
ment and ripening of fermented products (Rai and Chikindas, 2011). LAB produces inhibi-
tory substances, namely, metabolic acid products, which are classified as low-molecular-mass
compounds. Examples of those antimicrobial products are organic acids, hydrogen perox-
ide, carbon dioxide, diacetyl, and high-molecular-mass compounds such as bacteriocins.
The production of LAB inhibits spoilage organisms through lowering of pH, thus
creating a hostile environment (Yang, 2000). Examples of such inhibitory substances
are b acteriocins, which are proteinaceous in nature. They are grouped into four classes,
namely small peptides (e.g., nisin), small heat-stable peptides, large heat-labile proteins, and
complex bacteriocins. The following genera comprise the LAB: Lactobacillus, Leuconostoc,
Pediococcus, Streptococcus, Vagococcus, and Enterococcus (Yang, 2000).
3.4.2 Actinomycetes as producers of antimicrobial compounds

Actinomycetes comprise an extensive and diverse group of microorganisms, and in addi-
tion to the production of biologically active substances, they play a role in soil cycles. The
classification of actinomycetes is wrought with controversy due to possession of morpho-
logical features similar to fungi and biochemical and physiological features similar to bac-
teria (Muiru, 2000). This led to describing actinomycetes as bacteria that have the ability to
form branching hyphae at some stages of development. The actinomycetes exhibit a very
wide range of morphological forms extending from coccus forms through fragmenting
hyphal forms to permanent and highly differentiated branched mycelium. Some actino-
mycetes form spores that include motile zoospores and specialized structures that resist
desiccation and mild heat (Dhanasekaran et al., 2009).
Actinomycetes are the most important source of antibiotics, producing over two-
thirds of all the known antibiotics. The antibiotics from actinomycetes have high commer-
cial value including medical applications for the treatment of human, animal, and plant
diseases. Streptomyces and Micromonospora are the most commonly isolated genera of acti-
nomycetes, and consequently most of the metabolites identified from screening programs
have been identified from the two genera. However, there has been a concerted effort to
screen for bioactive compounds from minor groups of actinomycetes. Other genera in acti-
nomycetes that produce antibiotics are Nocardiaceae, Pseudonocardiaceae, and Actinomadura.
Most of the soil actinomycetes have preference for neutral and alkaline conditions
for optimal growth; however, some grow under extreme conditions of alkaline and acidic
conditions, and these are the alkalophilic and acidophilic actinomycetes, respectively. The
occurrence of actinomycetes is greatly influenced by environmental conditions such as
temperature, humidity, vegetation, and pH. Halotolerant actinomycetes are adapted to
saline environment and have mostly been isolated from marine habitats. Knowledge on
the requirements for growth of various actinomycetes, especially the rare ones, is essential
to develop isolation conditions that can help in detection of such species. This will help
expand the range of isolated actinomycetes and consequently the bioactive metabolites
produced by these species.
Actinomycetes are known to produce valuable antibiotics such as novobiocin, ampho-
tericin, vancomycin, neomycin, gentamicin, chloramphenicol, tetracycline, erythromycin,
and nystatin, among others (Sharma, 2014). In agriculture, actinomycetes are used as plant
growth–promoting agents in the production of the plant growth hormone indole-3-acetic
acid, as biocontrol tools, as biopesticide agents, as antifungal compounds, and as a source
of agroactive compounds. They are also important in soil biodegradation and humus
formation as they recycle the nutrients associated with recalcitrant polymers, such as
chitin, keratin, and lignocelluloses (Bull and Stach, 2007). Industrially, actinomycetes play
a significant role in the production of various antimicrobial agents, enzymes, and other
industrially important substances (Sharma, 2014).
Other groups of actinomycetes namely Thermonosporaceae and Mycobacteriaceae
and other unclassified species such as Actinosporangium, Frankia, and Sebekia are reported
to produce bioactive metabolites (Sharma, 2014).
3.4.2.1 Streptomyces
The genus Streptomyces is classified in the family Streptomycetaceae, and it is an aerobic
spore-forming actinomycete. The classification is based on the morphological and cell-wall
chemotaxonomic characters. Streptomyces includes aerobic, gram-positive bacteria that pro-
duce the extensively branched substrate mycelium and aerial hyphae (Liras and Martin,
2005). Streptomyces is the most important actinomycete genus in the production of antibiot-
ics, and over two-thirds of the clinically important antibiotics are produced by this genus.
Streptomyces are also some of the most abundant soil microorganisms and are found
in a wide range of habitats under a wide variety of conditions. The taxon has more than
500 species and subspecies with around 4000 antibiotics isolated and identified from this
genus. This number of antibiotics represents around 70% of all known antibiotics. Apart
from conventional methods, molecular–systematic methods in the phylogenetic analysis
are increasingly being used in Streptomyces systematics (Liras and Martin, 2005).
The production of antibiotics by Streptomyces is greatly influenced by factors such as
nutritional source (carbon, nitrogen, and minerals) and environmental factors (incubation
period, pH, and temperature). In artificial conditions, optimization of culture conditions
is essential to obtain high yields of the antimicrobial metabolites (Ozgur et al., 2008).
Abamectin is a macrocyclic lactone metabolite produced by Streptomyces avermitilis,
and it is used in seed treatment against nematodes in tomato plants. It kills nematode-
infected larvae and arrests egg hatching and RNA synthesis. Abamectin has been shown
to be highly active against Pratylenchus spp. and has significant effect on Hoplolaimus
galeatus, M. javanica, Radopholus similis, Ditylenchus dipsaci, Aphelenchoides fragariae, and
Tylenchorhynchus dubius, all of them being plant parasitic nematodes.
3.4.2.2 Micromonosporaceae
The genus has been reported to synthesize a variety of bioactive compounds (Igarashi
et al., 2011). It produces a wide range of antibiotics with over 300 being described. This
group of actinomycetes produces several antibiotics with varied chemical properties and
applications (Carro et al., 2013). Micromonospora coerulea strain A058 produces a glutarim-
ide antibiotic named streptimidone, which has been found to be effective in inhibiting
the following plant pathogenic organisms: Didymella bryoniae, Phytophthora capsici, Botrytis
cinerea, and Magnaporthe grisea. Greenhouse tests showed high efficacy in the management
of M. grisea, P. capsici, and B. cinerea on rice, pepper, and cucumbers, respectively. The
antibiotic was equally as effective as metalaxyl and had no phytotoxicity at relatively high
concentrations (Seok et al., 1999). Other antibiotics produced by this genus are gentamicins
from M. purpurea and M. echinospora, fortimicin from M. olivoasterospora, rosamicin from
M. rosaria, and omicin from M. inyoensis, among others (Badji et al., 2013).
Two Micromonospora strains namely Micromonospora aurantiaca and Micromonospora
coriariae have been reported in actinorhizal nodules from Casuarina and Coriaria plants,
respectively. In addition, Micromonospora was reported to be widespread in nodules of
legumes such as lupine (Trujillo et al., 2006) and peas.
3.4.2.3 Pseudonocardia
Pseudonocardia have been reported to produce bioactive metabolites that have properties
such as antifungal, antibacterial, neuroprotective, and enzyme inhibitors. For instance,
phenazostatin, which is a phenazine derivative, acts as a neuroprotective substance (Maskey
et al., 2003), azureomycins A and B from P. azurea have antimicrobial activity (Omura et al.,
1997), and Dekker et al. (1998) reported quinolone compounds from Pseudonocardia sp. that
have selective and potent activity against Helicobacter pylori (Mangamuri et al., 2011).
3.4.2.4 Actinomadura
Actinomadura are slow-growing organisms that take 10–14 days under optimal conditions
to form spore-bearing aerial mycelium. Soil is their natural habitat, and conditions for
growth are similar to those of other actinomycetes except that chitin and xylan are not
utilized. Actinomadura is one of the most predominant actinomycete genus in extreme
environments and an important target in screening programs for bioactive metabolites
(Berdy, 2005). It belongs to the family Thermomonosporaceae and currently has 37 s pecies
including 2 subspecies (Zhang et al., 2001). Actinomadura are reported to produce over 250
antibiotics, the most common ones being polyether ionophoric antibiotics. Examples of
such antibiotics are maduramicins produced by A. yumanensis and cationomycin produced
by A. azure. Some species, such as A. roseoviolacea, produce antitumor anthracyclines such
as carminomycins. The genus does not produce classical antibacterial macrolides but
produces structurally similar products, the macrolactams (Badji et al., 2013).
The genus is reported to produce other bioactive metabolites that are antagonistic to
microorganisms. For example, daunomycin, which is an antifungal metabolite and anthra-
cycline type of antibiotic, has activity against Phytophthora capsici and Rhizoctonia solani,
both of which are plant pathogenic organisms. This metabolite also demonstrated activity
against Saccharomyces cerevisiae and gram-positive bacteria (Beom et al., 2000).
Four active compounds were elucidated from bioactive metabolites from Actinomadura
species isolated from Algerian Saharan soil. They showed strong antifungal activity
against pathogenic and toxinogenic fungi. These compounds were shown to differ from
the known antibiotics produced by Actinomadura species, and they possessed aromatic
rings substituted by aliphatic chains (Badji et al., 2013).
3.4.3 Fungi as producers of antimicrobial compounds

3.4.3.1 Fungal endophytes
Endophytic fungi represent an important and quantifiable component of fungal diversity,
with an estimate of hundreds of species. Endophytes spend all or part of their life cycle
inter- and intracellularly colonizing healthy tissues of their host plants such as the epider-
mal cell layers and cause no apparent harm or negative effect to the host (Yu et al., 2010).
Endophytes are ubiquitous with over one million species reported and are found in all plant
species. They form inconspicuous infections within tissues of healthy plants for all or nearly
all their life cycle (Limsuwan et al., 2009). They are known to produce substances that pro-
vide protection and ultimate survival of the plant, and different species of plants are a host
to one or more endophytes (Strobel, 2003). Endophytes and their host plants share a complex
relationship with endophytes indirectly benefiting plant growth by producing special sub-
stances, mainly secondary metabolites, to prevent the growth or activity of plant pathogens
(Gutierrez et al., 2012).
Endophytes have proven to be a new and potential source of novel natural p roducts
for antimicrobial metabolites isolated from endophytes belonging to diverse struc-
tural classes, including alkaloids, peptides, steroids, terpenoids, phenols, q uinones,
flavonoids, lignans, lactones, isocoumarins, and phenylpropanoids (Zhao et al., 2010).
Metabolites of endophytes have been reported to inhibit a number of microorganisms,
and, therefore, these metabolites or their derivatives have chemotherapeutic value and
are used as antifungal and antibacterial products (Yu et al., 2010). Apart from possess-
ing antimicrobial activity, some of the active secondary metabolites have immuno-
suppressant properties and are used as anticancer compounds. Currently, more than
140 fungal metabolites have been confirmed to possess antitumor activity and have
the potential to be used in the treatment of several types of cancer (Wang et al., 2011;
Gutierrez et al., 2012).
Examples of endophytes that produce antimicrobial metabolites are Fusarium oxy-
sporum NFX06 isolated from the leaf of the medicinal plant Odulisporium foetida and
Phomopsis spp. that produce pyrenocines with good antifungal, antibacterial, and algi-
cidal properties (Hussain et al., 2012a). Other endophytes reported are Seimatosporium sp.,
which produces acaranoic acids, named seimatoporic acids A and B, together with six
other compounds (Hussain et al., 2012b), whereas Pichia guilliermondii Ppf9 derived from
the medicinal plant Paris polyphylla var. yunnanensis produces three steroids and one nor-
dammarane triterpenoid (Zhao et al., 2010). An endophytic Penicillium sp. from the palm
tree produced metabolites that showed antimicrobial properties against Staphylococcus
aureus, M. luteus, and Escherichia coli. Some of the active metabolite extracted included the
rare indole alkaloid glandicoline B (Koolen et al., 2012).
Due to the increasing threats of resistance to drugs by the human, animal, and plant
pathogens, it is necessary to investigate more sources of antimicrobial producers, and this
means endophytes can increase the chance of finding novel antimicrobial natural prod-
ucts, thus solving the problem of resistance. More research needs to be conducted since
endophytes are a rich and reliable source of genetic diversity (Huang et al., 2007).
3.4.3.2 Trichoderma
This is a free-living fungi that are highly interactive in root, soil, and foliar environ-
ments with antagonistic properties against plant pathogens. Due to this antagonis-
tic potential, they are being applied commercially as biological control agents against
fungal pathogens. The strains commonly used as biological control agents are from
the following species: Trichoderma harzianum, T. viride, T. virens, and T. koningii. They
have been effectively used in the management of plant diseases caused by Sclerotium
cepivorum, Pyrenophora tritici-repentis, Sclerotinia sclerotiorum, Pythium ultimum, and
Rhizoctonia solani.
Trichoderma uses different modes of action to control the development of plant dis-
eases, and one of these modes is the production of antimicrobial metabolites. In addition,
Trichoderma spp. isolated from suppressive soil have shown extreme antagonism toward
P. cinnamomi during the saprophytic stage via antibiosis and mycoparasitism (Keen and
Vancov, 2010). T. koningii SMF2 produces antimicrobial metabolites with antimicrobial
activity against a range of gram-positive bacterial and fungal phytopathogens (Xiao-Yan
et al., 2006). Other types of antibiotics produced by Trichoderma are viridian, pyrones, glio-
toxin, peptaibols, and gliovirin, among others. Trichoderma are also reported to produce
over 180 antimicrobial peptides called peptaibols, which have been reported to inhibit
spore germination and hyphal elongation of plant pathogenic fungi.
3.4.3.3 Diaporthe
Lignicolous fungi are fungi with the ability to degrade fiber from seaweed, rotten wood,
mangrove plants, and marine algae. They live on limited nutritional sources, and they
produce bioactive compounds with antimicrobial properties as a competitive means.
Phomopsidin, neomangicols A–C, mangicols A, and humicolone are some of the bioactive
compounds reportedly produced by Diaporthe species. Some of these bioactive compounds
have cytotoxic activity against cell lines (Xin et al., 2006).
3.4.3.4 Aspergillus
Aspergillus produces a diverse group of secondary metabolites, organic acids, antibi-
otics, polyketides, and ribosomal and nonribosomal peptides (Andersen et al., 2013).
Some of these are bioactive with antibacterial, antifungal, and antitumor properties.
Aspergillus species have been reported to produce metabolites with antimicrobial activ-
ity against Candida albicans. After isolation and purification, structural elucidation of
these metabolites yielded three metabolites with the following structures: 5,6-dihydro-
5(S)-acetoxy-6(S)-(1,2-trans-epoxypropyl)-2H-pyran-2-one (asperline (1), compound 1);
5,6-dihydro-5(S)-acetoxy-6(S)-(1,2-trans-propenyl)-2H-pyran-2-one (compound 2); and 5,6-
dihydro-5(R)-acetoxy-6(S)-(1,2-trans-epoxypropyl)-H-pyran-2-one (compound 3) (Mizuba
et al., 1975).
Other metabolites produced by Aspergillus are amino acid–derived metabolites such
as echinocandins, which are lipopeptide antifungal agents produced by A. nidulans and
A. rugulosus (Badji et al., 2013). A. terreus is a prolific producer of secondary metabolites
with the following compounds being reportedly produced: geodin, itaconate, lovastatin,
questrin, sulochrin, terrecyclic and asterric acids, asterriquinone, butyrolactone I, citrinin,
emodin, and aspulvinone.
3.4.3.5 Penicillium
The genus comprises more than 200 species distributed throughout the world in different
habitats. Members of this genus are known to produce secondary metabolites with anti-
microbial properties (Kang et al., 2007). In addition, they produce immunosuppressants,
antitumor drugs, antiviral drugs, and cholesterol-lowering agents (Koolen et al., 2012).
Some of the most well-known and economically important metabolites are produced by
P. chrysogenum, such as penicillins. Penicillins are derived from a tripeptide chain and have
been used in the treatment of human diseases caused by various plant pathogenic organ-
isms (Badji et al., 2013). Other metabolites produced by Penicillium are compactins produced
by P. solitum and mycophenolic acid produced by P. brevicompactum. In addition, members
of this genus are known to produce mycotoxins, and many of these mycotoxins such as
citrinin and penicillic acids have antimicrobial activity (Gharaei-Fathabad et al., 2009).
While some species, such as P. citrinum, produces citrinin as the only mycotoxin, other
species, such as P. aurantiogriseum, produce citrinin and penicillic acid simultaneously
(Petit et al., 2004). Some species of Penicillium, specifically P. waksmanii, produce different
metabolites namely alkaloids, pyrones, sulfur-containing dioxopiperazines, and griseoful-
vin (Petit et al., 2004). Two novel tryptoquivaline-like metabolites, tryptoquialanine A (1)
and tryptoquialanine B (2), have been isolated from Penicillium digitatum (Ariza et al., 2002).
3.4.3.6 Nematophagous fungi

3.4.3.6.1 Arthrobotrys This nematode-trapping fungi has been reported to pro-
duce antimicrobial compounds with nematicidal activities against Meloidogyne incognita
and Caenorhabditis elegans. The nematicidal compound was identified as linoleic acid
and was isolated from two species of Arthrobotrys namely Arthrobotrys conoides and
Arthrobotrys oligospora (Jansson and Thiman, 1992). Production of this antimicrobial com-
pound increased with the number of traps formed in both Arthrobotrys oligospora and
Arthrobotrys conoides (Anke et al., 1995).
3.4.3.6.2 Nematoctonus This group of nematophagous fungi is known to produce

antimicrobial metabolites. Two species namely Nematoctonus concurrens and Nematoctonus
robustus produce dihydropleurotinic acid, leucopleurotin, and pleurotin (Anke et al., 1995).
3.4.3.7 Ascomycetous fungi

Some species of ascomycetes produce antimicrobial compounds with nematicidal activi-
ties, for instance, Chlorosplenium species produces linoleic acid, Neobulgaria pura produces
14-epicochlioquinone B, and Daldinia produces naphthalenes derived from the melanin
biosynthetic pathway. Lachnum papyraceum produces more than 30 metabolites with chlo-
rine or bromine incorporation depending on the culture conditions (Anke et al., 1995).
3.4.3.8 Marine fungi

These have been reported to produce diverse antimicrobial metabolites that have
activity against fungal, bacterial, viral, and protozoan infections. Some of these
metabolites namely avrainvillamide, sargassamide, and halimide possess anticancer
properties. Hypoxysordarin, isolated from Hypoxylon croceum, and 1-hydroxy-6-methyl-
8-(hydroxymethyl)xanthone, isolated from Ulocladium botrytis, have potent antifungal
activities. Lactone metabolites from Phoma sp. possess antifungal properties against

Cryptococcus neoformans, Aspergillus fumigates, and Candida albicans. Some have been
reported to produce metabolites that can inhibit protozoans such as the malarial parasite
P. falciparum (Bhadury et al., 2006).
3.5 Detection/assay of microorganisms
producing antagonistic metabolites
The detection of an antimicrobial effect of a crude extract of the culture broth is the
first step needed in the discovery of new bioactive compounds. This is followed by the
identification of the bioactive compound and finally the elucidation of the structure of
potent metabolite (Menpara, and Chanda, 2013). In search for new a ntimicrobial metab-
olites, thousands of microbial strains are isolated, but only a few produce useful
metabolites. Microbial antagonism is the basis of selecting organisms that produce such
metabolites. Primary screening of isolates is done to ascertain the potential of strains
with respect to production of antimicrobial secondary metabolites (Monisha et al., 2011).
During primary screening process, a large number of isolates are screened against a
range of sensitive strains. On the basis of primary screening results, isolates showing
substantial antimicrobial activities are selected for subsequent secondary screening pro-
grams; hence, the methods to be used should allow rapid screening of many organisms
(Yang, 2000; Ryu et al., 2000).
The commonly used bioassay techniques are “bicultures,” “dual cultures,” “paired
cultures,” or “cross cultures” of the potential antagonist and the test pathogen. The
potential antagonist produces metabolites that diffuse through the media causing antag-
onism to the test pathogen (Sethi et al., 2013). Antagonism is indicated if zone of inhibi-
tion develops between the two organisms (Han et al., 2005). The culture media should
favor the growth and antibiotic production of potential antagonist and the growth of the
test pathogen.
The size of the inhibition zone between the test pathogen and the potential antagonist
is taken as a measure of antimicrobial activity, but it should be borne in mind that other
factors such as exhaustion of nutrients around a colony or inhibitory pH can result to such
zones. The production of the antimicrobial metabolites can be confirmed by testing the
cell-free culture filtrates of organisms that cause growth inhibition.
3.6 Production and detection of antimicrobial

metabolites in shaken liquid media
The organisms that show antagonistic properties are cultured in liquid media in a mechan-
ical shaker or in stationary cultures. Synthetic media or complex organic media can be
used. The components of the media, speed of agitation, and duration of incubation are
determined empirically. The organism in the shake cultures is grown to obtain sufficient
growth. The resultant broth contains a mixture of the antimicrobial metabolites, water,
microbial cells, and residues of the nutrients used in the media. Filtration or centrifugation
is done to obtain the metabolite from the crude culture filtrate (Vijayakumari et al., 2013),
which is then subjected to bioassays.
The evaluation or bioassays of antimicrobial substances depend on factors such as
nature of the antibiotic substance, composition of the medium employed, selection of assay
organisms, time of action, and the environment conditions for carrying out the tests. The
bioassays of these antibiotic substances may be accomplished through biological assay
methods or through nonbiological assay methods.
3.7 Factors affecting the bioassay of antibiotic substances

Since the size of the inhibition zones is usually used as an estimation of the quantity of the
antimicrobial metabolites produced, the measurement of these zones should be done with
a high degree of precision and accuracy. The inhibition zone should be due to the inhibi-
tion of the test organism by the antimicrobial metabolite diffusing through the medium,
but other factors affect the range, precision, and sensitivity of an assay. The following fac-
tors determine the size of the inhibition zone.
3.7.1 Ingredients of the assay medium

The important factors in the media are viscosity and depth in the petri dishes and sub-
stances in the media such as peptone, tryptone, yeast extract, and agar. These substances
may vary in their mineral content, and this may influence the activity of the antimicro-
bials. Calcium, magnesium, and iron affect the sizes of zones produced by gentamicin.
Addition of glucose may cause a reduction in the zone of inhibition in some antibiotics,
while others such as nitrofurantoin, ampicillin, and carbohydrates may enhance the zones
of inhibition. The diffusion of unused nutrients from the zone of the inhibition may cause
an enhancement of growth at the edge (Loo et al., 1945).
3.7.2 Choice of the medium and agar thickness

Plates should be poured flat with an even depth of media throughout and all contain-
ing the same volume (Collins et al., 1989). This is achieved by supporting the plates so
that the plates are level and adding measured volumes of molten agar. Thin layers give
larger clear zones with distinct margins, while thick layers give cone-shaped zones
with poorly defined margins. Nonuniformity in the thickness causes error in zone
size. The use of uninoculated agar below and above the inoculated layer enables the
growth layer to be made thin but has the disadvantage of the diffusion of the antimi-
crobial metabolites into the uninoculated layer and hence causing modification outside
the zone.
3.7.3 Effect of pH
pH has an effect on the size of inhibition zone produced by some antibiotics (Han et al.,
2005). Since the agar has very little buffer capacity, it is necessary to control the pH
of the samples to be assayed. Activity or zone size is enhanced in alkaline media for
aminoglycosides and streptomycin. The activity of tetracyclines is enhanced in acid
medium.
3.7.4 Temperature of incubation

Nonuniform incubation temperature is the major cause of inaccurate diffusion assays.
The temperature of incubation influences both the zone size and the slope of the dose–
response curve. Also, the rate of heating of the contents of the plate influences the zone
size and shape. Size of inhibition zone may be increased by delayed incubation. Delayed
incubation is achieved by holding plates at a low temperature (4°C) to delay the growth
of the test organism while allowing the antibiotic to diffuse into the medium and reach
inhibitory concentrations.
3.7.5 Volume of the sample

The volume of the sample applied to the paper disc in the case of paper disc method is of
critical importance. The volume applied should be sufficient to saturate the disc but not to
cause flooding. Accurate and rapid delivery of this amount of sample can be obtained by
using a calibrated pipette.
3.7.6 Nature of inocula propagules and amount of inoculum

The use of spore suspensions in place of vegetative propagules seems to give very repro-
ducible results. This is due to the time required for germination of the other types of inocu-
lum propagules. Higher concentrations of antimicrobial metabolites are needed to prevent
growth when small fragments of fungus mycelium are used to seed the media compared
to when fungus spores are used. Heavy inocula reduce inhibition zones to some extent,
and the ideal inoculum is the one that gives an even dense growth without being confluent.
3.8 Secondary screening for antimicrobial compounds

After the detection of antimicrobial-producing organisms and culturing in the appropri-
ate media, the active metabolites have to be recovered from the crude mixture screened
for bioactivity and characterized accordingly. Active metabolites are recovered from the
culture broth by fermentation, and production of metabolite can be increased by opti-
mizing the conditions during fermentation. The fermented broth is filtered through a
membrane filter (0.20–0.45 μm pore size) or Whatman No. 1 filter to separate the cellular
components from the culture filtrate. This can directly be used for the determination of
antimicrobial activity against the sensitive organisms or bioactive compounds recovered.
Bioactive compounds can be recovered from filtrate by organic solvent extraction method
(Badji et al., 2013).
In recovery of active metabolites from filtrate by organic solvent extraction method,
culture supernatants are extracted with an equal volume of appropriate organic solvent.
The organic fraction obtained is allowed to evaporate under vacuum to dryness using a
rotary evaporator to remove all traces of the organic solvent. Stock solution is prepared
from the residue and used for antimicrobial analyses to test for bioactivity and character-
ization of bioactive compounds.
3.9 Identification and characterization

of antimicrobial metabolites
Crude culture filtrates containing antimicrobial metabolites are usually mixtures of dis-
similar components. Even where the crude culture filtrate contains a single antibiotic, this
antibiotic is a very heterogenous group of biologically active compounds. A method that
is simple and rapid with applicability to easily prepared samples of crude antimicrobial
metabolites is of great importance to help identify new bioactive compounds (Badji et al.,
2013). The initial step in identifying unknown antimicrobial metabolites is the determi-
nation of movement in specific solvent systems and the nature of microbiological spec-
trum. Final identification depends on further physical, chemical, and microbiological tests
(Jaganathan et al., 2014).
Ideally, to obtain a complete characterization, antimicrobial metabolites should be iso-
lated in pure form and as a single component (Yang, 2000). However, this is not possible
especially in a screening program. The following properties and techniques are useful in
characterizing antimicrobial metabolites: solubility in different solvents, stability at dif-
ferent temperatures, pH ranges, storage duration, color reactions and fluorescence, light
absorption, paper chromatography of the whole antibiotic and of decomposition products,
electrophoresis, countercurrent distribution, elementary analysis of physical constant, and
mass spectrometry (MS). Characterization of antimicrobial metabolites is important since
the ultimate application and the methods of purification are determined by the stability of
the metabolites (Lavermicocca et al., 2000).
Bioautography is a method used for the detection of antimicrobial metabolites on
paper and thin chromatograms. Bioautography has been used in the classification, search,
and identification in search of new antibiotics, development of isolation procedures for
unknown antibiotics, preparative chromatography, separation of mixtures of new antibi-
otics, systematic analysis, and determination of the optimum harvest time.
The paper chromatography is inferior compared to the thin layer in that it has low
resolution power resulting in failure to identify macrolide antibiotics and to differentiate
closely related antimicrobial metabolites such as antibiotics. In contrast, thin-layer chro-
matography (TLC) has higher resolution power; thus, it gives an efficient separation and
differentiation and also gives results rapidly.
3.9.1 Thin-layer chromatography

TLC is a simple, quick, and inexpensive procedure that tells how many compounds are in
a mixture. TLC can also be used to support the identity of a compound in a mixture when
the retention factor (Rf) value of a compound is compared with the Rf value of a known
compound. Rf value is the retention factor or how far up a plate the compound travels, and
it is defined as the distance traveled by the compound divided by the distance traveled by
the solvent. Rf values can provide corroborative evidence as to the identity of a compound,
and the unknown compounds are spotted together with the standard on a TLC plate side
by side or on top of each other. If the two substances have the same Rf value, they are likely
(but not necessarily) the same compound. Identity check must be performed on a single
plate because it is difficult to duplicate factors that influence Rf values exactly from experi-
ment to experiment.
After spotting a small amount of the mixture to be analyzed near the bottom of the
plate, this is placed in a shallow pool of solvent in a developing chamber so that only the
very bottom of the plate is in the liquid. This liquid or eluent is the mobile phase, and it
slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that
was applied, an equilibrium is established for each component of the mixture between the
molecules of that component that are adsorbed on the solid and the molecules that are in
the solution. Different components differ in solubility and in the strength of their adsorp-
tion to the adsorbent, and some components will be carried further up the plate than the
others. When the solvent has reached the top of the plate, the plate is removed from the
developing chamber, dried, and the separated components of the mixture are visualized
using UV lamp.
3.9.2 High-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a chromatographic technique used to
separate a mixture of compounds with the purpose of identifying, quantifying, and puri-
fying the individual components of the mixture. HPLC is used in the analysis of unknown
compounds against the reference standard. HPLC utilizes special instruments designed
to separate, quantify, and analyze components of a chemical mixture. Samples of inter-
est are introduced to a solvent flow path, carried through a column packed with special-
ized materials for component separation, and component data are obtained through the
combination of a detection mechanism coupled with a data-recording system. Chemical
separation using HPLC is accomplished by utilizing the fact that certain compounds have
different migration rates in a particular column and mobile phase. The extent or degree of
separation is mostly determined by the choice of stationery phase and mobile phase. The
components being separated or identified using HPLC should have a characteristic peak
under certain chromatographic conditions. Chromatographic conditions such as the kind
of mobile phase can be adjusted to allow adequate separation and collection of the extract
or the desired compound as it elutes from the stationery phase.
In order to identify any compound by HPLC, a detector must first be selected and
set to optimal settings. The parameters of separation assay must be developed, and this
should allow a clear peak of the known sample to be observed from the chromatograph.
The identifying peak should have a reasonable retention time and should be well sepa-
rated from extraneous peaks at the detection levels that the assay is performed. The
retention time of a compound can be altered by manipulating the choice of a column,
mobile phase, and the flow rate. To identify an unknown compound by HPLC, a sample
of a known compound must be utilized in order to assure identification of the unknown
compound.
HPLC can also be used to quantify or determine the unknown concentrations of a
compound in a known solution (Grabley and Thiericke, 1999). This involves injecting a
series of known concentrations of the standard compound solution onto the HPLC for
detection. The chromatographs of these known concentrations give a series of peaks that
correlate to the concentration of the compound injected.
3.9.3 Sephadex columns

Sephadex resins are highly specialized gel filtration and chromatographic media that are
composed of macroscopic beads synthetically derived from the polysaccharide dextran.
The organic chains are cross-linked to give a 3D network having functional ionic groups
attached by either linkages to glucose units of the polysaccharide chains. These resins
are used to separate (fractionate) a mixture of compounds into its components. When a
mixture of molecules and ions are dissolved in a solvent and applied in a column with
sephadex, the smaller molecules and ions are distributed through a larger volume of sol-
vent than is available to the large molecules; consequently, the larger molecules move more
rapidly through the column enabling the separation of the mixtures.
3.9.4 Other methods of identifying antimicrobial metabolites

Techniques such as nuclear magnetic resonance, spectroscopy, and MS have been used
to identify antimicrobial compound from Lactobacillus and Pediococcus strains where
2-pyrrolidone-5-carboxylic acid has been identified. These metabolites were further sepa-
rated and purified by chromatographic methods (Yang, 2000). MS is normally used for the
determination of molecular mass.
3.9.5 Minimum inhibitory concentration test

Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial
compound that inhibits the visible growth of a sensitive strain after the appropriate incu-
bation period. It is the lowest concentration of residue at which there is no visible growth
of the pathogenic strain. MIC is determined in vitro, by means of agar and broth dilution
methods (Kanna et al., 2011; Koolen et al., 2012).
3.10 Utilization of antimicrobial compounds

3.10.1 Use in pharmaceutical industries
Chemical synthetic drugs have been used for a long time in combating human, plant,
and animal diseases; however, cases of increased resistance have made it necessary to
explore antimicrobial metabolites as an alternative to the use of synthetics (Yu et al., 2010).
Pharmacological properties of microbial metabolites have been recognized and exploited
for their activity as enzyme inhibitors, receptor agonists, hormone-like regulators, and
neurotransmitters (Grabley and Thiericke, 1999). Some antimicrobial metabolites have
shown antitumor properties. Antitumor activity of beauvericin to human leukemia cells
has been demonstrated (Qinggui and Lijian, 2012). Beauvericin also possesses strong
antibacterial activity against human and animal pathogenic bacteria with no selectivity
between gram-positive and gram-negative bacteria. It also has antiviral activity against
HIV (Rattanachaikunsopon and Phumkhachorn, 2010).
Reutericyclin, a lactic acid antibiotic produced by Lactobacillus reuteri, has a broad-
spectrum activity against fungi, protozoa, and a wide range of bacteria including both
gram-positive and gram-negative bacteria. E. coli, S. aureus, S. epidermidis, and Candida albi-
cans (Li et al., 2008) are strongly inhibited by endophytes belonging to the Streptomyces
genus. These metabolites possessing activity against various organisms have been used
to develop and formulate pharmaceutical products to combat human, animal, and plant
pathogenic diseases.
3.10.2 Food industry

Food spoilage and contamination with microorganisms and their associated mycotox-
ins are a major problem in food industry. Production of high-quality and safe food free
from pathogens relies on antimicrobial metabolites that inhibit spoilage microorganism.
Antimicrobial metabolites from bacteria, algae, and fungi are in use in food industry (Rai
and Chikindas, 2011). Naturally produced antimicrobial metabolites are used as biocides
to kill and nullify the effects of contaminants. Biocides are used as disinfectants, antisep-
tics, and food preservatives.
A range of antimicrobial metabolites such as lactic, acetic, and propionic acids pro-
duced during the fermentation process by the LAB have a preservative action since they
create unfavorable environment for the growth of many spoilage and pathogenic organ-
isms. Acids act against microbes by interfering with the cell membrane, inhibiting active
transport, and inhibiting a variety of other metabolic functions. Some acids, such as pro-
pionic acid produced by propionic acid bacteria, have antimicrobial activity against yeast
and moulds (Yang, 2000).
3.10.3 Crop protection

Hundreds of antimicrobial metabolites have demonstrated antagonistic activity against
phytopathogenic fungi and bacteria, and consequently a number of them have been
used to formulate products for use in crop protection. Extracts from Streptomyces sp. col-
lected from Allium fistulosum showed the potential to suppress infection of Alternaria
brassicicola on Chinese cabbage seedlings. Some actinomycete strains of plant origin pro-
duce herbicidal antibiotics, for instance, herbicidin H is a metabolite of Streptomyces sp.
strain, SANK 63997, that was isolated from the leaves of Setaria viridis var. pachystachys.
Another strain of Microbispora sp., SANK 62597, recovered from Carex kobomugi produced
γ-glutamylmethionine sulfoximine in culture broth. A strain of Dactylosporangium sp. iso-
lated from Cucubalus sp. was found to produce streptol and two plant growth inhibitors
that inhibit germination of Brassica rapa (Hasegawa et al., 2006).
Beauvericin produced by fungi, such as Beauveria bassiana and Fusarium spp., is a
hexadepsipeptide mycotoxin and has a strong insecticidal activity against a broad spectrum
of insect pests (Qinggui and Lijian, 2012). In addition, it has antiviral and cytotoxic activi-
ties. Antimicrobial metabolites from bacterial endophytes have exhibited activity against
various plant pathogenic organisms. Endophytes from Bacillus subtilis from wheat roots
showed strong activity against Fusarium graminearum, Rhizoctonia cerealis, Botrytis cinerea,
and Macrophomina kuwatsukai, among others. Endophytes from B. pumilus and B. licheniformis
from balloon flower showed antifungal activity against Pythium ultimum, Rhizoctonia solani,
Fusarium oxysporum, and Phytophthora capsici (Menpara and Chanda, 2013).
3.10.4 Other applications

Apart from production of antimicrobial metabolites, Bacillus also produces industrial
enzymes such as subtilisins, cellulases, and amylases used in laundry. Antimicrobial com-
pounds and the organisms producing them have been used in promoting plant growth.
Endophytes colonizing inside plants usually get nutrition and protection from the host
plants. In return, they confer profoundly enhanced fitness to the host plants by producing a
variety of bioactive metabolites. Among other mechanisms, they stimulate growth of plants
through biocontrol of phytopathogens through production of antibiotics or siderophores,
nutrient competition, and induction of systemic disease resistance. Some of these metabo-
lites directly affect physiology of the host plants, but others do so indirectly by affecting
the microbial population by antibiosis and/or competition (Raajmakers and Mazzola, 2012).
Pteridic acids A and B from the fermentation broth of an endophytic Streptomyces
hygroscopicus TP-A045 (isolated from Pteridium aquilinum) as plant growth promoters with
auxin-like activity. These compounds accelerated formation of adventitious roots in hypo-
cotyls of kidney beans at 1 nM as effectively as indole acetic acid (Hasegawa et al., 2006).
Actinomycetes play a role in ecological balance and are responsible for much of
the digestion of resistant carbohydrates such as chitin and cellulose. Some of them are
renowned as degraders of toxic materials and have been shown to degrade hydrocarbons,
explosives, chlorinated solvents, and plastics and, as a result, are used in bioremediation
(Sharma, 2014). Pseudonocardia can utilize hydrocarbons, methyl sulfides, and tetrahydro-
furan as growth substrates (Mahendra and Alvarez-Cohen, 2005). Adaptation to harsh
environments such as high temperatures allows actinomycetes to be used for composting
purposes (Sharma, 2014).
3.11 Challenges in the utilization of antimicrobial

metabolites and future prospects
Due to the emergence of drug-resistant infections, there is a need to identify and develop
new antibiotics. Despite the critical need for new antibiotics, very few new antibiotics are
being developed. Although several hundreds of compounds with antibiotic activity have
been isolated from microorganisms over the years, at present only 1% of the microbial
world have been explored, so there is still a need to tap the vast reservoir of microbial com-
munity for their antimicrobial potential.
Other factors that limit utilization of antimicrobial metabolites are that although some
microbes produce antimicrobial metabolites, some of these have unspecific toxicity and
they are toxic to human beings along with pathogenic organisms; thus, they cannot be
used, and some have moderate antimicrobial activity and cannot be used as medicine
(Menpara and Chanda, 2013). Hence, more work needs to be done to come up with bioac-
tive substances without any side effects to humans and plants. Studies of regulatory gene
in the synthesis of antimicrobial compounds can be manipulated to increase the yield of
these metabolites, and by modification of the structure, one can enhance antimicrobial
activity, thereby improving the efficacy and reducing the toxicity, thus decreasing the side
effects (Menpara and Chanda, 2013; Lancini and Lorenzetti, 1993).
References
Adhikari, T.B., Joseph, C.M., Yang, G., Phillips, D.A., and Nelson, L.M. 2001. Evaluation of bacteria
isolated from rice for plant growth promotion and biological control of seedling disease of
rice. Can. J. Microbiol. 47(10), 916–924.
Ahil, S.B., Ameer, B., Govardhanam, R., Mallela, V., Nagesh, K., Yukthi, S., Jagannath, V.P., Yuhei, T.,
and Yoshinori, F. 2014. Isolation and characterization of antimicrobial cyclic dipeptides from
Pseudomonas fluorescens and their efficacy on sorghum grain mold fungi. Chem. Biodivers. 11(1),
92–100.
Andersen, M.R., Jakob Nielsen, J.B., Andreas, K., Lene, M.P., Zachariasen, M., Tilde, J.H., and Blicher,
L.H. 2013. Accurate prediction of secondary metabolite gene clusters in filamentous fungi.
Proc. Natl. Acad. Sci. 110(1), E99–E107.
Anke, H., Stadler, M., Mayer, A., and Sterner, O. 1995. Secondary metabolites with nematicidal and
antimicrobial activity from nematophagous fungi and Ascomycetes. Can. J. Bot. 73(S1), 9 32–939.
doi:10.1139/b95-341.
Ariza, M.R., Larsen, T.O., Petersen, B.O., Duus, J.Q., and Barrero, B.F. 2002. Penicillium digitatum
metabolites on synthetic media and citrus fruits. J. Agric. Food Chem. 50, 6361–6365.
Awais, M., Pervez, A., Asim, Y., and Shah, M.M. 2010. Production of antimicrobial metabolites by
Bacillus subtilis immobilized in polyacrylamide gel. Pak. J. Zool. 42(3), 267–275.
Badji, B., Mostefaoui, A., Sabaou, N., Mathieu, F., and Lebrihi, A. 2013. Identification of a new strain
of Actinomadura isolated from Saharan soil and partial characterization of its antifungal com-
pounds. Afr. J. Biotechnol. 10, 13878–13886.
Basilio, A., Gonzalez, I., Vicente, M.F., Gorrochategui, J., Cabello, A., Gonzalez, A., and Genilloud,
O. (2003). Patterns of antimicrobial activities from soil actinomycetes isolated under different
conditions of pH and salinity. J. Appl. Microbiol. 95(4), 814–823.
Beom, S., Surk, S.M., and Byung, K.H. 2000. Structure elucidation and antifungal activity of an
anthracycline antibiotic, daunomycin, Isolated from Actinomadura roseola. J. Agric. Food Chem.
48(5), 1875–1881.
Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58(1), 1–26.
Berleman, J.E. and Kirby, J.R. 2009. Deciphering the hunting strategy of a bacterial wolfpack. FEMS
Microbiol. Rev. 33, 942–957.
Bhadury, P., Mohammad, B.T., and Wright, P.C. 2006. The current status of natural products from
marine fungi and their potential as anti-infective agents. J. Ind. Microbiol. Biotechnol. 33, 325–337.
Bull, A.T. and Stach, J.E. 2007. Marine actinobacteria: New opportunities for natural product search
and discovery. Trends Microbiol. 15, 491–499.
Buysens, S., Heungens, K., Poppe, J., and Hofte, M. 1996. Involvement of pyochelin and pyoverdin
in suppression of Pythium-induced damping-off of tomato by Pseudomonas aeruginosa 7NSK2.
Appl. Environ. Microbiol. 62, 865–871.
Carro, L., Pujic, P., Trujillo, M.E., and Normand, P. 2013. Micromonospora is a normal occupant of
actinorhizal nodules. J. Biosci. 38, 685–693.
Collins, C.H., Lyne, P.M., and Grenge, J.M. 1989. Microbiological Methods. Butterworth and Company
Publishers Ltd., London, U.K.
Davies, J. 1990. What are antibiotics? Archaic functions for modern activities. Mol. Microbiol. 4,
1227–1232.
Dekker, K.A., Inagaki, T., Gootz, T.D., and Huang, L.H. 1998. New quinolone compounds from
Pseudonocardia sp. with selective and potent anti-Helicobacter pylori activity. J. Antibiot. 51,
145–152.
Dhanasekaran, D., Selvamani, S., Panneerselvam, A., and Thajuddin, N. 2009. Isolation and charac-
terization of actinomycetes in Vellar estuary, Annagkoil, Tamil Nadu. Afr. J. Biotechnol. 8(17),
4159–4162.
Gharaei-Fathabad, E., Tajick-Ghanbary, M.A., and Shahrokhi, N. 2009. Antimicrobial properties
of Penicillium species isolated from agricultural soils of Northern Iran. Res. J. Toxins 6(1), 1–7.
Grabley, S. and Thiericke, R. (Eds.). 1999. Drug Discovery in Nature. Springer-Verlag, Berlin,
Germany.
Gunatilaka, A. 2006. Natural products from plant-associated microorganisms: Distribution,
structural diversity, bioactivity, and implications of their occurrence. J. Nat. Products 69,
509–526.
Gutierrez, R.M., Gonzalez, A.M., and Ramirez, A.M. 2012. Compounds derived from endophytes: A
review of phytochemistry and pharmacology. Curr. Med. Chem. 19(18), 2992–3030.
Haas, D. and Keel, C. 2003. Regulation of antibiotic production in root-colonizing Pseudomonas spp.
and relevance for biological control of plant disease. Annu. Rev. Phytopathol. 41, 117–153.
Han, J.S., Cheng, J.H., Yoon, T.M., Song, J., Rajkarnikar, A., Kim, W.G., Yoo, I.D., Yang, Y.Y., and Suh,
J.W. 2005. Biological control agent of common scab disease by antagonistic strain Bacillus sp.
sunhua. J. Appl. Microbiol. 99, 213–221.
Hasegawa, S., Akane, M., Masafumi, S., Tomio, N., and Hitoshi, K. 2006. Endophytic Actinomycetes
and their interactions with host plants. Actinomycetologica 20, 72–81.
Huang, W.Y., Cai, Y.Z., Hyde, K.D., Corke, H., and Sun, M. 2007. World J. Microbiol. Biotechnol. 23(9),
1253–1263.
Hussain, H., Ahmed, I., Schulz, B., Draeger, S., and Krohn, K. 2012a. Pyrenocines J-M: Four new
pyrenocines from the endophytic fungus. Fitoterapia 83(3), 523–526.
Hussain, H., Krohn, K., Schulz, B., Draeger, S., Nazir, M., and Saleem, M. 2012b. Two new antimi-
crobial metabolites from the endophytic fungus, Seimatosporium sp. Nat. Prod. Commun. 7(3),
293–294.
Igarashi, Y., Yanase, S., Sugimoto, K., Enomoto, M., Miyanaga, S., Trujillo, M.E., Saiki, I., and
Kuwahara, S. 2011. Lupinacidin C, an inhibitor of tumor cell invasion from Micromonospora
lupini. J. Nat. Prod. 74, 862–865.
Jansson, H.B. and Thiman, L. 1992. A preliminary study of chemotaxis of zoospores of the nematode
parasitic fungus Catenaria anguillulae. Mycologia 84, 109–112.
Jeganathan, P., Rajasekaran, K.M., and Asha Devi, N.K. 2014. Antimicrobial activity and character-
ization of marine bacteria in coastal sea water. Sch. Acad. J. Pharm. 3(1), 73–78.
Kang, S.W., Park, C.H., Hong, S.I., and Kim, S.W. 2007. Production of penicillic acid by Aspergillus
scleorotiorum CGF. Bioresour. Technol. 98, 191–197.
Kanna, M., Solanki, R., and Lal, R. 2011. Selective isolation of rare actinomycetes producing novel
antimicrobial compounds. Int. J. Adv. Biotechnol. Res. 2(3), 357–375.
Keen, B. and Vancov, T. 2010. Phytophthora cinnamomi suppressive soils. In: Méndez, A., ed., Current
Research Technology and Education Topics in Applied Microbiology and Microbial Biotechnology,
Vol. 1, Formatex Microbiology Series Publication, Formatex Research Center, Badajoz, Spain,
pp. 239–250.
Koolen, H.H.F., Soares, E.R., da Silva, F.M.A., de Almeida, R.A., and de Souza, A.D.L. 2012. An anti-
microbial alkaloid and other metabolites produced by Penicillium sp. an endophytic fungus
isolated from Mauritia Flexuosa L. f. Quim. Nova 35(4), 771–774.
Kumar, S.N., Siji, J.V., Ramya, R., Nambisan, B., and Mohandas, C. 2012. Improvement of anti-
microbial activity of compounds produced by Bacillus sp. associated with a Rhabditid sp.
(entomopathogenic nematode) by changing carbon and nitrogen sources in fermentation
media. J. Microbiol. Biotechnol. Food Sci. 1(6), 1424–1438.
Lancini, G. and Lorenzetti, R. 1993. Biotechnology of Antibiotics, and Other Bioactive Microbial
Metabolites. Plenum Press, New York.
Lavermicocca, P., Valerio, F., Evidente, A., Lazzaroni, S., Corsetti, A., and Gobbetti, M. 2000.
Purification and characterization of novel antifungal compounds from the sourdough
Lactobacillus plantarum strain 21B. Appl. Environ. Microbiol. 66(9), 4084–4090.
Li, J., Zhao, G., Chen, H., Wang, H., Qin, S., Zhu, W., Xu, L., Jiang, C., and Li, W. 2008. Antitumour and
antimicrobial activities of endophytic Streptomycetes from pharmaceutical plants in rainforest.
Lett. Appl. Microbiol. 47, 574–580.
Limsuwan, S., Trip, E.N., Kouwenc, T., Piersmac, S., Hiranrat, A., and Mahabusarakam, W. 2009.
Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa.
Phytomedicine 16, 645–651.
Liras, P. and Martin, J.F. 2005. Assay methods for detection and quantification of antimicrobial
metabolites produced by Streptomyces clvuligerus. Methods Biotechnol. 18, 149–164.
Loo, Y.H., Skell, P.S., Thornberry, H.H., Ehrlich, J., McGuire, J.M., Savage, G.M., and Sylvester, J.C.
1945. Assay of streptomycin by the paper disc plate method. J. Bact. 50, 701–709.
Mahendra, S. and Alvarez-Cohen, L. 2005. Pseudonocardia dioxanivorans sp. nov., a novel actinomy-
cete that grows on 1,4-dioxane. Int. J. System. Evolution. Microbiol. 55, 593–598.
Mangamuri, U.K., Poda, S., Kamma, S., and Muvva, V. 2011. Optimization of culturing conditions
for improved production of bioactive metabolites by Pseudonocardia sp. VUK-10. Mycobiology
39(3), 174–181.
Maskey, R.P., Kock, I., Helmke, E., and Laatsch, H. 2003. Isolation and structure determination of
phenazostatin D, a new phenazine derivative from a marine actinomycete isolate Pseudonocardia
sp. B6273. Z. Naturforsch. B. J. Chem. Sci. 58, 692–694.
Menpara, D. and Chanda, S. 2013. Endophytic bacteria—Unexplored reservoir of antimicrobials for
combating microbial pathogens. Microbial Pathogens and Strategies for Combating Them: Science,
Technology and Education (Méndez-Vilas, A., Ed.), pp. 1095–1103.
Mizuba, S., Lee, K., and Jiu, J. 1975. Three antimicrobial metabolites from Aspergillus caespitosus. Can.
J. Microbiol. 21(11), 1781–1787.
Monisha, K., Renu, S., and Rup, L. 2011. Selective isolation of rare Actinomycetes producing novel
antimicrobial compounds. Int. J. Adv. Biotechnol. Res. 3, 357–375.
Muiru, W.M. 2000. Isolation of soil actinomycetes, characterization and screening of their antibiotics
against economically important plant pathogens. MSc thesis, University of Nairobi, Nairobi,
Kenya.
Nowak-Thompson, B., Gould, S.J., Kraus, J., and Loper, J.E. 1994. Production of 2,4-diacetylphloro-
glucinol by the biocontrol agent Pseudomonas fluorescens Pf-5. Can. J. Microbiol. 40, 1064–1066.
Omura, S., Tanaka, H., Tanaka, Y., Spiri-nakagawa, P., Oiwa, R., Takahashi, Y., Matsuyama, K., and
Iwai, Y. 1997. Studies on bacterial cell wall inhibitors. VII. Azureomycins A and B new antibiot-
ics produced by Pseudonocardia azurea nov. sp. taxonomy of the producing organism, isolation,
characterization and biological properties. J. Antibiot. 32, 985–994.
Ozgur, C., Gulten, O., and Aysel, U. 2008. Isolation of soil Streptomyces as source antibiotics active
against antibiotic-resistant bacteria. Eurasia. J. Biol. Sci. 2, 73–82.
Paulsen, I.T., Press, C.M., and Ravel, J. 2005. Complete genome sequence of the plant commensal
Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23, 873–878.
Petit, K.E., Mondeguer, F., Roquebert, M.F., Biard, J.F., and Pouchus, Y.F. 2004. Detection of griseoful-
vin in a marine strain of Penicillium waksmanii by ion traps spectrometry. J. Microbiol. Methods
58, 59–65.
Qinggui, W. and Lijian, X. 2012. Beauvericin, a bioactive compound produced by fungi: A short
review. Molecules 17, 2367–2377.
Raaijmakers, J.M., Vlami, M. and de Souza, J.T. 2002. Antibiotic production by bacterial biocontrol
agents. Antonie Van Leeuwenhoek. 81, 537–547.
Raaijmakers, J.M. and Mazzola, M. 2012. Diversity and natural functions of antibiotics produced by
beneficial and plant pathogenic bacteria. Ann. Rev. Phytopathol. 50, 403–424.
Rai, M. and Chikindas, M. 2011. Natural Microbials in Food Safety and Quality. CABI International,
London, U.K.
Rattanachaikunsopon, P. and Phumkhachorn, P. 2010. Lactic acid bacteria: Their antimicrobial com-
pounds and their uses in food production. Ann. Biol. Res. 1(4), 218–228.
Ripa, F.A., Nikkon, F., Zaman, S., and Khond, P. 2009. Optimal conditions for antimicrobial metab-
olites production from a new Streptomyces sp. RUPA-08PR isolated from Bangladeshi Soil.
Mycobiology 37, 211–214.
Romero, D., Traxler, M.F., Lopez, D., and Kolter, R. 2011. Antibiotics as signal molecules. Chem. Rev.
111, 5492–5505.
Ryu, J., Lee, S., Lee, Y., Lee, S., Kim, D., Cho, S., Ba, D., Park, K., and Yun, H. 2000. Screening and
identification of antifungal Pseudomonas sp. that suppresses balloon flower root rot caused by
Rhizoctonia solani. J Microbiol. Biotechnol. 10, 435–440.
Seok, K.B., Surk, S.M., and Byung, K.H. 1999. Isolation, antifungal activity, and structure elucidation
of the glutarimide antibiotic, streptimidone, produced by Micromonospora coerulea. J. Agric.
Food Chem. 47, 3372–3380.
Sethi, S., Ravi, K., and Saksham, G. 2013. Antibiotic production by microbes isolated from soil. Int.
J. Pharm. Res. 4, 2967–2973.
Sharma, M. 2014. Actinomycetes: Source, identification, and their applications. Int. J. Curr. Microbiol.
Appl. Sci. 3(2), 801–832.
Strobel, G. 2003. Endophytes as sources of bioactive products. Microbes Infect. 5, 535–544.
Trujillo, M., Kroppenstedt, R., Schumann, P., Carro, L., and Martínez-Molina, E. 2006. Micromonospora
coriariae sp. nov., isolated from root nodules of Coriaria myrtifolia. Int. J. Syst. Evol. Microbiol. 56,
2381–2385.
Vijayakumari, S.J., Sasidharannair, N.K., and Nambisan, B. 2013. Optimization of media and tem-
perature for enhanced antimicrobial production by bacteria associated with Rhabditis sp. Iran.
J. Microbiol. 5, 136–141.
Wang, L.W., Zhang, Y.L., Lin, F.C., Hu, Y.Z., and Zhang, C.L. 2011. Natural products with antitumor
activity from endophytic fungi. Mini Rev. Med. Chem. 11, 1056–1074.
Xiao-Yan, S., Qing-Tao, S., Shu-Tao, X., Xiu-Lan, C., Cai-Yun, S., and Yu-Zhong, Z. 2006. Broad-
spectrum antimicrobial activity and high stability of trichokonins from Trichoderma koningii
SMF2 against plant pathogens. FEMS Microbiol. Lett. 260(1), 119–125.
Xin, L., Yaojian, H., Meijuan, F., Jianfeng, W., Zhonghui, Z., and Wenjin, S. 2006. Cytotoxic and anti-
microbial metabolites from marine lignicolous fungi, Diaporthe sp. FEMS Microbiol. Lett. 251(1),
53–58.
Yang, Z. 2000. Antimicrobial compounds and extracellular polysaccharides produced by lactic acid
bacteria: Structures and properties. Academic dissertation, Department of Food Technology,
University of Helsinki, Helsinki, Finland.
Yu, H., Lei, Z., Lin, L., Zheng, C., Lei, G., Wenchao, L., Peixin, S., and Luping, Q. 2010. Recent develop-
ments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol.
Res. 165, 437–449.
Zhao, J., Yan, M., Tijiang, S., Yan, L., Ligang, Z., Mingan, W., and Jingguo, W. 2010. Antimicrobial
metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris polyphylla var.
yunnanensis. Molecules 15, 7961–7970.
Zhang, Z., Kudo, T., Nakajima, Y., and Wang, Y. 2001. Clarification of the relationship between the
members of the family Thermomonosporaceae on the basis of 16S rDNA, 16S–23S rRNA internal
transcribed spacer and 23S rDNA sequences and chemotaxonomic analyses. Int. J. Syst. Evol.
Microbiol. 51, 373–383.
chapter four
Animal fecal actinomycetes

A new source for the discovery of drug leads
Yi Jiang, Li Han, Xueshi Huang, and Chenglin Jiang
Contents
4.1 Introduction...........................................................................................................................54
4.2 Isolation methods of animal fecal actinomycetes............................................................ 56
4.2.1 Collection and pretreatment of samples............................................................... 56
4.2.2 Isolation medium for actinobacteria (per liter)..................................................... 56
4.2.2.1 HV medium................................................................................................ 56
4.2.2.2 YIM 171........................................................................................................ 56
4.2.2.3 YIM 212........................................................................................................ 56
4.2.2.4 YIM 47.......................................................................................................... 56
4.2.2.5 YIM 601........................................................................................................ 56
4.2.3 Inhibitors.................................................................................................................... 56
4.2.4 Effect of isolation media.......................................................................................... 57
4.2.5 Key points for isolating actinobacteria from animal feces................................. 58
4.3 Diversity of animal fecal actinomycetes............................................................................ 58
4.3.1 Identification of pure cultivated actinobacteria................................................... 58
4.3.2 Diversity of actinobacteria....................................................................................... 59
4.3.2.1 Hoolock gibbon (Hylobates hoolock).......................................................... 59
4.3.2.2 Yunnan snub-nosed monkey (Rhinopithecus bieti)................................. 59
4.3.2.3 Assamese macaque (Macaca assamensis)................................................. 59
4.3.2.4 Bengal tiger (Panthera tigris)...................................................................... 59
4.3.2.5 Manchurian tiger (Panthera tigris altaica)................................................. 60
4.3.2.6 Giant panda (Ailuropoda melanoleuca)...................................................... 60
4.3.2.7 Red panda (Ailurus fulgens)....................................................................... 62
4.3.2.8 Civet (Viverra zibetha)................................................................................. 62
4.3.2.9 Asiatic black bear (Ursus thibetanus)........................................................ 62
4.3.2.10 Shanxi sika (Cervus nippon)....................................................................... 62
4.3.2.11 Sambar (Rusa unicolor)...............................................................................63
4.3.2.12 Vicuna (Vicugna pacos)...............................................................................63
4.3.2.13 Common wildebeest (Connochaetes taurinus)..........................................63
4.3.2.14 Rhino (Rhinoceros sondaicus).....................................................................63
4.3.2.15 Indian elephant (Elephas maximus)...........................................................63
4.3.2.16 Chinese bamboo rat (Rhizomys sinensis)..................................................64
4.3.2.17 Peacock (Pavo cristatus)..............................................................................64
4.3.2.18 Common black-headed gull (Larus ridibundus)......................................64
4.3.2.19 Red-crowned crane (Grus japonensis)......................................................64
53
4.3.3 Diverse features of animal fecal actinomycetes...................................................65

4.3.4 Comparison of actinomycete diversity in different habitats.............................. 67
4.4 Bioactivities of animal fecal actinomycetes...................................................................... 68
4.4.1 Antimicrobial activity.............................................................................................. 68
4.4.2 Enzymatic activities................................................................................................. 69
4.4.3 Antitumor activities................................................................................................. 71
4.4.4 Genes encoding the biosynthetic enzymes of five antibiotics........................... 72
4.4.5 Toxins of two streptomycetes to mice.................................................................... 73
4.5 Discovery of bioactive compounds.................................................................................... 73
4.6 Effects of microbial manure on Notoginseng disease.................................................... 73
4.7 Conclusion............................................................................................................................. 76
Acknowledgments.........................................................................................................................77
References........................................................................................................................................77
4.1 Introduction
The clinical demand for new drugs worldwide is substantial and extremely urgent, reflect-
ing the rapid expansion of dangerous diseases, including influenza, SARS, helopyra,
tuberculosis, and HIV, the frequency of multiple common ailments (cancer, hypertension,
diabetes, hyperlipidemia, etc.), the emergence of new diseases without known causes, and
the evolution and spread of antibiotic-resistant pathogens (Payne et al., 2007; Jiang et al.,
2008; Goodfellow et al., 2010).
Based on the most recent statistics obtained in 2012, the success rate for the devel-
opment of new drugs from synthetic compounds is only 0.005%, while the rate for the
development of whole natural products is 0.6% and that for the development of natural
microbial products is 1.6% (Bérdy, 2012). Most of the drugs currently available on the mar-
ket are those obtained from the synthesis or semisynthesis of natural products. Thus,
microorganisms remain an important source for the development of new drugs. The bio-
active compounds recovered from microbes can be modified using gene manipulation to
obtain more superior or ideal drugs, which are subsequently produced on a large scale.
Actinomycetes (Actinobacteria) have recently received much attention as these bacteria
produce a variety of natural drugs and other bioactive metabolites, including antibiotics,
enzyme inhibitors, and enzymes. More than 22,000 bioactive secondary metabolites (includ-
ing antibiotics) from microorganisms have been identified and published in the scientific
and patent literature, and approximately half of these compounds are produced by actino-
mycetes. Currently, approximately 160 antibiotics have been used in human therapy and
agriculture, and 100–120 of these compounds, including streptomycin, erythromycin, gen-
tamicin, and avermectin, are produced by actinomycetes (Bérdy, 2005). However, the use of
general approaches to develop new drugs from actinomycetes growing in common habitats is
extremely difficult (Jiang et al., 2009). Although several microorganisms have been identified,
described, screened, and used in many applications, more than 90% of all microorganisms
remain unknown (Jennifer et al., 2001; Kaeberlein et al., 2002; Zengler et al., 2002; Joseph et al.,
2003; Chiao, 2004; Handelsman 2004; Patrick and Handelsman, 2005; Lior 2007; Ibrahim et al.,
2009), and these unknown microbes might offer hope for the development of new drugs.
To overcome the challenges of drug development from microbial metabolites, new
concepts based on genomics have been described, that is, “new habitats, new methods, new
species, new gene clusters, new products, and new uses” (Jiang et al., 2009; Jensen, 2010; Xu
et al., 2010). Thus, novel microbes should contain new gene clusters that synthesize novel sec-
ondary metabolites to obtain new bioactive compounds (Jiang et al., 2009). Many laboratories
Chapter four: Animal fecal actinomycetes 55
and companies have focused on new actinomycete resources from new habitats, such as
oceans (Bull and Stach, 2007; Abdelmohsen et al., 2010; Blunt, 2011; Jiang et al., 2011; Blunt
et al., 2013; Subramani and Aalbersberg, 2013), extreme environments (Jiang et al., 2006; Javad
et al., 2013), and plants, for the development of new drugs. Generating pure cultured micro-
organisms from uncultured sources might provide information concerning new drug leads.
Actinomycetes remain an important source for the development of new natural drugs. Baltz
(2008) proposed a “Renaissance in antibacterial discovery from actinomycetes.”
Animal intestinal and fecal microorganisms (fecal microbiota) have been studied for
decades (Savage, 1977). A large number of microbes exist in the gastrointestinal tract and
feces of animals. The intestinal microbial community, comprising 1013 to 1014 microorgan-
isms, outnumbers somatic and germ cells by at least one order of magnitude (Simpson
et al., 2002). However, most of these microorganisms have not been cultured (Daly et al.,
2001; Greetham et al., 2002; Simpson et al., 2002; Suchodolski et al., 2004, 2008; Ritchie et al.,
2008; Durso et al., 2010; Run-chi et al., 2010; Zhang and Chen, 2010). Exploring and utiliz-
ing the enormous beneficial resources from fecal microbiota are a tempting challenge.
For example, probiotics as dietary supplements containing friendly bacteria have been
widely used to recover microbial balance, improve intestinal and overall health, and pro-
tect against disease (Falagas et al., 2006; Meyer et al., 2006).
Every species of animal possesses a specific intestinal microbial community (intesti-
nal or fecal microbiota), reflecting coevolution and natural selection between microbes and
hosts. However, the relationship between microorganisms and hosts remains elusive due
to the complexity of the internal ecological system (Hooper and Gorden, 2001; Curtis and
Sperandio, 2011). Notably, animals are sterile before birth. However, after birth, the combined
effects of the living environment (food, air, water, climate, etc.) contribute to the establish-
ment of relationships with beneficial, harmful, anaerobic, and aerobic microorganisms; sub-
sequent adaptation between the microorganism and its host results in the gradual formation
of an extremely complex and relatively stable microbial flora in these animals. The microbial
flora gradually changes with increasing age and changing habitat. For example, the intesti-
nal microbiota changes when animals are treated with antibiotics and subsequently slowly
returns to normal after the treatment is completed. Indeed, the relationships among hosts and
microbes and between different microbes are extremely complex and continuously evolving.
Notably, all animals eat dirty and raw foods every day but rarely become sick. This phe-
nomenon is quite common, suggesting that animals have strong antibacterial and immune
mechanisms, and the microorganisms in the intestinal tract are important components of
these immune mechanisms. With respect to the host, microorganisms play an important role
in food digestion, absorption, immunity, antimicrobial defenses, and health maintenance, pro-
ducing a variety of bioactive substances (such as antibiotics, inhibitor agents, immune inhibi-
tors, vitamins, various enzymes, and enzyme inhibitors). These roles reflect the coevolution
and natural selection between microbes and hosts. Indeed, these microorganisms and their
bioactive products are nontoxic to the host, generated through a long-term toxic test involving
the symbiosis between microorganisms and hosts. Thus, nontoxic microbes provide an impor-
tant advantage for the development of medicines, pesticides, and health products.
Actinomycetes, as human and animal pathogens, have been widely studied (Beman,
1983). However, until recently, there have been few studies concerning the use of fecal acti-
nomycetes as a source for the discovery of novel drug. To obtain more unknown actino-
mycetes for the discovery of new bioactive metabolites, fecal samples from 48 species of
carnivorous, herbivorous, and omnivorous animals, including primates, mammals (peris-
sodactyl, artiodactyl, and ruminant), birds, amphibians, fishes, and insects, were collected.
The fecal actinomycetes were isolated, cultivated, and identified. The antimicrobial and
enzymatic activities and synthesis of five antibiotics from selected strains were determined.
The metabolites produced from selected highly bioactive strains were studied. A mixture of
microbial manure containing fecal streptomycetes was applied for the preventive treatment
of soil-borne notoginseng diseases in the field. Some results are reported herein.
4.2 Isolation methods of animal fecal actinomycetes

4.2.1 Collection and pretreatment of samples
Fresh fecal samples were collected from 48 selected animal species in the Yunnan Wild
Animal Park, Kunming, and South China Sea, China. Some samples were collected from
the original animal habitats. The samples were immediately transferred to sterile dishes
and dried for 10 days at 28°C. Two grams of each dried sample was pretreated at 80°C for
1 h and subsequently dissolved in 18 mL of sterile water containing 0.1% Na4P2O5, followed
by shaking at 220 rpm for 60 min. The suspension was treated with ultrasound waves for
40 s at 150 W before coating (Jiang et al., 2010). The suspension was diluted from 10 –1 to 10 –8,
and three dilutions, 10 –5, 10 –6, and 10 –7, were used for isolating actinomycetes.
4.2.2 Isolation medium for actinobacteria (per liter)

4.2.2.1 HV medium
(Hayakawa and Nonomura, 1987)
4.2.2.2 YIM 171

Glycerol 10 g, asparagine 1 g, K2HPO4·H2O 1 g, MgSO4·7H2O 0.5 g, CaCO3 0.3 g, vit mixture
of HV medium 3.7 mg, and agar 15 g; pH 7.2
4.2.2.3 YIM 212

Mycose 5 g, proline 1 g, (NH4)2SO4 1 g, NaCl 1 g, CaCl2 2 g, K2HPO4 1 g, MgSO4·7H2O 1 g,
vit mixture of HV medium 3.7 mg, and agar 15 g; pH 7.2
4.2.2.4 YIM 47
Na2HPO4 0.5 g, KCl 1.7 g, MgSO4·7H2O 0.05 g; FeSO4·7H2O 0.01 g, CaCl2 1 g, soy bean flour
0.2 g, lignin 1 g, vit mixture of HV medium 3.7 mg, soil extract 100 mL, and water 900 mL;
pH 7.5
4.2.2.5 YIM 601

Solution starch 10 g, casein 0.3 g, KNO3 2 g, MgSO4·7H2O 0.05 g, NaCl 2 g, K2HPO4 2 g,
CaCO3 0.02 g, FeSO4 10 mg, vit mixture of HV medium 3.7 mg, and agar 15 g; pH 7.2 ~ 7.4
4.2.3 Inhibitors
All media were supplemented with four filter-sterilized mixtures or single solutions con-
taining inhibitors against fungi and gram-negative bacteria (per liter): (1) 50 mg cyclohexi-
mide, 50 mg nystatin, 20 mg nalidixic acid, and 3 mg penicillin; (2) 100 mg cycloheximide,
100 mg nystatin, 40 mg nalidixic acid, and 5 mg penicillin; (3) 50 mg K2Cr2O7 and 5 mg
penicillin; and (4) 75 mg K2Cr2O7 and 5 mg penicillin.
The plate dilution method was used to isolate actinobacteria from the sample suspen-
sion. Approximately 0.1 mL of each sample (10 –5, 10 –6, and 10 –7 dilutions) was used to coat
the plates and cultivated for 7−35 days at 28°C. Subsequently, the colonies were counted,
and a single Actinobacteria colony was picked to inoculate a slant with the same isolation
medium. The pure strains were cultured at 4°C and in 20% of glycerol at −20°C.
4.2.4 Effect of isolation media

The quantity of cultivable actinomycetes in mixed samples containing eight species of
animal feces was 7–21 × 109, and that of other bacteria was 6 × 109; mostly, the growth of
gram-negative bacteria was suppressed using inhibitors. The optimum fecal suspension
dilutions for isolating actinobacteria were 10 –6 and 10 –7, in which approximately 22–133
colonies were observed on the isolation plates (Table 4.1). However, the optimum concen-
tration for each animal fecal sample should be determined in advance.
Table 4.2 shows the results of the selective isolation of actinobacteria with five media
from fecal samples obtained from 13 species of animals. In Table 4.1, about 123 pure
Table 4.1 Cfu/ga of actinobacteria on YIM 171 isolation medium at different dilutions
Actinobacteria
Dilution Mixture fecal samples of Fecal sample of Other
times seven species of animal Vicugna pacos bacteria Fungi
4th 1634 × 10 5 1324 × 10 5 132 × 10 5 0
5th 204 × 106 188 × 106 87 × 106 0
6th 133 × 107 98 × 107 32 × 107 0
7th 66 × 108 22 × 108 11 × 108 0
8th 21 × 109 7 × 109 6 × 109 0
CKb 17 × 108 486 × 108 11 × 107
a cfu/g = colony-forming units.
b CK = 7th dilution without inhibitors, and single actinomycete colonies were not obtained.
Table 4.2 Effect of the selective isolation of actinobacteria from the fecal samples of 17 animal
species using five different media (number of strains obtained)
YIM medium No.
Sample source HV 47 171 212 601 Total
Hylobates hoolock 19 6 15 11 16 67
Panthera tigris 12 9 22 23 32 98
Panthera tigris altaica 11 6 15 21 19 72
Ailuropoda melanoleuca 15 16 18 21 17 87
Viverra zibetha 12 10 12 19 9 62
Cavnlvara zlrsidae 19 9 18 33 12 91
Vicugna pacos 38 39 27 36 16 156
Rhinoceros sondaicus 43 8 34 33 22 140
Buceros bicornis 11 7 20 14 12 64
Aceros undulatus 12 4 15 6 20 57
Testudo elephantopus 3 7 7 3 0 20
Ursus thibetanus 19 9 18 33 12 91
Cervus nippon 33 22 18 36 19 128
Total 247 152 239 289 206 1123
cultivated strains of actinomycetes were isolated. Among all the strains, 156 and 140 were
isolated from Vicugna pacos and Rhinoceros sondaicus, respectively. Only 20 strains were iso-
lated from Testudo elephantopus. YIM 212, HV, and YIM 171 media were better for isolating
actinobacteria, resulting in the identification of 289, 247, and 239 strains of actinobacteria,
respectively.
4.2.5 Key points for isolating actinobacteria from animal feces

The abundance of gram-negative bacteria in animal feces presents a major challenge for
the isolation of fecal actinobacteria. To eliminate gram-negative bacteria and fungi and to
obtain more unknown actinobacteria for discovering novel lead compounds, some key
points for sampling and isolation should be considered.
First, based on the results of previous experiments, it is best to collect fresh fecal sam-
ples from wild animals living in original habitats; second, the fresh samples should be
dried at 25°C–28°C for 7–10 days; third, the dried samples should be treated for 60 min at
80°C, and the fecal suspension should be treated with ultrasound waves for 40 s at 150 W
before coating (Jiang et al., 2010); fourth, potassium bichromate 50 and 5 mg penicillin or
nystatin 50 mg, nalidixic acid 20 mg, and 5 mg penicillin should be added per liter of iso-
lation medium to inhibit the growth of fungi and gram-negative bacteria; fifth, the sam-
ples should be diluted to 10 –5, 10 –6, and 10 –7, and the optimum dilution concentration for
each animal fecal sample should be determined in advance; sixth, YIM 212, YIM 171, and
HV media are better for the isolation of fecal actinobacteria, and these media should be
improved and constantly updated with respect to different samples; and seventh, all exper-
iments should be performed under strict sterile conditions for avoiding spread of pathogen.
Animal fecal actinomycetes represent a new field of study. The physiological features
of these bacteria are not understood. Therefore, the method for the selective isolation of
actinomycetes (including the isolation media, pH, inhibitors, sample pretreatment, and
culture temperatures) should be different from those used to isolate bacteria from soil,
sea, and plant samples, and this method should be continually improved and updated for
isolation from different fecal samples.
Some actinobacteria were the pathogen of humans and animals (Beman, 1983). For
example, Cellulosimicrobium funkei, which was first discovered in a clinic, was an oppor-
tunistic pathogen, close to Oerskovia in taxonomy, and mainly infected people who have
immune dysfunction or inflammation (Brown et al., 2006; Petkar et al., 2011); this was
found in our study, too. Therefore, the fresh fecal samples have to be collected from health
bodies. The whole length of research work should be carried under strict sterile conditions
for avoiding pathogens to interference researchers.
4.3 Diversity of animal fecal actinomycetes

4.3.1 Identification of pure cultivated actinobacteria
A total of 3049 pure strains were isolated from the feces samples obtained from 48 animal
species; 1869 strains were obtained after eliminating duplicate strains based on morpho-
logical and cultural characteristics. The DNA was extracted from pure strains for 16S rDNA
analysis (Orsini and Romano-Spica, 2001). PCR amplification of the 16S rDNA, followed by
purification and sequencing of the PCR products, was performed as previously described
(Cui et al., 2001). The forward primer F8 (8 ± 27; 5′-GAG AGT TTG ATC CTG GCT CAG-3′)
and the reverse primer (1510 ± 1492; 5′-GGT TAC CTT GTT ACG ACT T-3′) were used.
The resulting sequences were manually aligned using the sequences from available, public
databases. Phylogenetic trees were inferred using neighbor-joining (Saitou and Nei, 1987)
and maximum-likelihood methods (Felsenstein, 1981). All pure cultivated strains were
identified at a genus and species level.
4.3.2 Diversity of actinobacteria

The actinomycete communities in 19 of the 48 fecal samples are described.
4.3.2.1 Hoolock gibbon (Hylobates hoolock)

The hoolock gibbon is a member of the Hylobatidae primates. This primate is classified as
a class I protected species according to the Law of the People’s Republic of China on the
Protection of the Wildlife (LPW), the Convention on International Trade in Endangered
Species of Wild Fauna and Flora (CITES), and the International Union for Conservation
of Nature and Natural Resources (IUCN). Fresh fecal samples from two individual pri-
mates were collected at three different times, and a total of 108 pure cultured strains were
isolated. After eliminating several duplicate strains based on morphological and cultural
characteristics, 76 strains were selected, and the 16S rDNA sequences were determined.
A phylogenetic analysis was performed. The strains were identified at the genus and
species levels. The isolated strains comprised 12 genera of actinobacteria: Arthrobacter,
Cellulosimicrobium, Corynebacterium, Dietzia, Gulosibacter, Kocuria, Microbacterium, Nocardia,
Oerskovia, Rhodococcus, Streptomyces, and Zimmermannella. The molinate-degrading actino-
mycete Gulosibacter, initially identified by Célia et al. (2004), currently contains three spe-
cies. Ten other bacteria, Acinetobacter, Bacillus, Jeotgalicoccus, Kurthia, Leuconostoc, Planococcus,
Psychrobacter, Pseudomonas, Psychrobacillus, and Rummeliibacillus, were also identified.
4.3.2.2 Yunnan snub-nosed monkey (Rhinopithecus bieti)

The rare Yunnan snub-nosed monkey belongs to Cercopithecidae primates. This monkey
is classified as class I protected animal of China according to the CITES and the IUCN.
Fresh fecal samples from three individuals were collected at three different times. A total
of 122 strains were isolated, and 66 strains were identified, belonging to 13 genera of acti-
nobacteria: Agrococcus, Arthrobacter, Cellulosimicrobium, Citricoccus, Corynebacterium, Gordonia,
Jiangella, Kocuria, Microbacterium, Mobilicoccus, Oerskovia, Rhodococcus, and Streptomyces.
Jiangella are distributed in various habitats including deserts, alkaline soils, caves, and plants
(Song et al., 2005; Lee, 2008; Qin et al., 2009; Kampfer et al., 2011; Tang et al., 2011). Other bacte-
ria, namely Bacillus, Enterococcus, Leuconostoc, Paenibacillus, Catellibacterium, and Myroides, were
also identified.
4.3.2.3 Assamese macaque (Macaca assamensis)

The Assamese macaque is a member of the Cercopithecidae primates. This primate is a
class I protected animal in China. Ninety-three selected strains were identified. These
Actinobacteria belonged to eight genera, including Acinetobacter, Corynebacterium, Kocuria,
Luteococcus, Microbacterium, Nocardiopsis, Rhodococcus, and Streptomyces. About 11 other
bacteria, namely, Achromobacter, Acinetobacter, Bacillus, Bordetella, Enterococcus, Escherichia,
Jeotgalicoccus, Methylobacterium, Planococcus, Pseudomonas, and Shigella, were also identified.
4.3.2.4 Bengal tiger (Panthera tigris)

The Bengal tiger is a member of the family Felidae of the order Carnivore and has been clas-
sified as a class I protected animal of China according to the CITES and the IUCN. These
animals inhabit the same type of forest as the Manchurian tiger. Around 258 strains of
actinomycetes were isolated from fresh fecal samples, and 177 of these strains, belong-
ing to 13 genera, namely, Arthrobacter, Corynebacterium, Dietzia, Enteractinococcus, Kocuria,
Microbacterium, Nocardia, Nocardiopsis, Oerskovia, Promicromonospora, Saccharomonospora,
Streptomyces, and Yaniella, were identified. The novel genus Enteractinococcus was described
in the International Journal of Systematic and Evolutionary Microbiology (Cao et al., 2012).
The genus Yaniella is typically found in saline soil (Li et al., 2004, 2008). Streptomycetaceae
occupied 64% of actinobacteria observed, representing the predominant strain, followed
by Micrococcaceae with 7% (Figures 4.1 and 4.2).
4.3.2.5 Manchurian tiger (Panthera tigris altaica)

The Manchurian tiger is classified as a class I protected animal in China according to the
CITES and the IUCN, and this animal is a member of the carnivorous Felidae family. Fresh
fecal samples from four Manchurian tigers were collected, and 302 pure cultured actino-
mycete strains were isolated. Among these, 107 strains were identified. Nine genera of acti-
nobacteria were identified from these fecal samples, namely, Arthrobacter, Enteractinococcus,
Microbacterium, Nocardia, Oerskovia, Promicromonospora, Saccharomonospora, Streptomyces, and
Yaniella. The fecal actinomycete component in the Manchurian tiger fecal samples was less
four genera than that of the Bengal tigers, although the nine identified genera were the same.
4.3.2.6 Giant panda (Ailuropoda melanoleuca)

The giant panda is a rare, national treasure in China, classified as a class I protected animal
according to the IUCN and the CITES. This panda only inhabits a limited area of northern
Sichuan, China, and is a member of the family Ailuridae (Ailuropodidae) of the order Carnivora;
its main food is bamboo. Five individual feces were sampled. A total of 330 pure cultured
actinomycete strains were isolated, and 133 of these strains were identified through phylo-
genetic analysis of the 16S rRNA gene sequences. These strains belonged to 13 genera of acti-
nobacteria: Agrococcus, Arthrobacter, Cellulomonas, Cellulosimicrobium, Janibacter, Micrococcus,
Micromonospora, Mycobacterium, Oerskovia, Patulibacter, Rhodococcus, Streptomyces, and
Verrucosispora. The genus Janibacter was identified in 1997 (Martin et al., 1997). Verrucosispora
are members of the family Micromonosporaceae, and these bacteria were isolated from a
% of family
4%
5% 2% 7%
2% 3% Micrococcaceae
5% 5% Nocardiaceae
Dietziaceae
3%
Streptomycetaceae
Nocardiopsaceae
Corynebacteriaceae
Pseudonocardineae
Promicromonosporaceae
Microbacteriaceae
Cellulomonadaceae
64%
Figure 4.1 Composition of actinobacteria in Panthera tigris feces.

95 Kocuria rosea DSM 20447T (X87756)

100 YIM 100298 (JQ669805)
93 Kocuria polaris CMS 76orT (AJ278868)
Arthrobacter koreensis CA15-8T(AY116496)
100
YIM 100297 (JQ669804)
58
92 Arthrobacter luteolus CF25T(AJ243422)
70 Yaniella flava YIM 70178T (AY684123)
100 Enteractinococcus fodinae G5T(FJ871122)
100 Enteractinococcus coprophilus YIM 100590T(JF507603)
YIM 100602 (JQ669809)
97 Microbacterium esteraromaticum DSM 8609T(Y17231)
100 YIM 100717 (JQ669813)
Microbacterium arabinogalactanolyticum IFO 14344T(AB004715)
61 98 Promicromonospora xylanilytica YIM 61515T(FJ214352)
100 YIM 100715 (JQ669812)
97 Promicromonospora aerolata V54AT(AJ487303)
100 Oerskovia paurometabola DSM 14281T(AJ314851)

YIM 100718 (JQ669814)
51 Oerskovia turbata NCIMB 10587T(X79454)
100 YIM 100296(JQ669806)

Streptomyces phaeochromogenes NBRC 3180T(AB184738)
64 Streptomyces ederensis NBRC 15410T(AB184658)
100 Nocardiopsis synnemataformans IMMIB D-1215T(Y13593)

YIM 100589 (JQ669808)
92 Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (ABUI01000017)
99 Saccharomonospora azurea NA128T (Z38017)

100
YIM 100713 (JQ669811)
Saccharomonospora xinjiangensis DSM 44391T (AJ306300)
YIM 100593 (JQ669810)
78 100
Corynebacterium freneyi ISPB 6695110T (AJ292762)
Corynebacterium xerosis DSM 20743T (X84446)
85
91 Dietzia cercidiphylli YIM 65002T(EU375846)
100 YIM 100291 (JQ669803)
Dietzia psychralcaliphila JCM 10987T(AB159036)

52 100 Nocardia pseudobrasiliensis ATCC 51512T (X84857)
YIM 100342 (JQ669807)
60 Nocardia altamirensis OFN S17T (EU006090)
Figure 4.2 Neighbor-joining tree showing the phylogenetic relationships based on the 16S rRNA
gene sequences of culturable actinomycetes isolated from the fecal samples of Panthera tigris. The
sequences identified in the present study are bolded. The bootstrap values (expressed as percent-
ages of 1000 replications) > 50% are indicated at the nodes. Bar = 1 nt substitution per 100 nt.
peat bog near Gifhorn, Germany, and they typically inhabit peat bogs, lakes, and oceans
(Goodfellow et al., 2012b). Patulibacter belongs to the order Solirubrobacterales (Gundlapally
and Ferran, 2009).
4.3.2.7 Red panda (Ailurus fulgens)

The red panda belongs to the family Procyonidae of the order Carnivora. This panda is clas-
sified as a class II protected animal in China. About 66 selected strains were identified,
belonging to 10 genera of actinobacteria, namely, Agrococcus, Arthrobacter, Corynebacterium,
Gulosibacter, Leucobacter, Microbacterium, Micrococcus, Pseudonocardia, Rhodococcus, and
Streptomyces. Six genera of other bacteria, namely Aerococcus, Brevundimonas, Jeotgalicoccus,
Planomicrobium, Pseudomonas, and Psychrobacter, were identified.
4.3.2.8 Civet (Viverra zibetha)

Civet (small Indian civet, Viverra indica, Viverricula malaccensis thai, Viverricula hanensis,
Viverricula pallida taivana, and Viverra pallida) lives in tropical and subtropical rain forests
and evergreen broadleaf forests. This omnivorous species are classified as class II pro-
tected animals in China according to the IUCN and the CITES. The civet is named for
producing musk. Civet fecal samples were collected from three individuals in a wild ani-
mal park in Malaysia twice. A total of 88 strains of Actinobacteria were isolated using five
different media. Fifty-eight strains were identified, comprising of 15 genera: Arthrobacter,
Cellulomonas, Cellulosimicrobium, Corynebacterium, Curtobacterium, Enteroactinococcus, Isop
tericola, Kocuria, Leucobacter, Microbacterium, Micrococcus, Rhodococcus, Saccharopolyspora,
Sanguibacter, and Streptomyces. Curtobacterium were rarely observed in the feces of
these animals. Cellulosimicrobium were identified as inhabitants of termites (Bakalidou
et al., 2002; Stackebrantd et al., 2004). Other bacteria, including Bacillus, Enterococcus,
Flavobacterium, Hansschlegelia, Methylobacterium, Ochrobactrum, Pseudomonas, Sporosarcina,
and Stenotrophomonas, were also identified from the civet fecal samples.
4.3.2.9 Asiatic black bear (Ursus thibetanus)

Asiatic black bears (moon bear) are omnivorous animals widely distributed throughout
the forests of broad areas from north to south in the eastern half sphere of China, Russia,
Iran, Pakistan, Laos, Japan, and India. These bears are classified as class II protected ani-
mals in China according to the IUCN and the CITES. Fecal samples of three individu-
als were collected twice. One hundred twelve strains of actinobacteria were isolated, and
32 strains were identified. They belong to 11 genera: Cellulosimicrobium, Dietzia, Kribbella,
Gordonia, Microlunatus, Microbacterium, Nocardiopsis, Promicromonospora, Rhodococcus,
Saccharomonospora, and Streptomyces. Five genera of other bacteria, namely, Massilia,
Myroides, Methylobacterium, Rhizobium, and Stenotrophomonas, were identified.
4.3.2.10 Shanxi sika (Cervus nippon)

Shanxi sika (sika, sika deer, spotted deer) is a class I protected animal in China according to
the IUCN. These phytophagous animals are widely distributed throughout forests and grass-
lands. Notably, the prevalence of these animals is rapidly decreasing. Fecal samples were
obtained from 12 individuals in Kunming and Shenyang, China, and 337 pure strains were
isolated. Among these, 117 strains were identified, belonging to 16 genera of actinobacteria:
Actinocorallia, Agrococcus, Arthrobacter, Cellulosimicrobium, Citricoccus, Isoptericola, Kocuria,
Leucobacter, Microbacterium, Mycobacterium, Promicromonospora, Nocardiopsis, Rhodococcus,
Salinibacterium, Streptomyces, and Tsukamurella. Only two other bacteria, namely Bosea and
Stenotrophomonas, were identified.
4.3.2.11 Sambar (Rusa unicolor)

Sambars are members of the family Cervidae of the order Artiodactyla, and these animals are
classified as class II protected animal in China according to the IUCN. A total of 33 selected
strains were identified, including eight actinobacteria, namely, Actinotalea, Arthrobacter,
Corynebacterium, Dietzia, Kocuria, Micrococcus, Streptomyces, and Tessaracoccus, and seven
other bacteria, including Bacillus, Desemzia, Paracoccus, Planococcus, Planomicrobium,
Psychrobacillus, and Sphingomonas.
4.3.2.12 Vicuna (Vicugna pacos)

Vicunas were originally identified in frigid zones from 3650 to 4800 m above sea
level in the Andes Mountains. Humans have long reared these camels, for it produce
high-grade hair. Vicunas belong to the family Camelidae of the order Artiodactyla.
Three individuals were imported from the Republic of Chile into the Yunnan Wild
Animal Park. Fecal samples were obtained from these animals, and 87 strains of acti-
nomycetes were isolated. Among these, 66 strains were identified, which belong to
15 genera, namely, Arthrobacter, Brevundimonas, Cellulosimicrobium, Corynebacterium,
Dietzia, Enteractinococcus, Gordonia, Isoptericola, Kocuria, Microbacterium, Micrococcus,
Nocardiopsis, Rhodococcus, Saccharomonospora, and Streptomyces. The number of
Arthrobacter arilaitensis was 80 × 105/g in the fresh fecal sample. Eleven other bacte-
ria, including Achromobacter, Advenella, Ancylobacter, Jeotgalicoccus, Kurthia, Lysobacter,
Methylobacterium, Ornithinibacillus, Psychrobacillus, Shigella, and Solibacillus, were also
isolated and identified.
4.3.2.13 Common wildebeest (Connochaetes taurinus)

Common wildebeests are typically found living in the grasslands in Africa and were
imported to the Yunnan Wild Animal Park. The wildebeest belongs to the family Bovidae of
the order Artiodactyla. Fecal samples were obtained from four healthy individuals, and 96
purified strains were isolated. Among these, 43 strains were selected for identification. Only
five genera of actinobacteria, namely, Citricoccus, Microbacterium, Micrococcus, Rhodococcus,
and Streptomyces, were identified. These actinomycetes are commonly found in nature.
Other bacteria, including Stenotrophomonas and Methylobacterium, were also identified.
4.3.2.14 Rhino (Rhinoceros sondaicus)

The rhino is a rare nationally treasured animal in China, classified as a class I protected ani-
mal according to the IUCN. The rhino belongs to both Rhinocerotidae and Perissodactyla. Fecal
samples were collected from two individuals in Indonesia. About 202 strains were isolated,
and 112 strains were identified. Fourteen genera were identified: Arthrobacter, Brevundimonas,
Corynebacterium, Dietzia, Gulosibacter, Kocuria, Microbacterium, Micromonospora, Nocardiopsis,
Promicromonospora, Pseudoclavibacter, Rhodococcus, Streptomyces, and Tessaracoccus. Gulosibacter
and Tessaracoccus are rarely observed in nature. One strain (YIM 100770), identified as
a novel genus, was characterized using polyphasic taxonomic procedures (Xu et al.,
2007). Nineteen other bacteria, including Achromobacter, Alcaligenes, Ancylobacter, Bacillus,
Hansschlegelia, Kurthia, Luteimonas, Methylobacterium, Massilia, Novosphingobium, Paracoccus,
Pseudomonas, Psychrobacillus, Rhizobium, Shigella, Solibacillus, Sphingobacterium, Staphylococcus,
and Stenotrophomonas, were also identified.
4.3.2.15 Indian elephant (Elephas maximus)

The Indian elephant is typically found in south China and Asia, and the prevalence
of this rare, nationally treasured animal is rapidly decreasing. Thus, this elephant is
classified as a class I protected animal in China according to the IUCN. The Indian ele-
phant belongs to the family Elephantidae of the order Proboscidea. Fresh fecal samples
were obtained from four individuals in the Xiaomemgyang National Natural Protect
area and Yunnan Wild Animal Park. One hundred twenty-one strains were isolated, and
68 strains were identified, comprising 15 genera of actinobacteria, including Arthrobacter,
Cellulomonas, Cellulosimicrobium, Citricoccus, Janibacter, Kocuria, Leucobacter, Microbacterium,
Micrococcus, Micromonospora, Promicromonospora, Rhodococcus, Sphaerobacter, Streptomyces,
and Verrucosispora. Three genera of gram-negative bacteria, namely, Bacillus, Devosia, and
Planococcus, were identified.
4.3.2.16 Chinese bamboo rat (Rhizomys sinensis)

Humans have long reared the Chinese bamboo rat for producing high protein. The bamboo
rat is a member of the family Rhizomyidae of the order Rodentia. Fresh fecal samples were
obtained from six individuals, and actinomycetes were isolated from these samples at two
different times. A total of 306 strains were isolated, and 104 strains were identified, belong-
ing to 13 genera, namely, Agrococcus, Arthrobacter, Brachybacterium, Corynebacterium, Dietzia,
Gordonia, Labedella, Microbacterium, Oerskovia, Rhodococcus, Sanguibacter, Streptomyces, and
Williamsia. Three genera of gram-negative bacteria, namely Comamonas, Flavobacterium,
and Psychrobacter, were identified.
4.3.2.17 Peacock (Pavo cristatus)

The peacock, a member of the family Phasianidae of the order Galliformes, is classified as a
class I protected animal according to the LPW, the IUCN, and the CITES. Fecal samples
were obtained from 12 individuals, and the actinomycetes were isolated for 3 times. One
hundred eighty-eight strains were isolated, and 118 selected strains were identified, com-
prising 18 genera of actinobacteria, namely, Arthrobacter, Brevibacterium, Cellulosimicrobium,
Curtobacterium, Dietzia, Gordonia, Isoptericola, Janibacter, Kineococcus, Kocuria, Leucobacter,
Microbacterium, Nocardiopsis, Oerskovia, Rhodococcus, Pseudonocardia, Sanguibacter, and
Streptomyces, representing the largest genus observed in the present study. Other bacte-
ria, including Bacillus, Devosia, Lysinibacillus, Methylobacterium, Planococcus, Planomicrobium,
Shigella, and Staphylococcus, were also identified.
4.3.2.18 Common black-headed gull (Larus ridibundus)

The common black-headed gull belongs to the family Laridae of the order Ciconiiformes.
It is listed in the directory by LPW and IUCN. The common black-headed gull, a typical
migrant bird, is typically found in Siberia, Russia. Over 30,000 Larus ridibundus migrate
to Dian Lake, Kunming, from Siberia between December and March every year. A total
of 37 selected strains were identified from the fecal samples of these birds, belonging to
10 genera of actinobacteria, namely, Arthrobacter, Blastococcus, Devosia, Microbacterium,
Oerskovia, Paracoccus, Plantibacter, Promicromonospora, Pseudoclavibacter, and Streptomyces.
4.3.2.19 Red-crowned crane (Grus japonensis)

The red-crowned crane belongs to the family Gruidae of the order Gruiformes. This bird has
been classified as a class I protected animal according to the LPW, the IUCN, and the CITES.
Forty-six selected strains were identified from the feces of these birds. Nine genera of
actinobacteria were identified: Arthrobacter, Blastococcus, Cellulosimicrobium, Microbacterium,
Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and Yaniella. Actinomycete composi-
tions of a total of 48 species of animal feces were studied in our laboratories recent years.
Related results for rest of the 29 species of animals are not shown here.
4.3.3 Diverse features of animal fecal actinomycetes

A total of 222 genera, 53 families, and 23 orders have been collected and described in
Bergey’s Manual of Systematic Bacteriology (Goodfellow et al., 2012a). Fifty-one genera of pure
culturable Actinobacteria were isolated and identified in fecal samples collected from only
48 animal species. These Actinobacteria belong to 27 families of 12 orders, representing 24%
of 222 genera, 51% of 53 families, and 52% of 23 orders. An incertae sedis, Sphaerobacter, was
isolated from Indian elephant feces. Thus, these results showed that the diversity of animal
fecal actinobacteria is very rich. The actinobacteria identified herein are listed in Table 4.3.
Forty-eight other bacteria, including Achromobacter, Acinetobacter, Advenella, Aerococcus,
Alcaligenes, Ancylobacter, Aurantimonas, Bacillus, Bordetella, Bosea, Brevundimonas, Brochothrix,
Catellibacterium, Desemzia, Devosia, Enterococcus, Escherichia, Flavobacterium, Hansschlegelia,
Table 4.3 Component of the phylum Actinobacteria in the fecal samples of 48 animal species
Family Genus
Class I Actinobacteria
Corynebacteriales Corynebacteriaceae Corynebacterium
Dietziaceae Dietzia
Mycobacteriaceae Mycobacterium
Nocardiaceae Gordonia, Nocardia, Rhodococcus, Williamsia
Tsukamurellaceae Tsukamurella
Frankiales Frankiaceae Blastococcus
Geodermatophilaceae Mobilicoccus
Jiangellales Jiangellaceae Jiangelle
Kineosporiales Kineosporiaceae Kineococcus
Micrococcales Micrococcaceae Arthrobacter, Citricoccus, Enteractinococcus,
Kocuria, Micrococcus, Yaniella
Brevibacteriaceae Brevibacterium
Cellulomonadaceae Cellulomonas
Dermabacteraceae Brachybacterium
Intrasporangiaceae Janibacter
Promicromonosporaceae Cellulosimicrobium, Isoptericola, Promicromonospora
Microbacteriaceae Agrococcus, Curtobacterium, Gulosibacter, Labedella,
Leucobacter, Microbacterium, Plantibacter,
Pseudoclavibacter, Salinibacterium, Zimmermannella
Cellulomonadaceae Oerskovia
Sanguibacteraceae Sanguibacter
Incertae sedis Actinotalea
Micromonosporales Micromonosporaceae Micromonospora, Verrucosispora
Propionibacteriales Propionibacteriaceae Luteococcus, Microlunatus, Tessaracoccu
Pseudonocardiales Pseudonocardiaceae Pseudonocardia, Saccharomonospora,
Saccharopolyspora
Streptomycetales Streptomycetaceae Streptomyces
Streptosporangiales Thermomonosporaceae Actinocorallia
Nocardiopsaceae Nocardiopsis
Class II Thermoleophilia
Solirubrobacterales Patulibacteriaceae Patulibacter
Incertae sedis Incertae sedis Sphaerobacter
Jeotgalicoccus, Kurthia, Lactococcus, Luteimonas, Leuconostoc, Lysinibacillus, Lysobacter, Massilia,

Methylobacterium, Myroides, Novosphingobium, Ochrobactrum, Ornithinibacillus, Paenalc ligenes,
Paenibacillus, Paracoccus, Planococcus, Planomicrobium, Pseudomonas, Psychrobacter, Psychrobacillus,
Shigella, Solibacillus, Sphingobacterium, Sphingomonas, Sporosarcina, Staphylococcus, Stenotrophomon
as, and Rhizobium, were also identified.
Eighteen genera of actinobacteria were identified from fecal samples of peacock, and
the actinomycete component was the complexest; the second was 16 genera in Shanxi sika
deers; the third was 15 genera in civet, vicuna, and Indian elephants; and the least was
only 3 genera from Tragelaphus buxtoni and Python reticulates (unknown).
Members of the genus Streptomyces were isolated from 100% samples, representing the
most predominant microbes, and the colony-forming units (cfu)/g fresh sample ranged from
2 × 105 to 176 × 107 in different fecal samples. Streptomyces albus, S. albidoflavus, S. griseus, S.
hygroscopicus, S. rutgersensis, S. tendae, and S. violaceoruber were observed at a high frequency.
Members of Rhodococcus were isolated and identified in the fecal samples from all
animal species, representing the second most prevalent and widely distributed genus.
Rhodococcus coprophilus, Rh. corynebacterioides, Rh. equi, Rh. pyridinivorans, and Rh. zopfii
were most frequently observed.
The genome sizes of Streptomyces and Rhodococcus, up to 9 × 107 base pairs, are the largest
genomes in actinobacteria, and some species contain 20 or more natural product biosynthetic
gene clusters (Ōmura et al., 2001; Bentley et al., 2002; Mcleod et al., 2006). These results are con-
sistent with the idea that the function of actinomycetes in the host intestinal tract is primarily
influenced through bioactive substances produced by the members in these two genera.
Members of Arthrobacter, Microbacterium, and Micrococcus were identified from most of
the animal feces examined. Twenty-six genera belong to the order Micrococcales and eight
genera belong to the order Corynebacteriales in the 48 tested animal feces.
Based on these results, two conclusions can be drawn: first, members of both
Streptomyces and Rhodococcus exhibited the widest distribution and contained the larg-
est numbers, and second, the composition of actinobacteria with chemotypes IV–IX
(Lechevalier and Lechevalier, 1970, 1980), globose, and bacilliform shapes, particularly the
order Micrococcales, exhibited the richest diversity and were detected at a high frequency
in most of animal fecal samples. These distinct features of the fecal actinobacterium com-
munity differ from those of the soil, marine, and plant communities.
Some members of rare actinobacteria, such as Yaniella (Li et al., 2004, 2008), were identi-
fied from the feces of two species of tigers. A strain (YIM 100708) of Jiangella (Tang et al.,
2011), a genus widely distributed in saline and alkaline soil, desert, indoor wall material,
caves, and plant stems, was also isolated from the feces of Rhinopithecus bieti. The members
of a novel genus, Enteractinococcus, belonging to Micrococcaceae, were isolated and charac-
terized from three species of tigers (Cao et al., 2012).
Members of Micromonospora were isolated from the feces of Ailuropoda melanoleuca,
Rhinoceros sondaicus, Elephas maximus, and Cygnus melancoryphus (not shown). Interestingly,
Nocardia was only isolated from Panthera tigris altaica and Grus japonensis; Saccharopolyspora
was only isolated from Viverra zibetha; and Verrucosispora was only isolated from Ailuropoda
melanoleuca. Actinomycetes possessing cell wall chemotypes II (e.g., Actinoplanes and
Dactylosporangium) and III (Actinomadura and Streptosporangium) are commonly found in soil
and lakes, but these genera are not isolated from the animal fecal samples examined herein.
It is worth to notice that among the 1869 sequenced strains, 16S rDNA sequence simi-
larities of 106 strains with valid published species were below 98.5%. Thus, nearly 6% pure
cultivated strains were unknown, potentially representing novel species (Stackebrandt
and Gorbel, 1994; Xu et al., 2007).
4.3.4 Comparison of actinomycete diversity in different habitats

In previous studies of the actinomycetes diversity, 17 genera were isolated from soil
samples collected from primeval forests in Grand Shangri-La, southwest China (Cao
et al., 2009); 13 genera were isolated from soil samples from subtropical every green
forest in Gulin, Sichuan (Cao et al., 2010); 26 genera were isolated from soil samples
from tropical rain forests in Xishuangbanna, southwest China (Jiang et al., 2013); and
16 genera were isolated from soil samples from hypersaline soil in Qinghai, west China
(Jiang et al., 2006, 2011) (Table 4.4). The aerobic mycelium of these soil actinomycetes was
abundant. Fifteen genera of actinomycetes were identified from samples collected from
the Baltic Sea, and the members of Micromonospraceae were the most (Jiang et al., 2011).
Until recently, approximately 48 genera of actinobacteria were isolated and identified
from marine habitats worldwide (Goodfellow and Fiedler, 2010). In the present study,
Table 4.4 Comparison of actinomycete diversity in different habitats
Habitat Composition of actinobacteria References
Primeval forest Actinomadura, Actinopolymorpha, Agromyces, Arthrobacter, Cao et al.
soil in Grand Dactylosporangium, Kocuria, Lentzea, Mycetocola, Nocardia, (2009)
Shangri-La Nocardioides, Oerskovia, Promicromonospora, Pseudonocardia,
Rhodococcus, Streptomyces, Streptosporangium, Tsukamurella
Subtropical Actinomadura, Actinopolymorpha, Micromonospora, Mycobacterium, Cao et al.
evergreen Nocardia, Nocardioides, Nonomuraea, Promicromonospora, (2010)
forest soil in Pseudonocardia, Rhodococcus, Saccharomonospora, Streptomyces,
Sichuan Verrucosispora
Tropical rain Actinomadura, Actinoplanes, Actinopolymorpha, Agrococcus, Jiang et al.
forest soil in Agromyces, Arthrobacter, Citricoccus, Dactylosporangium, (2013)
Xishuangbanna Friedmanniella, Kribbella, Lentzea, Microbacterium,
Micromonospora, Mycobacterium, Nocardia, Nocardioides,
Nonomuraea, Oerskovia, Planosporangium, Promicromonospora,
Pseudonocardia, Rhodococcus, Saccharopolyspora,
Sphaerisporangium, Streptomyces, Streptosporangium
Hypersaline soil Citricoccus, Corynebacterium, Isoptericola, Jiangella, Marinococcus, Jiang et al.
in Qinghai Myceligererans, Nesterenkonia, Nocardiopsis, Prauserella, (2006)
Rhodococcus, Saccharomonospora, Salinimicrobium,
Streptomonospora, Streptomyces, Yaniella, Zhihengliuella
Baltic Sea Actinomadura, Actinoplanes, Amycolatopsis, Arthrobacter, Jiang et al.
Cellulomonas, Isoptericola, Kocuria, Micromonospora, (2011)
Microbacterium, Myceligenerans, Mycobacterium, Nocardiopsis,
Promicromonospora, Rhodococcus, Streptomyces
48 species of Actinocorallia, Actinotalea, Agrococcus, Arthrobacter, This study
animal feces Brachybacterium, Brevibacterium, Brevundimonas, Cellulomonas,
Cellulosimicrobium, Citricoccus, Corynebacterium, Curtobacterium,
Dietzia, Enteractinococcus, Gordonia, Gulosibacter, Isoptericola,
Janibacter, Jiangella, Kineococcus, Kocuria, Labedella, Leucobacter,
Luteococcus, Microbacterium, Micrococcus, Micromonospora,
Mobilicoccus, Mycobacterium, Nocardia, Nocardiopsis, Oerskovia,
Patulibacter, Plantibacter, Promicromonospora, Pseudoclavibacter,
Pseudonocardia, Rhodococcus, Saccharomonospora, Saccharopolyspora,
Salinibacterium, Sanguibacter, Sphaerobacter, Streptomyces,
Tessaracoccus, Tsukamurella, Verrucosispora, Williamsia, Yaniella,
Zimmermannella
51 genera of actinobacteria were isolated from only 48 species of animal feces, and 23%
of 222 genera, 51% of 53 families, and 52% of 23 orders were classified in Bergey’s Manual.
Therefore, actinomycete communities of animal feces are not only complex but also dif-
ferent from those in soil, extreme environments, and marine environments. More than
one million animal species have been identified worldwide. Thus, animal fecal actino-
mycetes are tremendous natural resources.
4.4 Bioactivities of animal fecal actinomycetes

4.4.1 Antimicrobial activity
The strains were fermented in nutrient broth (YIM 61, soybean meal 20 g, glucose 10 g,
peptone 4 g, K2HPO4 1 g, MgSO4 · 7H2O 0.5 g, NaCl 1 g, CaCO3 2 g, water 1000 mL,
pH 7.8), shaking for 7 days at 28°C. The agar diffusion method was used to determine
the
antimicrobial activities against Bacillus subtilis (DSM 3258T), Staphylococcus aureus
(DSM 30501T), Mycobacterium tuberculosis avium (unpathogen from Dr. Lixin Zhang),
Candida albicans (DSM 24506T), and Aspergillus niger (IAM 190). The results from the experi-
ments performed with animal fecal actinomycetes are shown in Table 4.5.
Table 4.5 Antimicrobial activities of some fecal actinobacteria

Number
of test Bacillus Staphylococcus Mycobacterium Candida Aspergillus
Source of Strains strains subtilis aureus tuberculosis albicans niger
Hylobates hoolock 26 4 1 1 0 11
Rhinopithecus bieti 29 4 1 1 3 14
Panthera tigris altaica 21 4 0 0 2 1
Panthera tigris tigris 13 1 0 0 2 0
Ailuropoda melanoleuca 28 2 3 2 2 0
Viverricula indica 7 5 4 1 0 4
Helarctos malayanus 3 0 0 0 0 0
Elephas maximus 32 5 8 0 12 6
Cervus Nippon 43 5 3 0 7 1
Cervus elaphus 32 3 6 0 3 10
Elaphurus davidianus 18 4 1 0 6 0
Giraffa camelopardalis 7 0 0 0 3 0
Connochaetes taurinus 6 0 1 0 1 0
Vicugna pacos 18 4 1 0 7 1
Oryx leucoryx 9 4 2 0 4 0
Tragelaphus buxtoni 7 2 3 0 5 0
Aceros undulatus 3 0 0 0 0 0
Pavo cristatus 3 0 0 0 0 0
Cygnus cygnus 16 8 2 3 4 2
Struthio camelus 5 1 1 1 1 3
Indotestudo elongata 11 1 0 0 1 2
Python reticulates 47 27 15 14 13 6
Total 384 84 52 23 76 61
% 22 13 6 20 16
The antimicrobial activities of 384 strains isolated from 22 species of animal feces
were determined using the agar diffusion method (Table 4.5). Eighty-four strains (22%)
showed inhibitory activity against Bacillus subtilis, 52 strains (13%) showed activity
against Staphylococcus aureus, 23 strains (6%) showed activity against Mycobacterium
tuberculosis, 76 strains (20%) showed activity against Candida albicans, and 61 strains
(16%) showed activity against Aspergillus niger. Notably, 13–55% of the 47 strains iso-
lated from Python reticulates, which primarily included streptomycetes, exhibited
higher inhibitory activities against all five tested microbes. Fewer anti–Mycobacterium
tuberculosis strains (6%) exhibited antimicrobial activity. Three strains isolated from
Helarctos malayanus, Aceros undulatus, and Pavo cristatus feces did not exhibit antimicro-
bial activities. These results showed that actinobacteria from animal feces have wide
antimicrobial activities.
4.4.2 Enzymatic activities

The activities of 19 enzymes were determined using the API ZYM Kit (Biomèrieux). The
hydrolysis of cellulose and chicken hair were determined using the methods of Shirling
and Gottlieb.
The enzymatic activities of 233 strains from feces were determined using the API
ZYM Kit (Figure 4.3). More than 90% of the tested strains showed activity for five enzymes,
namely, alkaline phosphatase, acid phosphatase, leucine arylamidase, naphthol-AS-BI-
phosphohydrolase, and α-glucosidase. A total of 10%–90% of strains showed activity for 12
other enzymes. Less than 10% of the strains produced α-mannosidase and α-fucosidase.
No strains showed β-glucuronidase activity (Figure 4.3). The strains isolated from 13 spe-
cies of animal feces were able to hydrolyze cellulose and chicken hair; however, those
isolated from Panthera tigris altaica, a carnivorous animal, were not able to hydrolyze cel-
lulose. More than 90% of strains from Cervus elaphus, Giraffa camelopardalis, Vicugna pacos,
Elaphurus davidianus, and Oryx leucoryx were able to hydrolyze chicken hair. Approximately
80% of strains from Giraffa camelopardalis, Equus burchelli, and Oryx leucoryx were able to
hydrolyze cellulose. Only a few strains isolated from Panthera tigris altaica and Ailuropoda
melanoleuca showed activity for the hydrolysis of chicken hair, and a few strains from
% of positive strains
120
100
80
60
%
40
20
0
A B C D E F G H I J K L M N O P Q R S
Enzyme
Figure 4.3 Enzyme activities of some actinomycetes isolated from animal feces: A = alkaline
phosphatase, B = esterase (C4), C = esterase lipase (C8), D = lipase (C14), E = leucine arylamidase,
F = valine arylamidase, G = cysteine arylamidase, H = trypsinase, I = α-chymotrypsinase, J = acid
phosphatase, K = naphthol-AS-BI-phosphohydrolase, L = α-galactosidase, M = β-galactosidase,
N = β-glucuronidase, O = α-glucosidase, P = β-glucosidase, Q = N-acetyl-β-glucosaminidase, R =
α-mannosidase, and S = α-fucosidase.
Hypoblasts hoolock, Rhinopithecus bieti, Ailuropoda melanoleuca, and Elephas maximus showed
activity for the hydrolysis of cellulose (Figure 4.4).
The activity of 233 strains for the hydrolysis of cellulose and chicken hair was deter-
mined (Figure 4.4). The results showed that all strains were able to hydrolyze cellulose
and chicken hair, except those isolated from the carnivorous animal Panthera tigris altaica.
More than 90% of strains isolated from Cervus elaphus, Giraffa camelopardalis, Vicugna pacos,
Elaphurus davidianus, and Oryx leucoryx were able to hydrolyze chicken hair. Approximately
80% of strains from Giraffa camelopardalis, Equus burchelli, and Oryx leucoryx were able to
hydrolyze cellulose. The strains isolated from Panthera tigris altaica were unable hydrolyze
cellulose, and 20% of the strains isolated from Panthera tigris altaica and Ailuropoda melano-
leuca were able to hydrolyze chicken hair. Approximately 20% of the strains isolated from
Hypoblasts hoolock, Rhinopithecus bieti, Ailuropoda melanoleuca, and Elephas maximus were
able to hydrolyze cellulose (Figure 4.4). Two strains, YIM 100110 and YIM 100135, were able
to completely hydrolyze chicken hair at 28°C after 1 week (Figure 4.5). YIM 100118 was also
120
% of strain hydrolysing
cellulose
100
% of strain hydrolysing
chicken hair
80
60
%
40
20
0
a b c d e f g h i j k l m
Animal
Figure 4.4 Hydrolytic activity of some actinomycetes for cellulose and chicken hair: a = Hypoblasts
hoolock, b = Rhinopithecus bieti, c = Panthera tigris altaica, d = Ailuropoda melanoleuca, e = Elephas maximus,
f = Cervus Nippon, g = Cervus elaphus, h = Elaphurus davidianus, i = Giraffa camelopardalis, j = Equus
burchelli, k = Vicugna pacos, l = Oryx leucoryx, and m = Tragelaphus buxtoni.
Figure 4.5 Hydrolysis of chicken hair by YIM 100110, YIM 100135, and control (CK).
Figure 4.6 Hydrolysis of cellulose by YIM 100118 and control (CK).
able to completely hydrolyze cellulose under the same conditions (Figure 4.6). Thus, the
enzyme preparation developed from animal fecal actinomycetes was significant.
4.4.3 Antitumor activities

The antitumor activities of 238 fecal actinobacterial strains were determined using HL60,
HepG-2, Skov-3, A431, and K562 cell lines in vitro. The results are shown in Table 4.6.
Approximately 33% and 30% of the tested strains showed K562 and HL60 cell line inhi-
bition activity, respectively, and more than 50% of the strains from Rhinopithecus bieti
Table 4.6 Antitumor activities of some fecal actinomycete strains

K562 Cell line HL60 Cell line
Number of Number of
Number of strains >90% Number of strains >60%
Source of strains test strains inhibition test strains inhibition
Hylobates hoolock 19 12 16 4
Rhinopithecus bieti 24 12 24 13
Panthera tigris altaica 13 6 12 2
Ailuropoda melanoleuca 23 12 18 0
Elephas maximus 37 13 24 11
Cervus Nippon 25 4 42 14
Cervus elaphus 19 0 20 5
Giraffa camelopardalis 0 0 12 0
Elaphurus davidianus 19 5 23 11
Connochaetes taurinus 14 0 5 2
Vicugna pacos 22 7 11 0
Oryx leucoryx 13 5 25 9
Total 238 76 232 69
% 33 30
could inhibit K562 and HL60. The IC50 of the crude extracts from some strains was below
4 μg/mL. Several of the tested strains showing antitumor activities exhibited the distinct
features of actinomycetes from animal feces.
4.4.4 Genes encoding the biosynthetic enzymes of five antibiotics

The genes encoding the biosynthetic enzyme of five antibiotics developed from 201 strains
from 15 species of animal feces were analyzed. The genes encoding the biosynthetic
enzymes of type I and II polyketide synthases (PKSs), nonribosomal peptide synthetase
(NRPS), and polyene cytochrome P450 (CYP) hydroxylase were determined through PCR
using specific primers (Cao et al., 2010). The 3-amino-5-hydroxybenzoic acid (AHBA) gene
was determined using the method of Hui-tu et al. (2009), and the results are shown in
Figure 4.7.
The results from Figure 4.7 shows that 19%, 15%, 34%, and 22% of strains contained
PKS I, PKS II, NRPS, and CYP genes, respectively. Many of the strains isolated from
Rhinopithecus bieti, Panthera tigris altaica, Cavnlvara zlrsidae, Vicugna pacos, Rhinoceros son-
daicus, and Pavo cristatus possess these four genes. None of these genes were detected in
the strains from Cervus nippon and Equus burchelli. The gene encoding the biosynthetic
enzyme AHBA, involved in ansamycin biosynthesis, was determined; however, no posi-
tive strains were detected. These results showed that fecal actinobacteria contained vari-
ous genes encoding biosynthetic enzymes, implicating these species as a new source for
developing novel bioactive metabolites (Figure 4.5). The AHBA gene was detected in only
one strain (YIM 100801) isolated from the feces of Viverra zibetha, suggesting that this gene
is not well expressed in animal feces.
In previous studies of the antimicrobial activities of actinomycetes, we used the
five test strains described earlier. The study of soil actinomycetes from three areas in
Yunnan and Sichuan, southwest China (Cao et al., 2010), showed that 12%–15% strepto-
mycetes and 5%–12% rare actinomycetes presented antimicrobial activities and, in addi-
tion, 4.3%–21% actinomycetes from Baltic Sea presented antimicrobial activities. Fecal
actinomycetes showed a wide spectrum of antimicrobial activities. The antimicrobial
activities of 486 strains isolated from animal feces were detected (data not shown). The
results show that 15%–27% of strains could inhibit Bacillus subtilis, 8%–30% of strains
inhibited Staphylococcus aureus, 2%–9% of strains inhibited Mycobacterium tuberculosis
avium, 11%–20% of strains inhibited Candida albicans, and 2%–18% of strains inhib-
ited Aspergillus niger. Some strains generated large zones of inhibition, up to 60 mm.
% of positive strains
40
35
30
25
20
%
15
10
5
0
PKS I PKS II NRPS CYP AHBA
Figure 4.7 The genes encoding the biosynthetic enzyme of five compounds produced from fecal
actinomycetes.
Fecal actinomycetes also showed a wide spectrum of antitumor abilities. At least 30% of

fecal actinomycete strains could inhibit two types of tumor cell lines, and more than 50%
of the strains isolated from Rhinopithecus bieti could inhibit K562 and HL60. Moreover,
the fermented crude extracts from some of these strains showed high inhibition activi-
ties against tumor cells, with an IC50 below 4 μg/mL. Fecal actinomycetes also contained
various enzymes showing high activity for the degradation of difficult substances, such
as cellulose and chicken hair. These active symbiotic fecal actinomycetes could provide
enormous benefits to hosts, such as to improve food digestion and absorption, maintain
the microbial balance in the intestinal tract, provide resistance against pathogens and
tumors, and improve the overall health of hosts.
4.4.5 Toxins of two streptomycetes to mice

Fifteen mice were fed with two streptomycetes, T005 and T019, containing living cell
10 × 25.28/mL and 10 × 36.68/mL, respectively, for 15 days. All tested mice did not die, and
their body weights were not reduced, suggesting that streptomycetes are nontoxic to mice.
4.5 Discovery of bioactive compounds

More than 80 bioactive secondary metabolites have been isolated and characterized
from some fecal actinomycete strains, including 6-N,N-dimethyladenosine (Li et al.,
2012), abkhazomycin, actinomycin, AI 77B, akashin A, alazopeptin, apigenin, candicidin,
cosmomycin, cyclo(4-Hyp-Phe) (Li et al., 2012), desertomycin, desferrioxamine E, discoder-
molide, emodin, enopetin A/B, erythromycins, favofungin, geldanamycin, isostreptazo-
lin (Zheng et al., 2012), kasugamycin, kidamycins, leucomycin, longestin, panosialin-wA,
puromycin, rutamycin, rhodomycinone, sannaphenol (Zheng et al., 2012), sannastatin
(Yang et al., 2011), tirandamycin, vicenistatin, violapyrones A−G (Zhang et al., 2013), and
polyene macrolides. Some of these compounds are described in Figure 4.8.
These compounds showed complex and different structures and exhibited various
activities. Several novel compounds, such as sannastatin, a novel macrolactam polyketide
glycoside, produced by an unidentified Streptomyces sp. YIM 100282, had been identified
(Yang et al., 2011). The compounds displayed significant cytotoxicity against brine shrimp
nauplii (Artemia salina). A new compound with high activity to some targets of senile
dementia was obtained (unshown), and related study was carried out.
4.6 Effects of microbial manure on notoginseng disease

Notoginseng (Panax pseudoginseng var. Notoginseng) is a local and rare medicinal
herb from Wenshan, Yunnan, a main raw material used in many traditional Chinese
medicines. Annual yield of it in Yunnan is 90% in China. However, the cultivation of
notoginseng is severely affected by soil-borne diseases causing continuous cropping.
Nine streptomyces strains, which showed the strongest inhibitory activity against
many pathogens of notoginseng, were selected from actinomycete strains isolated
from animal feces. The nine strains were fermented, mixed with manure, and used
for the preventative treatment of soil-borne diseases affecting notoginseng fields in
Wenshan, Yunnan for 3 years. The morbidity of the plants at a dosage of 20 kg/Chinese
Mu (666 m 2) of microbial manure was 81% lower than that observed using agricultural
chemicals (Figure 4.9). These results suggest that microbial manure can be widely used
in large tracts of land.
O NH2
OH OH
O
O O
HO O O O OH OH OH O O OH
O OH O
O
O O NH
HO OH O
NH2 NH2 O
1. Candicidin 2. Geldanamycin
N
N N
HO N
O N
OH HO OH
O O O HN NH2
O NH OH HO O
OH HO O O
NH2 HO
O HO
OH OH O
4. Apigenin 5. AI 77B
3. Puromycin
OH OH O OH
O
HO O H
O O O OH O N O N
O H
OH O
HO O
N
6. Cosmomycin 7. Vicenistatin
(a)
Figure 4.8 Bioactive compounds produced from actinomycete strains from animal feces.
(Continued)
O H
N O N
O H H
H2N O N O
OH
OH O
OH
OH
8. New compound 9. Sannastatin (new compound)
O
O
O O
O
O
H3C CH3
HO OH N
H3C
H3C OH CH3
H3C N CH3
C2H5 O O HO O CH3
O
O O
O O
OCH3
CH3 O
CH3 O
O OH
CH3
10. Erythromycins 11. Spinosyns
(b)
HO
O O OH O O
Me O O Me Me
N O
Me OH
OH
HO HO Me
O Me Me O
O HO O Et O
OMe
HO HO O N
Me Et OH
OH Ac CH3 OH
12. Leuconolides 13. Erythronolide 14. Kidamycin
(c)
Figure 4.8 (Continued) Bioactive compounds produced from actinomycete strains from animal
feces.(Continued)
O
O
O N
O
N N
N N
O O
O HN
O HN
N O
NH NH O
O O O
O
N NH2
O O
15. Actinomycins
H3C CH3
NH2 N
HO OH
OH N N
O HO
HO O
N N
OH
O OH
O O
H2N
OH
H O
N OH
H3C
OH OH
16. Desertomycin 17. 6-N, N-dimethyladenosine
(d)
Figure 4.8 (Continued) Bioactive compounds produced from actinomycete strains from animal feces.
(a) (b) (c)
Figure 4.9 Effectiveness of using microbial manure to treat soil-borne notoginseng diseases:

(a) agricultural chemicals control, (b) 15 g/m2 microbial manure, and (c) 30 g/m2 microbial manure.
4.7 Conclusion
The results obtained from the 8-year study of animal fecal actinomycetes should be high-
lighted with several key points:
1. The diversity of animal fecal actinomycetes is rich. Fifty-one genera were identi-
fied from only 48 animal fecal samples. The members of the genera Streptomyces,
Rhodococcus, Microbacterium, and Micrococcineae (including the genus Arthrobacter)
were predominant. However, many of the actinomycetes present in animal feces
remain unknown.
2. The antimicrobial and antitumor activities of actinomycetes were strong and wide-
spread. The activities of many enzymes were also strong. Actinomycetes play impor-
tant roles in health, including improving food digestion and absorption, maintaining
the balance of microbial ecological system in intestinal tract, providing resistance to
various pathogens and tumors, and improving the overall health of the host.
3. Fecal actinomycetes produce multifarious secondary metabolites with bioactivities,
and these metabolites exhibit kaleidoscope structures. We further propose that the
metabolites produced by nonpathogen fecal actinobacteria should be not toxic or less
toxic to the hosts. This is a very important excellence comparing with the metabolites
of other microorganisms from other habitats. Thus, discovering new compounds
now should focus on unknown streptomycetes.
4. There are millions of species of animals on the earth. Similar to the actinomycetes
in other habitats (soil, sea, plant), animal fecal actinomycetes represent a tremendous
resource for the development of drug leads.
5. To discover much more ideal drug leads from animal fecal actinomycetes, first, the iso-
lation methods should be innovated, improved, and updated constantly to make uncul-
tivable into pure cultured actinomycete strains; second, fermentation is an important
prerequisite to produce bioactive substances, and thus studies on the improvement
of fermentation approaches are needed; third, new and unknown isolates should be
screened using new and highly individualized models; and fourth, a rapid, simple,
and accurate remove–repeat system in the early stage should be established.
Acknowledgments
This research was supported by the National Natural Science Foundation of China
(Nos 30900002, 31270001, and 21062028), the National Major Scientific and Technology
Special Projects (2009ZX09302–003), and the National Institutes of Health in the United
States (1P41GM086184–01A1). We thank Professor H. Laatsch and Dr. S. X. Yang (University
of Göttingen, Germany); Professor G. L. Challis and Dr. L. J. Song (University of Warwick,
United Kingdom) for the analysis of bioactive metabolites; Dr. Yanru Cao, Dr. Xiu Chen,
Dr. Dan Zheng, Dr. Jiang Liu, and Ms. Chunhua Yang for taking part in some works; and
Mr. Li Youlong and Ms. Shumei Qiu for collecting the test samples.
References
Abdelmohsen, U. R., Pimentel-Elardo, S. M., Hanora, A., Radwan, M., Abou-El-Ela, S. H., Ahmed, S.,
and Hentschel, U. 2010. Isolation, phylogenetic analysis and anti-infective activity screening of
marine sponge-associated Actinomycetes. Mar. Drugs, 8, 399–412.
Bakalidou, A., Kämpfer, P., Berchtold, M., and Kuhnigk, T. 2002. Cellulosimicrobium variable sp. nov.,
a cellulolytic bacterium from the hindgut of the termite Mastotermes darwiniensis. Int. J. Syst.
Evol. Microbiol., 52, 1185–1192.
Baltz, R. H. 2008. Renaissance in antibacterial discovery from actinomycetes. Curr. Opin. Pharmacol.,
8, 1–7.
Beman, B. L. 1983. Actinomycete pathogen. In: Goodfellow, M., Mordarski, M., and Williams, S. T.
(eds.), The Biology of the Actinomycetes. Academic Press, London, U.K., pp. 457–480.
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R., James, K. D., and
Hopwood, D. A. 2002. Complete genome sequence of the model actinomycetes Streptomyces
coelicolor A3(2). Nature, 417, 141–147.
Bérdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. Tokyo, 58, 1–26.
Bérdy, J. 2012. Thoughts and facts about antibiotics: Where we are now and where we are heading.
J. Antibiot., 65, 385–395.
Blunt, J. W. 2011. Marine natural products. Nat. Prod. Rep., 28, 196–268.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H., and Prinsep, M. R. 2013. Marine natural prod-
ucts. Nat. Prod. Rep., 30, 237–323.
Brown, J. M., Steigerwalt, A. G., Morey, R. E., Daneshvar, M. I., Romero, L. J., and McNeil, M. M.
2006. Characterization of clinical isolates previously identified as Oerskovia turbata: Proposal
of Cellulosimicrobium funkei sp. nov. and emended description of the genus Cellulosimicrobium.
Int. J. Syst. Evol. Microbiol., 56, 801–804.
Bull, A. T. and Stach, J. E. M. 2007. Marine actinobacteria: New opportunities for natural produce
search and discovery. Trends Microbiol., 15, 491–499.
Cao, Y., Jiang, Y., Wang, Q., Zhao, L., Jin, R., and Jiang, C. 2010. Diversity and some bioactivities of cul-
tured actinomycetes in four areas in Sichuan and Yunnan. Acta Microbiol. Sinica, 50(8), 995–1000.
Cao, Y. R., Jiang, Y., Jin, R. X., Han, L., He, W. X., Li, Y. L., and Xue, Q. H. 2012. Enteractinococcus
coprophilus gen. nov., sp. nov., of the family Micrococcaceae isolated from Panthera tigris amoy-
ensis feces, transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as
Enteractinococcus fodinae comb. nov. Int. J. Syst. Evol. Microbiol., 62, 2710–2716.
Cao, Y. R., Jiang, Y., and Xu, L. H. 2009. Actinomycetes composition and bioactivities in Grand
Shangri-La. Acta Microbiol. Sinica, 49, 105–109.
Célia, M. M., Balbina, N., Norbert, W., and Olga, C. N. 2004. Gulosibacter molinativorax gen. nov., sp.
nov., a molinate-degrading bacterium, and classification of ‘Brevibacterium helvolum’ DSM 20419
as Pseudoclavibacter helvolus gen. nov., sp. nov. Int. J. Syst. Evol. Microbiol. 54, 783–789.
Chiao, J. S. 2004. An important mission for microbiologist in the new century—Cultivation of the
un-culturable microorganism. Chin. J. Biotechnol., 20, 641–645.
Cui, X. L., Mao, P. H., Zeng, M., Li, W. J., Zhang, L. P., Xu, L. H., and Jiang, C. L. 2001. Streptomonospora
salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae. Int. J. Syst. Evol.
Microbiol., 51, 357–363.
Curtis, M. M. and Sperandio, V. 2011. A complex relationship: The interaction among symbiotic
microbes, invading pathogens, and their mammalian host. Mucosal Immunol., 4, 133–138.
Daly, K., Stewart, C. S., Flint, H. J., and Shirazi-Beechey, S. P. 2001. Bacterial diversity within the
equine large intestine as revealed by molecular analysis of cloned 16S rRNA genes. FEMS
Microbiol. Ecol., 38, 141–151.
Durso, L. M., Harhay, G. P., Smith, T. P., Bono, J. L., DeSantis, T. Z., Harhay, D. M., and Clawson, M. L.
2010. Animal-to-animal variation in fecal microbial diversity among beef cattle. Appl. Environ.
Microbiol., 76, 4858–4862.
Falagas, M., Betsi, G., and Athanasiou, S. 2006. Probiotics for prevention of recurrent vulvovaginal
candidiasis: A review. J. Antimicrob. Chemother., 58(2), 266–272.
Felsenstein, J. 1981. Evolutionary trees from DNA sequences: A maximum likelihood approach.
J. Mol. Evol., 17, 368–376.
Goodfellow, M. and Fiedler, H. P. 2010. A guide to successful bioprospecting: Informed by actino-
bacterial systematics. Antonie van Leeuwenhoek, 98, 119–142.
Goodfellow, M., Kämpfer, P., Busse, H. J., Trujillo, M. E., Suzuki, K., Ludwig, W., and Whitman, W. B.
2012a. Bergey’s Manual of Systematic Bacteriology, 2nd eds, vol. 5, The Actinobacteria, Part A, B.
Springer, New York.
Goodfellow, M., Stach, J. E., Brown, R., Bonda, A. N. V., Jones, A. L., Mexson, J., and Bull, A. T. 2012b.
Verrucosispora maris sp. nov., a novel deep-sea actinomycete isolated from a marine sediment
which produces abyssomicins. Antonie van Leeuwenhoek, 101(1), 185–193.
Greetham, H. L., Giffard, C., Hutson, R. A., Collins, M. D., and Gibson, G. R. 2002. Bacteriology of the
labrador dog gut: A cultural and genotypic approach. J. Appl. Microbiol., 93, 640–646.
Gundlapally, S. N. R. and Ferran, G. P. 2009. Description of Patulibacter americanus sp. nov., isolated
from biological soil crusts, emended description of the genus Patulibacter Takahashi et al. 2006
and proposal of Solirubrobacterales ord. nov. and Thermoleophilales ord. nov. Int. J. Syst. Evol.
Microbiol., 59, 87–94.
Handelsman, J. 2004. Metagenomics: Application of genomics to uncultured microorganisms.
Microbiol. Molecular Biol., 68, 669–685.
Hayakawa, M. and Nonomura, H. 1987. Humic acid-vitamin agar, a new medium for the selective
isolation of soil actinomycetes. J. Ferment. Technol., 65, 501–509.
Hooper, L. V. and Gorden, J. I. 2001. Commensal host-bacyrtial relationship in the gut. Science, 292,
1115–1118.
Hui-tu, Z., Ai-ming, L., Lin-zhuan, W., Gui-zhi, S., Feng, H., Qun-jie, G., and Yi-guang, W. 2009.
Screening of AHBA synthase gene and discovery of actinomycin D production in Streptomyces
violaceusniger 4353. J. Chin. Antibiotics, 34, 30–33.
Ibrahim, A. S. S., Al-Salamah, A. A., Hatamleh, A. A., El-Shiekh, M. S., and Ibrahim, S. S. S. 2012.
Tapping uncultured microorganisms through metagenomics for drug discovery. Afr. J.
Biotechnol., 11(92), 15823–15834.
Javad, H., Fatemeh, M., and Antonio, V. 2013. Systematic and biotechnological aspects of halophilic
and halotolerant actinomycetes. Extremophiles, 17, 1–13.
Jennifer, B. H., Jessica, J. H., Taylor, H. R., and Brendan, J. M. B. 2001. Counting the uncountable: Statistical
approaches to estimating microbial diversity. Appl. Environ. Microbiol., 67(10), 4399–4406.
Jensen, P. R. 2010. Linking species concepts to natural product discovery in the post-genomic era.
J. Ind. Microbiol. Biotechnol., 37, 219–224.
Jiang, Y. 2011. Diversity of cultured actinomycetes in the Baltic Sea. Acta Microbiol. Sinica, 51(11),
1461–1457.
Jiang, Y., Cao, Y. R., Wiese, J., Lou, K., Zhao, L. X., Imhoff, J. F., and Jiang, C. L. 2009. A new approach of
research and development on pharmaceuticals from actinomycetes. J. Life Science US, 3(7), 52–56.
Jiang, Y., Cao, Y. R., Zhao, L., Tang, S., Wang, Y., Li, W., and Xu, L. 2011. Large numbers of new bacte-
rial taxa found by Yunnan Institute of Microbiology. Chin. Sci. Bull., 56(8), 709–712.
Jiang, Y., Cao, Y. R., Zhao, L., Wang, Q., Jin, R., He, W., and Xue, Q. 2010. Treatment of ultrasonic to soil
sample for increase of the kind of rare actinomycetes. Acta Microbiol. Sinica, 50(8), 1094–1097.
Jiang, Y., Chen, X., Cao, Y. R., and Ren, Z. 2013. Diversity of cultivable actinomycetes in tropical rainy
forest of Xishuangbanna, China. Open J. Soil Sci., 3, 9–14.
Jiang, Y., Li, W. J., Xu, P., Tang, S. K., and Xu, L. H. 2006. Study on actinomycete diversity under salt
and alkaline environments. Acta Microbiol. Sinica, 46, 191–195.
Jiang, Y., Xu, P., Lou, K., Xu, L., and Liu, Z. 2008. Problem and countermeasure on development of
pharmaceuticals from actinomycete resources. Microbiology, 35(2), 272–274.
Joseph, S. J., Hugenholtz, P., Sangwan, P., Osborne, C. A., and Janssen, P. H. 2003. Laboratory cultiva-
tion of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol., 69(12),
7210–7215.
Kaeberlein, T., Lewis, K., and Epstein, S. S. 2002. Isolating “unculturable microorganisms” in pure
culture in a simulated natural environment. Science, 296, 1127–1129.
Kampfer, P., Schafer. J., Lodders, N., and Martin, K. 2011. Jiangella muralis sp. nov., from an indoor
environment. Int. J. Syst. Evol. Microbiol., 61, 128–131.
Lechevalier, M. P. and Lechevalier, H. A. 1970. Chemical composition as a criterion in the classifica-
tion of aerobic Actinomycetes. Int. J. Syst. Bacteriol., 20, 435–443.
Lechevalier, M. P. and Lechevalier, H. A. 1980. The chemotaxonomy of Actinomycetes. In: Dietz, A. and
Thayer, D. W. (eds.), Actinomycete Taxonomy, special publications No. 6. Society for Industrial
Microbiology, Arlington, VA, pp. 227–291.
Lee, S. D. 2008. Jiangella alkaliphila sp. nov., an actinobacterium isolated from a cave. Int. J. Syst. Evol.
Microbiol., 58, 1176–1179.
Li, W. J., Chen, H. H., Xu, P., Zhang, Y. Q., Schumann, P., Tang, S. K., and Jiang, C. L. 2004. Yania halo-
tolerans gen. nov., sp. nov., a novel member of the suborder Micrococcineae isolated from saline
soil in China. Int. J. Syst. Evol. Microbiol., 54, 525–531.
Li, W. J., Zhi, X. Y., and Euzéby, J. P. 2008. Proposal of Yaniellaceae fam. nov., Yaniella gen. nov. and
Sinobaca gen. nov. as replacements for the illegitimate prokaryotic names Yaniaceae Li et al.
2005, Yania Li et al. 2004, emend Li et al. 2005 and Sinococcus Li et al. 2006, respectively. Int. J.
Syst. Evol. Microbiol., 58, 525–527.
Li, X., Han, L., Cao, Y. R., Jiang, Y., Huang, X. S. 2012. Separation and identification of chemical
constituent from a Streptomyces sp. isolated from Sika deer feces. Chemistry & Bioengineering,
39(6), 31–49.
Lior, P. 2007. Interpreting the unculturable majority. Nat. Methods, 4, 479–480.
Martin, K., Schumann, P., Rainey, F. A., and Schuetze, B. 1997. Janibacter limosus gen. nov., sp. nov., a New
Actinomycete with meso-uiaminopimelic acid in the cell wall. Int. J. Syst. Bacteriol., 47, 529–534.
McLeod, M. P., Warren, R. L., Hsiao, W. W., Araki, N., Myhre, M., Fernandes, C., and Eltis, L. D. 2006.
The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.
Proc. Natl Acad. Sci. USA, 103(42), 15582–15587.
Meyer, A., Micksche, M., Herbacek, I., and Elmadfa, I. 2006. Daily intake of probiotic as well as con-
ventional yogurt has a stimulating effect on cellular immunity in young healthy women. Ann.
Nutr. Metab., 50(3), 282–289.
Ōmura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M., and Hattori, M. 2001.
Genome sequence of an industrial microorganism Streptomyces avermitilis: Deducing the abil-
ity of producing secondary metabolites. Proc. Natl Acad. Sci. USA, 98(21), 12215–12220.
Orsini, M. and Romano-Spica, V. 2001. A microwave-based method for nucleic acid isolation from
environmental samples. Lett. Appl. Microbiol., 33, 17–20.
Patrick, D. S. and Handelsman, J. 2005. Metagenomics for studying unculturable microorganisms:
Cutting the Gordian knot. Genome Biol., 2005, 6, 229–302.
Payne, D. J., Gwynn, M. N., Holmes, D. J., and Pompliano, D. L. 2007. Drugs from bad bugs:
Confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov., 6, 29–40.
Petkar, H., Li, A., Bunce, N., Duffy, K., Malnick, H., and Shah, J. J. 2011. Cellulosimicrobium funkei:
First report of infection in a nonimmunocompromised patient and useful phenotypic tests
for differentiation from Cellulosimicrobium cellulans and Cellulosimicrobium terreum. J. Clin.
Microbiol., 49(3), 1175–1178.
Qin, S., Zhao, G. Z., Li, J., Zhu, W. Y., Xu, L. H., and Li, W. J. 2009. Jiangella alba sp. nov., an endophytic
actinomycete isolated from the stem of Maytenus austroyunnanensis. Int. J. Syst. Evol. Microbiol.,
59, 2162–2165.
Ritchie, L. E., Steiner, J. M., and Suchodolski, J. S. 2008. Assessment of microbial diversity along the
feline intestinal tract using 16S rRNA gene analysis. FEMS Microbiol. Ecol., 66, 590–598.
Run-chi, G., Zun-xi, H., Chang-fei, W., Bo, X., and Xiao-yan, W. 2010. Culture-independent analysis
of microflora in gayals (Bos frontalis) feces. Afr. J. Biotechnol., 9, 2774–2788.
Saitou, N. and Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylo-
genetic trees. Mol. Biol. Evol., 4, 406–425.
Savage, D. C. 1977. Microbial ecology of gastrointestinal tract. Annu. Rev. Microbiol., 31, 107–133.
Simpson, J. M., Martineau, B., Jones, W. E., Ballam, J. M., and Mackie, R. I. 2002. Characterization of fecal
bacterial population in canines: Effects of age, breed and dietary fiber. Microb. Ecol., 44, 186–197.
Song, L., Li, W. J., Wang, Q. L., Chen, G. Z., Zhang, Y. S., and Xu, L. H. 2005. Jiangella gansuensis gen.
nov., sp. nov., a novel actinomycete from a desert soil in north-west China. Int. J. Syst. Evol.
Microbiol., 55, 881–884.
Stackebrandt, E., Schumann, P., and Cui, X. L. 2004. Reclassication of Cellulosimicrobium variabile
Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol., 54,
685–688.
Subramani, R. and Aalbersberg, W. 2013. Culturable rare Actinomycetes: Diversity, isolation and
marine natural product discovery. Appl. Microbiol. Biotechnol., 97, 9291–9321.
Suchodolski, J. S., Camacho, J., and Steiner, J. M. 2008. Analysis of bacterial diversity in the canine,
duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis. FEMS
Microbiol. Ecol., 66, 567–578.
Suchodolski, J. S., Ruaux, C. G., Steiner, J. M., Fetz, K., and Williams, D. A. 2004. Application of
molecular fingerprinting for qualitative assessment of small-intestinal bacterial diversity in
dogs. J. Clin. Mictobiol., 42, 4702–4708.
Tang, S. K., Zhi, X. Y., Wang, Y., Shi, R., Lou, K., Xu, L. H., and Li, W. J. 2011. Haloactinopolyspora alba
gen. nov., sp. nov., a halophilic filamentous actinomycete isolated from a salt lake, with pro-
posal of Jiangellaceae fam. nov. and Jiangellineae subord. nov. Int. J. Syst. Evol. Microbiol., 61, 194–200.
Xu, L. H., Li, W. J., Liu, Z. H.,and Jiang, C. L. 2007. Actinomycete Taxonomy. Academic Press, Beijing,
China, pp. 202–208.
Xu, L. H., Zhang, H., Zhang, L. P., Xue, Q. H., Zhang, L. X., and Xiong, Z. 2010. Microbial Resource
Science, 2nd ed. Academic Press, Beijing, China.
Yang, S. X., Gao, J. M., Zhang, A. L., and Laatsch, H. 2011. Sannastatin, a novel toxic macrolactam
polyketide glycoside produced by actinomycete Streptomyces sannanensis. Bioorg. Medical Chem.,
21, 3905–3908.
Zengler, K., Toledo, G., Rappé, M., Elkins, J., Mathur, E. J., Short, J. M., and Keller, M. 2002. Cultivating
the uncultured. Proc. Natl Acad. Sci. USA, 99(24), 15681–15686.
Zhang, H. H. and Chen, L. 2010. Phylogenetic analysis of 16S rRNA gen sequences reveals distal gut
bacterial diversity in wild wolves (Canis lupus). Mol. Biol. Res., 37, 4013–4023.
Zhang, J., Jiang, Y., Cao, Y., Liu, J., Zheng, D., Chen, X., and Huang, X. 2013. Violapyrones A−G,
α-pyrone derivatives from streptomyces violascens isolated from Hylobates hoolock feces.
J. Natural Products., 76(11), 2126–2130.
Zheng, D., Han, L., Li, Y., Li, J., Rong, H., Leng, Q., and Huang, X. 2012. Isostreptazolin and sanna-
phenol, two new metabolites from Streptomyces sannanensis. Molecules, 17, 836–842.
chapter five
Potentially novel Actinobacteria-

derived antibiotics from unique
microenvironments
Contents
5.1 Introduction...........................................................................................................................83
5.2 Hurdles in commercialization of Actinobacteria antibiotics..........................................84
5.2.1 Successful culture methodologies for Actinobacteria.......................................... 85
5.2.2 Drug discovery and development of antibiotics.................................................. 86
5.3 Novel sources of Actinobacteria species and antibiotics................................................. 88
5.3.1 Sandy locations (tropical and temperate).............................................................. 88
5.3.2 Tropical locations with other soil types................................................................. 90
5.3.2.1 Terrestrial and tropical Actinobacteria antibiotics................................ 90
5.3.2.2 Role of animals in Actinobacteria growth and production of
antibiotics.................................................................................................... 91
5.3.2.3 Role of plants in Actinobacteria growth and production of
antibiotics: Endophytic species�� 93
5.4 Conclusions and future directions...................................................................................... 93
Acknowledgments......................................................................................................................... 94
References........................................................................................................................................ 94
5.1 Introduction
Antibiotics are natural or synthetic molecules able to inhibit the growth of bacteria and
display a diversity of targets including the cell wall, the cell membrane, protein synthesis,
and nucleic acid synthesis (Singh et al., 2012). Their production is not constant and is typi-
cally induced by a change in the environment such as lowered nutrient supply or tempera-
ture changes. Currently, some 75% of the world’s antibiotics are derived from terrestrial
Streptomyces bacteria, with the phyla to which they belong, the Actinobacteria, is thought
to produce as many as 12,000 bioactive secondary molecules (Raja and Prabakaran, 2011).
The remaining bacterial sources of antibiotics are from terrestrial species of Bacilli and
Pseudomonas (Singh et al., 2012).
Studies have shown that antibiotics are produced by these species in small amounts
when the growth of the colony begins to slow due to nutrient exhaustion (Challis and
Hopwood, 2003). Although possibly originally produced as signaling molecules able to
induce gene expression (Chater et al., 2010), antibiotics provide a key to bacterial survival
83
and dominance in natural environments since they remove competitors. This is, how-
ever, not without cost to the bacteria producing them since antibiotics often require large
numbers of enzymatic proteins involved in their assembly (Challis and Hopwood, 2003).
A good example is the bacterium Saccharomyces erythrea (formerly Streptomyces erythrea)
in which 60 kb of its DNA is involved in making the complex macrolide erythromycin
(Donadio et al., 1991).
First discovered to produce “chemicals able to kill other organisms” by Gasperini in
1892 (Waksman and Lechevalier, 1962), soil Actinobacteria are dominant species in most
microenvironments where they play a vital role as decomposers of organic substances
including chitin and cellulose (Hogan, 2010). Their ability to thrive in diverse habitats
is a function of several attributes. First, Actinobacteria produce resistant spores, which
allow them to survive changes in nutrient and water levels in many diverse habitats
(Schaechter, 2010). Second, researchers have shown that although Actinobacteria prefer
a neutral to acid pH, they are resistant to desiccation, allowing their colonization of
desert and sandy soils (Schaechter, 2010). They are especially important to the continued
fertility of soils, being the second group to appear after pigmented bacteria and are the
commonest bacteria in desert crusts (Kieft, 1991). Third, Actinobacteria have been shown
to exist in an extreme diversity of climates and are tolerant to heat (Kurapova et al.,
2012), cold (Augustine et al., 2012), as well as alkaline and saline soils (Zvyagintsev and
Zenova, 2007).
Actinobacteria species already produce an impressive group of bioactive molecules,
but it is apparent that they probably represent the mere tip of the biological iceberg.
First, studies have shown that between 20% and 50% of all terrestrial and aquatic spe-
cies, Actinobacteria are able to generate antibacterial and antifungal factors. Second, it is
probable that only around 10% of the Actinobacteria species in existence on our planet
have actually been isolated, despite intensive screening of soils by the pharmaceutical
industry for over 50 years (Enright, 2003). Given these parameters, Streptomyces species
that come from unique, biologically challenging locations such as tropical soils and arid
desert environments are also likely to be a potential source for novel antimicrobials. These
locations may also be seeded with the so-called “rare Actinomycetes,” most of which are
still uncharacterized as to their antimicrobial activity (Lazzarini et al., 2001). One group of
interest in this category is the genus Micromonospora, which is already a source of a variety
of macrolides (Lancini and Lorenzetti, 1993).
Given the current and also putative future capabilities of the Actinobacteria phylum,
the remaining goal of this chapter is twofold. First is to describe the hurdles that need to
be overcome in order to successfully identify and launch new Actinobacteria antibiotics.
Second is to survey the scientific literature as to the potential of antibiotics isolated from
two novel environments namely sandy (including temperate and tropical) and tropical
locales with other soil types, particularly the unique environment of Jamaica.
5.2 Hurdles in commercialization of Actinobacteria antibiotics

Although Actinobacteria have been studied since the 1940s, their methods for producing
antibiotics are still often poorly understood. The path from discovery to commercializa-
tion is also a difficult one, with several major barriers along the way. First is the ability to
both successfully culture and stabilize a novel antibiotic; second is the testing procedure,
which is often lengthy and may prove unsuccessful in terms of the overall, that is, nonbac-
terial, toxicity of the novel therapeutic; and third is the clinical testing (Silver, 2011). The
next sections explore each of these issues with a view to how each is being addressed.
Chapter five: Potentially novel Actinobacteria-derived antibiotics 85
5.2.1 Successful culture methodologies for Actinobacteria

The issue with recovery of antibiotics in culture is twofold. First, current culture methods
tend to be primarily a “one size fits all” model with the bacterium being separated out
of its natural environment. For over 40 years, since the first observations of Waksman in
1952, primary methods of Actinobacteria isolation have involved separating the individual
viable colonies from their habitat by plating diluted soil samples onto an albumen agar
medium and incubating at 30°C for 1 week (Boisvert-Bertrand et al., 2004). Although many
viable producers can be recovered this way, it tends to select for the more rapidly growing
colonies and, interestingly, researchers have developed new methods for recovery of new
types of antibiotics (Li, 1989). For example, air drying and heating soil, followed by culture
on streptomycin- and rifamycin-supplemented isolation media allowed slower growing
strains of Actinomadura and Microtetraspora Actinobacteria to grow and produce antibiotics
(Li, 1989). This has since paved the way for the discovery of over 200 antibiotics from these
genera (Lazzarini et al., 2001).
Another issue in generating antibiotics from Actinobacteria is that some produce anti-
biotics only in response to soil competition and not when grown in isolation. A notable
example of this was Streptomyces coelicor, which was originally believed not to be capable
of producing significant antibiotic activity (Marinelli, 2009). Genomic sequencing of the
bacterium uncovered 23 gene clusters capable of producing secondary metabolites with
only six ever having been recovered from growth media of the microbe (Marinelli, 2009).
With the recognition that the majority of Streptomyces species produce polyketide or poly-
peptide antibiotics, genes responsible for their production can be screened for, thus also
facilitating the identification of novel antibiotic-producing microbes (Busti et al., 2006).
More recently, it has also been recognized that horizontal gene transfer may also occur
when antibiotic-producing and non-antibiotic-producing Actinobacteria species are cocul-
tured (Kurosawa et al., 2008). Research on the bacterium Rhodococcus fascians demonstrated
that it possessed many genes involved in antibiotic synthesis, although the bacterium had
never been shown to release any under normal growth conditions (Kurosawa et al., 2008).
Only when cocultured with a second Actinobacteria species, Streptomyces padanus, did
R. fascians generate antibiotics (Kurosawa et al., 2008). Furthermore, the rhodostreptomy-
cins produced by R. fascians were found to belong to a novel class of aminoglycosides that
were distinct from the actinomycin that S. padanus generated (Kurosawa et al., 2008).
A second hurdle is that many of the Actinobacteria recovered grow only slowly in
culture and produce small amounts of viable product. This is particularly the case for
Actinobacteria recovered on humic acid agar, which may take as much as a month to pro-
duce viable isolates (Hayakawa, 2008). Researchers identifying glycopeptide antibiotics
also need to wrestle with the problems of low yield, mixtures of molecules at varying
stages of glycosylation, and lack of extrusion of the product from the mycelium (Nicolaou
et al., 1999). Being aware of the bacteria’s culture needs becomes even more important when
trying to isolate and identify antibiotic-producing Actinobacteria from unusual environ-
ments. Our own studies of Presque Isle’s temperate, sandy loam (Boisvert-Bertrand et al.,
2004) and Jamaica’s tropical and varied soils (Piechoski et al., 2012) were successful in iden-
tifying some effective colonies but may well have missed many other organisms requiring
nonclassical isolation strategies.
Others have also struggled in culturing Actinobacteria from unique environments.
Indeed, a recent study by Hozzein and colleagues (2008) demonstrated that desert
Actinobacteria are quite fastidious and instead of a rich medium require a special mini-
mal medium that includes glucose, yeast extract, and mineral salts. By using this medium,
Hozzein and his team (2008) were able to increase the recovery of Actinobacteria from the
Western and Eastern Egyptian deserts by as much as 10-fold and also expanded the diver-
sity of the population recovered. This study alone underlines the fact that many species
may be discovered from these locations given optimal isolation methodologies.
5.2.2 Drug discovery and development of antibiotics

Early drug discovery of natural products (1940s–1960s) typically involved empirical
screening of bacterial cultures for antibiotic activity and secondary testing for cytotoxicity
(Silver, 2011). Classic, standard qualitative procedures involved overlaying the colonies on
agar embedded with a bacterial pathogen and then defining antibiotic activity as a zone of
“no growth” of >3 mm (Atlas et al., 1995). Quantitative analysis of actual antibiotic activity
typically then involved a microassay in which extracts of growth inhibitory activity from
growth media were compared with the minimum inhibitory concentration of antibiotics
as to their ability to kill a panel of standard, clinical strains of bacteria (Zgoda and Porter,
2001). To ensure consistency, bacterial strains to be tested were usually obtained from ref-
erence libraries such as the American Type Culture Collection (ATCC) and then cultured
in rich media with continuous agitation to allow consistent oxygenation and maximum
growth. Secondary cytotoxicity testing then employed exposing brine shrimp to extracts
of the culture medium and estimating the LD50 of the bioactive moiety (Solis et al., 1993).
Unfortunately, the initial empirical-based screening process of identification of anti-
biotic activity did not distinguish between already-described antibiotic activities, and to
avoid replication, additional screening methods were next developed by the pharmaceu-
tical industry to identify the cellular target for the putative novel therapeutic (Hancock,
2007; Silver, 2011). The first screens addressed inhibitors of peptidoglycan (cell wall synthe-
sis) as this is an effective strategy to limit bacterial growth, and utilization of this proce-
dure identified several classes of antibiotics including the monobactams and carbapenems
(Gadebusch et al., 1992). In addition, it was also apparent that most successful individually
used antibiotics targeted multiple components within a bacterial cell, for example, mac-
rolides target bacteria with multiple copies of rRNA genes and so much focus was made
in their discovery (Silver, 2011). Finally, the arrival of genetic cloning and overexpression
technologies, as well as the use of microarray gene signatures, allowed researchers to iden-
tify genetic signatures and, in turn, to also understand more fully the mechanisms by
which antibiotics are effective (Hancock, 2007). Genetic analyses now estimate that there
are around 160 enzyme targets that are useful to bacteria and absent from human beings
(Payne et al., 2007), and Lange et al. (2007) list 16 enzyme classes that are already targets for
commercialized antibiotics. Such screenings, although increasing understanding of how
antibiotics work have, however, been notable in not yielding significant numbers of new
antibiotics other than a few like platensin and the fluoroquinolones (Silver, 2011).
Interestingly, despite the advances in genomic research and high-throughput screen-
ing of potential antibiotics, many companies lost faith in these rather generic methods and
current screening technologies typically involve either searching for novel natural prod-
ucts or reevaluating underexploited natural products (Brötz-Oesterhelt and Sass, 2010).
It seems that in the search for new marketable pharmaceuticals, there is a definite split
with at least five major companies (Wyeth, Sanofi-Aventis, Hoechst, Cubist, and Merck)
electing to choose this route (Brötz-Oesterhelt and Sass, 2010). Both strategies have proven
successful with the discovery of the natural products mannopeptimycins, friulimicin, and
plectasin and the reevaluation of usage for daptomycin and ramoplanin (Brötz-Oesterhelt
and Sass, 2010). This latter strategy was very successful for Bayer who used functional
genomics in combination with mode of action studies to identify two novel classes of bac-
terial translation inhibitors, dihydropyrimidinones and biphenomycins (Bandow et al.,
2003; von Nussbaum et al., 2006), and a new target in fatty acid biosynthesis, the pseudo-
peptide pyrrolidine diones (Freiberg et al., 2004). Dihydropyrimidinones are derived from
a natural product, Tan-1057, and bind to the large ribosomal subunit, thus inhibiting the
peptidyl transferase reaction (Bandow et al., 2003), while biphenomycins affect bacterial
protein synthesis (von Nussbaum et al., 2006). New derivatives of both these compounds
were able to increase the selectivity for gram-positive bacteria and remain well in the
range of efficacy reported for existing antibiotics (von Nussbaum et al., 2006).
Another technique used by pharmaceutical companies, anxious to optimize antibiotic
efficacy by decreasing toxicity, circumventing existing resistance, and increasing bioavail-
ability (as well as minimize costs), has turned to the modification of existing antibiotics
rather than the search for novel ones. Older Actinobacteria antibiotics, such as the ami-
noglycoside kanamycin, now have semisynthetic forms (amikacin and arbekacin) whose
altered chemistry has decreased the toxicity of these agents while maintaining their effi-
cacy against multidrug-resistant bacteria including methicillin-resistant Staphylococcus
aureus (MRSA), Pseudomonas aeruginosa, and enteric bacteria (Cordeiro et al., 2001; Falagas
et al., 2008).
For some Actinobacteria antibiotics, the generation of semisynthetic variants did not just
reduce toxicity, but it allowed their usage. This was the case for oxytetracycline (Terramycin)
initially discovered by Dr. Robert Woodward in 1950 and deemed highly since it prevented
protein synthesis by blocking aminoacyl tRNA entry (Nelson and Levy, 2011). Although
Terramycin was short-lived due to its many side effects, the discovery was to be important
because structural modifications to their polyketide structure resulted in the semisynthetic
antibiotics doxycycline and minocycline (Fuoco, 2012). Both doxycycline and minocycline
have been found to be effective against most gram-positive and gram-negative bacteria,
intracellular bacteria, mycoplasma, and spirochetes (Fuoco, 2012). Their use is widespread,
and revenues from their prescription as doxycycline hyclate netted $264 million in 2011
(Mylan Receives Final FDA Approval for First Generic Version of Doryx® Tablets, 150 mg).
Having obtained a potentially useful antibiotic, in terms of in vitro performance, it
must survive several phases of clinical trials as well as receive FDA approval. For one suc-
cessful drug to reach marketability, it is estimated that 10,000 compounds must be discov-
ered (Raja and Prabakarana, 2011). Indeed, current studies suggest that for around 60% of
the therapeutics that received FDA approval, their journey will end at or before phase III
trials when they are found to have toxic side effects in human subjects or in animal models
(Raja and Prabakarana, 2011). Many also cannot be effectively stabilized and will need to
be administered intravenously, which will limit their usage. At this point, pharmaceutical
companies have invested large sums of money, and although each new antibiotic can yield
annual profits, which may be in the several-hundred-million-dollar range, each may cost
as much as a billion dollars to produce (Raja and Prabakarana, 2011).
The costs are, however, worth the benefits. According to the most recent report by
the IMS Institute for Healthcare Informatics in 2011, azithromycin was ranked 8 of the
10 most popular drugs prescribed in the United States accounting for 56 million written
prescriptions (IMS, 2011). This represents an increase of nearly three million prescriptions
from 2010, with consumers enjoying the ease of the Z-Pak form of the antibiotic in which
the 5-day course is numbered for ease of memory. Savvy marketing has helped make this
antibiotic a household name, and a study in the Journal of Family Practice suggests that as
many as four out of five patients with upper respiratory infections will ask for the drug
directly by name (Hickner, 2007).
Despite the promise of an excellent market return on a successful product, many bio-
tech and pharmaceutical companies left the market to focus instead on the more lucrative
chronic diseases (Brötz-Oesterhelt and Sass, 2010). Consequently, the number of patent
applications has declined (Katz et al., 2006) along with the number of approved antibiot-
ics (Powers, 2004). This has led some scientists to believe that current trends will result
in there being no effective antibiotics for some microbes in the next 10 years (de Lima
Procópio et al., 2012).
The good news is that those biotechnology companies that remain investigated in
antibiotic drug discovery are demonstrating recent, renewed vigor in research (Brötz-
Oesterhelt and Sass, 2010). Armed with well-established protocols for identification of
antibiotic activity, as well as modeling the metabolism and pharmacokinetics of the active
moieties in highly predictive animal models, it can be anticipated that more drugs will
be developed (Brötz-Oesterhelt and Sass, 2010). Indeed, success rates from Investigational
New Drug applications (those associated with human trials) are now 30% for anti-infective
therapeutics (Powers, 2004). The majority (70%) of these novel antibiotics appear to be com-
ing from natural products, as suggested in a review of the literature by Brötz-Oesterhelt
and Sass (2010).
Natural products typically strongly surpass their synthetic counterparts in activ-
ity and structural diversity and in addition usually have more complex modes of action
(von Nussbaum et al., 2006). This is not surprising since antibiotic-producing strains have
coevolved to occupy and compete for an environmental niche, thus honing their products
accordingly. In addition to broad-spectrum antibiotics, many companies have also begun
searching for novel narrow-spectrum antibiotics, with concomitantly fewer side effects on
normal human flora (Brötz-Oesterhelt and Sass, 2010; de Lima Procópio et al., 2012). For
many researchers, these new narrow- and broad-spectrum antibiotics are to be sought
from the Actinobacteria indigenous to unique, diverse environments (Baltz, 2008).
5.3 Novel sources of Actinobacteria species and antibiotics

Although pharmaceutical companies have been harvesting natural Actinobacteria for over
50 years, it is likely that only a fraction of the terrestrial species have been identified, with
still fewer from nutrient-poor environments, such as sandy locations or very diverse tropi-
cal environments. Tropical environments are also home to many Actinobacteria, which
have only recently begun to be even described (Araragi, 1979; Janso and Carter, 2010;
Kumar et al., 2012a; Piechoski et al., 2012; Ambrose et al., 2013). Thus, in terms of trying to
identify novel, natural products, terrestrial locations with unique flora and soil structures
clearly represent the next leap forward in the natural product world. In the next two sec-
tions, two such biomes will be discussed: first, sandy locations where nutrients and water
are limited and second, tropical soils with soil types that are rich in humus and possess a
diverse microflora.
5.3.1 Sandy locations (tropical and temperate)

Sandy soils and deserts provide an extreme terrestrial environment where few micro-
bial species can survive, but they have been shown to be habitats for bacteria able to
form desiccation-resistant structures such as spores (Kieft, 1991). Actinomycetes in these
locations often use the cyanobacterium Microcoleus as a food source and are also typi-
cally associated with any plant life especially plant roots (Skujins, 1984). These species
tend to migrate further down into the subsoils in harsher climates and, therefore, be
more frequently isolated 2–15 cm down rather than at the surface (Cameron et al., 1970).
Although most Actinobacteria are primarily mesophilic species needing an adequate sup-
ply of water, thermophilic and thermotolerant Streptomyces, Micromonospora, Actinomadura,
and Streptosporangium (Kurapova et al., 2012) as well as alkaliphiles (Ali et al., 2009; Osman
et al., 2011) have also been described from desert environments.
Several reports exist of antibiotic-secreting Actinobacteria in tropical and subtropical
desert locations (Table 5.1). Though some are known Streptomyces, at least one new spe-
cies, S. atacamicus, has emerged and is capable of secreting a novel class of antibiotics, the
chaxamycins (Chaxamycins, 2011). Chaxamycins act on bacteria, particularly MRSA, by
attaching to the ATP-binding domain (Chaxamycins, 2011).
Three alkaline desert locations, two in Egypt and one in Bangladesh, have also yielded
successful novel antibiotics (Table 5.1). Alkaline deserts typically consist of dry, compacted
clay soils, which, unlike moist clay that is typically nutrient rich, are typically nutrient
poor (Chhabra, 1996). Life in these environments is, therefore, very extreme and competi-
tive, and many microbes may theoretically only exist in dormant forms (Skujins, 1984).
In the case of the Egyptian deserts, the first, Wadi El Natrun, is a saline, alkaline desert,
which yielded meroparamycin in 2006 (El-Naggar et al., 2006). This novel therapeutic comes
from a previously unidentified Streptomyces species and is highly effective against several
gram-positive and gram-negative bacterial species (El-Naggar et al., 2006). The second,
Table 5.1 Antibiotic-producing Actinobacteria from desert sand or sandy/loam areas

Location/soil type Species Antibiotic Antibacterial efficacy
Atacama, Chile1 Streptomyces sp. Chaxamycins MRSA, E. coli
(hyperarid)
Cairo, Egypt2 (sand) S. bikiniensis Unknown Broad-spectrum
aminoglycoside antibacterial
Falmouth, MA3 (sandy, Nocardia Neocitreamicin MRSA and VRE
temperate)
Iran (Eastern desert)4 Strong similarities to Unknown Broad spectrum
several Streptomyces
strains
Karnataka, India5 (black Actinomycetes Unknown Broad-spectrum
alkaline) (unknown) antibacterial
Keffi Metropolis, Nigeria6 Various Streptomyces Various, unknown Broad spectrum
(sandy, loam) and Actinomycetes
species
Presque Isle, PA (sandy, Actinomycetes Unknown Narrow spectrum;
loam, temperate)7 (unknown) S. aureus, P. aeruginosa,
and B. megaterium
Thar, Rajasthan8 S. hygroscopicus Unknown Vancomycin-resistant
(subtropical desert) subsp. ossamyceticus S. aureus and Klebsiella
Wadi El Natrun, Egypt9 S. coelicolor, S. flavius, Unknown Broad-spectrum
(saline, alkaline desert) S. griseoruber, and antibacterial and
S. plicatus antifungal
Wadi Araba, Egypt10 Nocardiopsis WA52 Macrolide Broad-spectrum
(alkaline desert) dansonvilli WA52 antibacterial
1 Chaxamycins (2011), 2 El-Khagawa and Megahed (2012), 3 Peoples et al. (2008), 4 Tabrizi et al. (2013), 5 Basavaraj
et al. (2010), 6 Makut and Abdalazeez (2012), 7 Boisvert-Bertrand et al. (2004), 8 Selvameena et al. (2009), 9 Osman
et al. (2011), and 10 Ali et al. (2009).
Wadi Araba, which is also an alkaline desert, yielded a macrolide, WA52, from a novel spe-
cies, Nocardiopsis dansonvilli (Osman et al., 2011). The Thar desert contained a black, alka-
line soil and yielded an S. hygroscopicus species able to secrete a broad-spectrum antibiotic
activity (Selvameena et al., 2009). The isolation of classes of antibiotics from previously
unidentified species from the Bangladesh and Egyptian desert alkaline Streptomyces may
therefore herald a whole series of new microbes and novel antibiotics.
Temperate locations have also proven relatively fruitful for the isolation of novel
Actinobacteria (Table 5.1). Neocitreamicins were isolated from Streptomyces species grow-
ing in sandy soil from Falmouth, Massachusetts (Peoples et al., 2008). They are chemically
closely related to citreamicins, which are polycyclic xanthones first isolated in the 1980s
and possess antibiotic activity against MRSA and vancomycin-resistant Enterococci (VRE)
(Carter et al., 1990). Although their exact antibacterial mode of action remains unclear,
they are known to be toxic for tumor cells by inhibiting cell division at G1 phase (Liu et al.,
2013). Our team was also able to identify narrow-spectrum Actinomycetes from sandy,
loam soil in Presque Isle, Pennsylvania, which were able to selectively inhibit Pseudomonas
aeruginosa, Bacillus species, and S. aureus (Boisvert-Bertrand et al., 2004). In this study, the
number of Actinobacteria species closely mirrored those of fungal isolates and S. aureus in
the soils, and these species thus have these microbes as their competitors for the microen-
vironment (Boisvert-Bertrand et al., 2004). Temperate sandy soils with their narrow com-
plement of microflora may therefore well provide suitable locations from which to obtain
novel antistaphylococcal and antifungal agents.
5.3.2 Tropical locations with other soil types

Tropical terrestrial environments are known to comprise many novel species (Janso and
Carter, 2010), and these organically nutrient-rich locations are home for thousands of bac-
terial species, each of which must compete for territory (Hawksworth and Colwell, 1992).
Studies by Araragi (1979) on fertile tropical Thai soil being used as farmland have sug-
gested that although many of these species are Streptomyces and Nocardia species, there is
also a differential distribution based on soil type. For example, soils with high water reten-
tion ability (grumusols) tend to have more Monospora species, whereas humus, bedrock
(rendzina), and iron-rich, forest soils (grey podzolic) tend to have more Nocardia (Araragi,
1979). More recent studies performed on the granite, loam, acidic soils in the tropical forest
in Western Ghats, India, have similarly suggested that Actinomycete species are typically
Streptomyces, Nocardia, and Micromonospora (Panaiyadiyan and Chellaia, 2011). In addition,
as is the case for all soil ecosystems, the species and dynamics of Actinobacteria growing
in these locations may be influenced by many biotic factors, including the plant species
growing on the soils (Ambrose et al., 2013) and the animal species (ants, earthworms, etc.)
living within it (Kumar et al., 2012b). The next section will deal with antibiotics that have
been identified in each of these three very separate tropical environments.
5.3.2.1 Terrestrial and tropical Actinobacteria antibiotics

Many reports of novel terrestrial tropical Actinobacteria exist including those harvested
from rich soils in Bangladesh (Al-Bari et al., 2005) and Jamaica (Piechoski et al., 2012).
Some of the diverse locations are shown in Table 5.2. Jamaica is a particularly diverse
location from which to sample in this respect since it possesses a tropical, maritime cli-
mate conducive to bacterial proliferation and diversity (Borneman and Triplett, 1997).
The primary bedrock soil is limestone, but the soil diversity ranges from erosion-resis-
tant upland plateaus to lowland sand and gravel. This variety in tropical soil construct
Table 5.2 Antibiotic-producing Actinobacteria from tropical, rich soils

Location/soil type Species Antibiotic Antibacterial efficacy
Jordanian red soils (clay) 1 Unknown Unknown Micrococcus luteus and
S. aureus
Karnataka, India2 Streptomyces sp. Unknown MRSA
(rhizosphere, college
campus, rich soil)
Kutumsar Cave, India3 S. prasinosporus, Unknown Escherichia coli,
(Limestone) S. roseus, S. aurantiacus, Staphylococcus aureus,
and S. longisporoflavus and Pseudomonas
aeruginosa
Natore, Bangladesh4 S. bangladeshensis bis-2-Ethylhexyl Broad spectrum
phthalate
Port Antonio, Jamaica5 Unknown Unknown Mycobacterium
(clay soil) Actinobacteria tuberculosis
Tamil Nadu, India6 Streptomyces sp. Unknown Narrow-spectrum
(Manakudy mangrove PVRK-1 MRSA
swamp)
Shola Soils, Kerala, India7 Streptomyces sp. Unknown Broad spectrum
(Tropical Forest)
Samed Island, Indonesia8 S. sporoclivatus Geldanamycin MRSA
(forest)
1 Falkingham et al. (2009), 2 Prashith Kekuda et al. (2010), 3 Rajput et al. (2012), 4 Al-Bari et al. (2005), 5 Piechoski
et al. (2012), 6 Kannan et al. (2011), 7 Varghese et al. (2012), and 8 Anansiriwattana et al. (2006).
has been previously shown to be previously associated with extreme microbial diversity
(Juo and Franzluebbers, 2003). In addition, the northwestern area of the island, around
the St. Ann’s district, is associated with the rare red soils (terra rossa) that are currently
being mined for copper, iron, silver, and gold ores (Wall Street Journal Market Watch,
2013). Red soils may be further sites for novel Actinobacteria, as evidenced by the studies
of Falkingham and colleagues (2009). Falgingham et al. (2009) sampled the Jordanian red
soils and demonstrated that they contained Actinobacteria with potent antibacterial activ-
ities. These soils have a long history of being used as topical treatments for diaper rash and
wounds, which led weight to their therapeutic content (Falkinham et al., 2009). Finally, the
coast, especially to the east, is surrounded by coral reefs, which are also known sources
of novel Actinobacteria. Our own studies showed the presence of Actinobacteria produc-
ing narrow-spectrum antibiotic activity against tuberculosis bacteria in soils from three
locations: Sunset Cove, where limestone is the predominant rock and bats are common;
Bonnie view, a former cholera burial site where clay soil predominates; and the sands of
Folly Ruin, under a piece of coral (Piechoski et al., 2012). In this particular case, soil abiotic
components and animal activity in the Jamaican soil isolates have not been delineated, but
this is not the case for all tropical, terrestrial environments. The next sections will there-
fore discuss the currently available information for the roles played by animals and plants
in Actinobacteria growth and production of antibiotics.
5.3.2.2 Role of animals in Actinobacteria growth and production of antibiotics

Environmentally, in both terrestrial and aquatic locations, the majority of the Actinobacteria
behave as strict saprophytes involved in maintaining the carbon cycle, but some may be par-
asitic or even form symbiotic associations with animals (Barke et al., 2010). An interesting
symbiosis of this nature was recently demonstrated in the fungus farming ant Acromyrmex
octospinosus in which the mutualist Actinobacteria species Streptomyces and Pseudonocardia
were able to generate antibiotic and antifungal agents, presumably to protect the colony
against microbial infection (Barke et al., 2010). Ants are common inhabitants of tropical
soils where they may encourage or even “farm” bacteria and fungi able to release antibiotic
substances (Barke et al., 2010; Mendes et al., 2013). Actinobacteria have been successfully
isolated from attine ants and include the species Pseudonocardia and Streptomyces (vander
Meer, 2012). Both have been shown to produce the antibiotic 6-deoxy-8-O-methylrabelo-
mycin, active against B. subtilis as well as anti-Candida urauchimicins from Streptomyces
species (Mendes et al., 2013). Our studies of Jamaican tropical soil also isolated Streptomyces
able to secrete effective anti-Candida activity from a location with heavy ant contamina-
tion (Folly View; Piechoski et al., 2012).
Studies by Kumar and colleagues (2012b) have also demonstrated the presence of vari-
ous antibiotic-secreting Actinobacteria in earthworm castings of Doon Valley, India, soils.
Earthworms have long been studied by scientists as they are known to be important vec-
tors for the transport of beneficial bacteria in soils (Doube et al., 1994), and these species
have now been found to include a variety of Actinobacteria species of Streptomyces were
found to be the dominant organisms and Actinobacteria were found to be more frequent
in worm casts originating in forest soils as compared to agricultural land (Kumar et al.,
2012b). Antibiotic production was seen in many Streptomyces species with S. rochei generat-
ing excellent antibacterial and antifungal activities (Kumar et al., 2012b).
Another tropical location, which has yielded novel and unique microbes, is limestone
caves including Kotumsar and Magura (Rajput et al., 2012; Tomova et al., 2013). Caves
typically possess higher humidity and more stable temperatures than outside locations,
and their floors are covered with bacteria derived from bat guano (E. coli, S. aureus, and
Pseudomonas) as well as Actinobacteria able to generate antibiotic activities to inhibit these
microbes (Ikner et al., 2007). As with other terrestrial locations, Streptomyces often pre-
dominate (Niyomvong et al., 2012), but for the Magura cave, they contain novel Arthrobacter
and Myroides species (Yucel and Yamac, 2010; Tomova et al., 2013). Interestingly, one of the
Jamaican locations that proved fruitful in our own studies was associated with bat guano
(Sunset Cove) and was the site of Streptomyces isolates with activity against both tubercu-
losis and Candida albicans (Piechoski et al., 2012).
Finally, animal associations with Actinobacteria are not limited to terrestrial loca-
tions. Two major studies have demonstrated the association of novel antibiotic-secreting
Actinobacteria associated with soft (Fu et al., 2013) and hard (scleractinian; Nithyand et al.,
2010) corals. Strepchloritides A and B, with activity against MRSA, were identified from
South China Sea soft corals (Fu et al., 2013) and S. akiyoshinensis associated with Gulf of
Mannar, Tamil Nadu scleractinian corals inhibited S. pyogenes biofilm formation associ-
ated with atopic dermatitis and impetigo (Nithyanand et al., 2010). Jamaica harbors more
than 60 species of coral including elk horn and stag horn species, and the reefs fringe up
to a mile out in the northern coasts, where they are commonest and more sporadically in
the south (Idjadi et al., 2006). Like many of the coral reef environments around the world,
Jamaican coral is endangered (Broad, 1994). Studies demonstrated that amounts decreased
from 1970s from 52% to 3% due to a combination of intense fishing for parrotfish, which
are the major grazers on algae that smothers coral, and hurricane damage. Some attempts
to reseed the coral seem successful as shown in Dairy Bull east of Discovery Bay, which
is again dominated by scleractinian corals (Idjadi et al., 2006). Given that these corals
may also have potential for novel, narrow-spectrum antibiotics, the preservation of these
unique locations is clearly of paramount importance on several levels.
5.3.2.3 Role of plants in Actinobacteria growth

and production of antibiotics: Endophytic species
Researchers have also recently identified several tropical endophyte Actinobacteria able
to secrete novel antibiotics (Ambrose et al., 2013). Endophyte bacteria are unusual in that
they spend their lives associated with plant species in a beneficially mutualistic relation-
ship that may also now extend to secreting antibiotics to protect them from infection
(Ambrose et al., 2013). At least three important classes of antibiotics have been described
from endophyte Actinobacteria: the munumbicins, the kakadumycins, and the xiamycins
(Ambrose et al., 2013).
Munumbicins are secreted by Streptomyces growing on snakevine plants in the tropi-
cal, Northern Territory of Australia and have their primary activity against gram-positive
bacteria such as Bacillus cereus, MRSA, VRE, and Mycobacterium tuberculosis (Castillo et al.,
2002). In this latter respect, munumbicin B antibiotic is particularly interesting since it
has a ctivity against multidrug resistant (MDR) forms of tuberculosis (Ambrose et al.,
2013) and may therefore represent a new therapeutic for the treatment of this bacterium.
Kakadumycins have been identified in cultures of endophytic Streptomyces (NRRL30566)
bacteria isolated from the grevillea tree (Grevillea pteridifolia, synonym: Grevillea
chrysodendron R.Br.) also native to the Northern Territory of Australia (Castillo et al., 2003).
Two kakadumycins have been identified: kakadumycin A is similar to echinomycin, a qui-
noxaline antibiotic, and kakadumycin B, which is similar in spectrum to the munumbicins
(Castillo et al., 2003). Xiamycins represent a third class of novel antibiotics and are novel
pentacyclicindolosesquiterpenes, obtained from Streptomyces sp. strain GT2002/1503, an
endophyte from the mangrove plant Bruguiera gymnorrhiza (Ding et al., 2010). In addition
to potent activity against MRSA and VRE, similar to that demonstrated by the munumbi-
cins, xiamycins also demonstrate selective effectiveness against the HIV (Ding et al., 2010).
These compounds, therefore, have promise in several treatment areas.
5.4 Conclusions and future directions

Even though drug discovery and development aim to maintain effective therapy by
replacing antibiotics to which bacteria have become resistant with new ones, few new
natural drugs of significance have made their appearance in the past 20 years (Raja and
Prabakarana, 2011). There are two major reasons for this shortfall: failure to match growth
conditions with antibiotic product and cost of therapeutic development. Since it is likely
that many of the more dominant Actinobacteria have already been isolated and identified
as antibiotic producers, many researchers are now beginning to scour the globe for more
remote, as yet untapped environments where novel species might yield novel drugs. Given
that many terrestrial Actinobacteria produce their antibiotics in response to external pres-
sures including both biotic (competitors, food) and abiotic (changes in temperature and
pH) factors, it would be anticipated that both hostile and nutrient-rich locales would yield
effective antibiotic-producing species. For example, in addition to the terrestrial, tropical
Actinobacteria, several researchers have identified tropical marine antibiotic-producing
Actinobacteria, which seem to represent a separate ecosystem rather than a subset of
terrestrial washings (Kumar et al., 2012a). Aquatic antibiotic-secreting bacteria belong-
ing primarily to the genera Streptomyces and Rhodococcus have been isolated from Indian
sediments and the Arabian Sea as well as the Bay of Bengal, Andhra Pradesh (Kumar
et al., 2012a). The majority of these antibiotics have not yet been characterized, and it is not
known if they are novel or represent rediscoveries of already known isolates.
Antibiotic-resistant bacteria are now our constant companions, and new strains are
emerging all the time, despite effective measures to limit the use of therapeutics (Levy,
1998). MDR tuberculosis is a case in point, affecting 500,000 annually, which requires
2 years of treatment and is effectively cured in only 50% of patients (de Lima Procópio
et al., 2012). Even more virulent is extensively drug-resistant tuberculosis (XDR-TB), which
has been reported in 58 countries (Mlambo et al., 2008). More worrying are genotyping
studies that suggest between 63% and 75% of MDR-TB strains can progress to becom-
ing XDR-TB (Mlambo et al., 2008). Tropical Actinobacteria may be capable of generating
antibiotics effective against this bacterium, as suggested by the work of researchers in the
Amazon Biotechnology Center (CBA) (de Lima Procópio et al., 2012) and in our own study
(Piechoski et al., 2012). Lack of toxicity of antibiotics from any of these sources could result
in new hope for many across the world.
Finally, a return to natural antibiotics may help allay mistrust by many in the ability
of “artificial medicines” to effectively treat disease. Synthetic and semisynthetic antibiot-
ics have received bad press recently with the limitations placed on telithromycin (Ketek®;
Echols, 2011) as well as dangerous side effects from trimethoprim/sulfa drugs (Antoniou
et al., 2011). Even the well-loved semisynthetic azithromycin (Z-Pak®) was suggested by
one set of authors to increase cardiovascular death, although others labeled the studies
inconclusive azithromycin and the risk of cardiovascular death (Ray et al., 2012). These
observations, published in reputable journals, reduce public confidence in the safety of
antibiotics and may have even been a contributory factor in the increased use of natu-
ral, herbal medicines, particularly in the United States (Bauer, 2003). Natural antibiot-
ics and their derivatives typically possess better bioavailability and capacity to bind to
cellular targets than their synthetic counterparts, again lending support for new thera-
peutics to come from natural sources (Wright, 2010). Given that many natural microbial
metabolites have been and will continue to be derived from Actinobacteria species, these
resilient bacteria are destined to remain unchallenged as primary industrial sources of
our antibiotics.
Acknowledgments
The authors thank Dr. Anne Bower and Dr. John Porter for their valuable scientific insights
into the Jamaican soil project and also Catherine Boisvert-Bertrand and Stefanie Emanuel
for their bench skills.
References
Al-Bari, M. A. A., Bhuiyan, M. S. A., Flores, M. E., Petrosyan, P., Garcia-Varela, M., and Islam, M. A. U.
2005. Streptomyces bandladeshensis sp. nov., isolated from soil, which produces bis-(2-ethylhexyl)
phthalate. Int. J. Syst. Evol. Microbiol., 55(5), 1973–1977.
Ali, M. I., Ahmad, M. S., and Hozzein, M. N. 2009. WA52 a macrolide produced by alkalophile
Nocardiensis dassonvillei WA52. Aus. J. Basic Appl. Sci., 3, 607–616.
Ambrose, C., Varghese, C., and Bhore, S. J. 2013. Endophytic bacteria as a source of novel antibiotics—
An overview. Pharmacogn. Rev., 7(13), 11–16.
Antoniou, T., Gomes, T., Mamdani, M. M., Yao, Z., Hellings, C., Garg, A. X., and Juurlink, D. N.
2011. Trimethoprim-sulfamethoxazole induced hyperkalaemia in elderly patients receiving
spironolactone: Nested case-control study. BMJ, 343, d5228.
Araragi, M. 1979. Actinomycete flora of tropical, upland farmland soil on the basis of genus compo-
sition and antagonistic property. Soil Sci. Plant. Nutr. 25, 514–523.
Atlas, R. M., Parks, L. C., and Brown, A. E. 1995. Screening for the isolation of an antibiotic pro-
ducer. In: Atlas, R. M., Parks, L. C., and Brown, A. E., eds., Laboratory Manual of Experimental
Microbiology, exercise 52, 2nd edn. Mosby Publishers, St. Louis, MO, pp. 425–426.
Augustine, N., Wilson, P. A., Kerkar, S., and Thomas, S. 2012. Arctic actinomycetes as potential
inhibitors of Vibrio cholera biofilm. Curr. Microbiol., 64, 338–342.
Baltz, R. H. 2008. Renaissance in antibacterial discovery from Actinomycetes. Curr. Opin. Pharmacol.,
8, 557–563.
Bandow, J. E., Brötz, H., Leichert, L. I. O., Labischinski, H., and Hecker, M. 2003. Proteomic approach
to understanding antibiotic action. Antimicrob. Agents Chemother., 50, 519–526.
Barke, J., Seipke, R. F., Grüschow, S., Heavens, D., Drou, N., Bibb, M. J., and Hutchings, M. I. 2010. A
mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant
Acromyrmex octospinus. BMC Biology, 8, 109–119.
Bauer, B. A. 2003. Herbal medicine the good, the bad and the ugly. SoCRA SOURCE, pp. 27–30.
Boisvert-Bertrand, C., Brendley, B., and Cundell, D. 2004. Searching for narrow spectrum antibiotics
from microbes in soil from Presque Isle, Pennsylvania. J. Young Invest., 10(4). Accessed January
13, 2014. http://legacy.jyi.org/volumes/volume10/issue4/articles/boisvert.html.
Borneman, E. W. and Triplett, J. 1997. Molecular microbial diversity in soils from eastern Amazonia:
Evidence for unusual microoorganisms and microbial population shifts associated with defor-
estation. Appl. Environ. Microbiol., 63, 2647–2453.
Broad, W. J. 1994. Coral reefs endangered in Jamaica. The New York Times, September 9.
Brötz-Oesterhelt, H. and Sass, P. 2010. Postegenomic strategies in antibacterial drug therapy. Future
Microbiol., 5, 1553–1579.
Busti, E., Monciardini, P., Cavaletti, L., Bamonte, R., Lazzarini, A., Sosio, M., and Donadio, S. 2006.
Antibiotic-producing ability by representatives of a newly discovered lineage of actinomy-
cetes. Microbiology, 152, 675–683.
Cameron, R. E., King, J., and David, C. N. 1970. Soil microbial ecology of Wheeler Valley, Antarctica.
Soil Sci., 109, 110–120.
Carter, G. T., Nietsche, J. A., Williams, D. R., and Borders, D. B. 1990. Citreamicins, novel antibiotics
from Micromonospora citrea: Isolation, characterization and structure determination. J. Antibiot.,
43, 504–512.
Castillo, U., Harper, J. K., Strobel, G. A., Sears, J., Alesi, K., Ford, E., and Teplow, D. 2003. Kakadumycins,
novel antibiotics from Streptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia. FEMS
Microbiol. Lett., 224, 183–190.
Castillo, U. F., Strobel, G. A., Ford, E. J., Hess, W. M., Porter, H., Jensen, J. B., and Yaver, D. 2002.
Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic
on Kennedia nigriscans. Microbiology, 148, 2675–2685.
Challis, G. L. and Hopwood, D. A. 2003. Synergy and contingency as driving forces for the evolution
and multiple secondary metabolite production by Streptomyces species. Proc. Natl Acad. Sci.,
100, 14555–14561.
Chater, K. F., Biró, S., Lee, K. J., Palmer, T., and Schrempf, H. 2010. The complex extracellular biology
of Streptomyces. FEMS Microbiol. Rev., 34, 171–198.
Chaxamycins, A. D. 2011. Bioactive ansamycins from a hyper-arid desert Streptomyces sp. J. Nat.
Prod., 74(6), 1491–1499.
Chhabra, R. 1996. Soil Salinity and Water Quality. Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi,
India (South Asian edition), p. 284.
Cordeiro, J. C. R., Reis, A. O., Miranda, E. A., and Sader, H. S. 2001. In vitro antimicrobial activity of
the aminoglycoside arbekacin tested against oxacillin-resistant Staphylococcus aureus isolated
in Brazilian hospitals. Braz. J. Infect. Dis., 5(3), 130–135.
de Lima Procópio, R. E., da Silva, I. R., Martins, M. K., de Azevedo, J. L., and de Araújo, J. M. 2012.
Antibiotics produced by Streptomyces. Braz. J. Infect. Dis., 16(5), 466–471.
Ding, L., Münch, J., Goerls, H., Maier, A., Fiebig, H. H., Lin, W. H., and Hertweck, C. 2010. Xiamycin,
a pentacyclic indolosesquiterpene with selective anti-HIV activity from a bacterial mangrove
endophyte. Bioorg. Med. Chem. Lett., 20(22), 6685–6687.
Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L. 1991. Modular organization of
genes required for complex polyketide biosynthesis. Science, 252, 675–679.
Doube, B. M., Stephens, P. M., Davoren, C. W., and Ryder, M. H. 1994. Interaction between earth-
worms, beneficial microbes and root pathogens. Appl. Soil. Ecol., 1, 3–10.
Echols, R. M. 2011. Understanding the regulatory hurdles for antibacterial drug development in the
post-Ketek world. Ann. NY Acad. Sci., 1241, 153–161.
El-Khagawa, M. A. and Megahed, M. M. M. 2012. Antibacterial and anti-insecticidal activity of acti-
nomycetes isolated from sandy soil of Cairo Egypt. Egypt Acad. J. Biolog. Sci., 4, 53–67.
El-Naggar, M. Y., El-Assar, S. A., and Abdul-Gawad, S. M. 2006. Meroparamycin production by
newly isolated Streptomyces sp. strain MAR01: Taxonomy, fermentation, purification and struc-
tural elucidation. J. Microbiol., 44, 432–438.
Enright, M. C. 2003. The evolution of a resistant pathogen—The case of MRSA. Curr. Op. Pharmacol.,
3, 474–479.
Falagas, M. E., Grammatikos, A. P., and Michalopoulos, A. 2008. Potential of old-generation antibiot-
ics to address current need for new antibiotics. Expert Rev. Anti Infect. Ther., 6(5), 593–600.
Falkinham, J. O., Wall, T. E., Tanner, J. R., Tawaha, K., Alali, F. Q., Li, C., and Oberlies, N. H. 2009.
Proliferation of antibiotic-producing bacteria and concomitant antibiotic production as the
basis for the antibiotic activity of Jordan’s red soils. Appl. Environ. Microbiol., 75(9), 2735–2741.
Freiberg, C., Brunner, N. A., Schiffer, G., Lampe, T., Pohlmann, J., Brands, M., and Ziegelbauer, K.
2004. Identification and characterization of the first class of potent bacterial acetyl-CoA car-
boxylase inhibitors with antibacterial activity. J. Biol. Chem., 279(25), 26066–26073.
Fu, P., Kong, F., Wang, Y., Wang, Y., Liu, P., Zuo, G., and Zhu, W. 2013. Antibiotic metabolites from the
coral-associated Streptomyces species OUCMDZ-1703. Chin. J. Chem., 31(1), 100–104.
Fuoco, D. 2012. Classification framework and chemical biology of tetracycline-structure-based
drugs. Antibiotics, 1, 1–13.
Gadebusch, H. H., Stapley, E. O., and Zimmerman, S. B. 1992. The discovery of cell wall active anti-
bacterial antibiotics. Crit. Rev. Biotechnol., 12, 225–243.
Hancock, R. E. W. 2007. The complexities of antibiotic action. Mol. Syst. Biol., 3, 142.
Hawksworth, D. L. and Colwell, R. R. 1992. Biodiversity amongst microorganisms and its relevance.
Biodiver. Conserv., 1, 221–345.
Hayakawa, M. 2008. Studies of the isolation and distribution of rare Actinomycetes in soil.
Actinomycetologica, 22, 12–19.
Hickner, J. 2007. Can you call in a Z-Pak for me? J. Family Pract., 56(4), 262.
Hogan, C. M. 2010. Bacteria. In: Draggan, S. and Cleveland, C. J., eds., Encyclopedia of Earth. National
Council for Science and the Environment, Washington, DC.
Hozzein, W. M., Ali, M. I. A., and Rabie, W. 2008. A new preferential medium for enumeration and
isolation of desert Actinomycetes. World J. Microbiol. Biotechnol., 24(8), 1547–1552.
Idjadi, J. A., Lee, S. C., Bruno, J. F., Precht, W. F., Allen-Requa, L., and Edmunds, P. J. 2006. Rapid
phase shift reversal on a Jamaican coral reef. Coral Reefs., 25, 209–211.
Ikner, L. A., Toomey, R. S., Nolan, G., Neilson, J. W., Pryor, B. M., and Maier, R. M. 2007. Culturable
microdiversity and the impact of tourism in Kartchner Caverns, Arizona. Microb. Ecol., 53,
30–42.
IMS Institute for Healthcare Informatics. 2011. The use of medicines in the United States: Review of
2011. Accessed January 14, 2014, http://www.imshealth.com/ims/Global/Content/Insights/
IMS%20Institute%20for%20Healthcare%20Informatics/IHII_Medicines_in_U.S_Report_2011.pdf.
Janso, J. E. and Carter, G. T. 2010. Phylogenetically unique endophytic actinomycetes from tropical
plants possess great biosynthetic potential. Appl. Environ. Microbiol., 77, 4377–4386.
Juo, A. S. and Franzluebbers, K. 2003. Tropical Soils: Properties and Management for Sustainable Agriculture
(Topics in Sustainable Agronomy), 1st edn. Oxford University Press, New York, p. 78.
Kannan, R.R., Iniyan, M.M., and Prakash, V.S.G. 2011. Isolation of a small molecule with anti-MRSA
activity from a mangrove symbiont Streptomyces sp PVRK-1 and its biomedical studies in
zebrafish embryos. Asian Pac. J. Trop. Biomed., 5, 341–347.
Katz, M. L., Mueller, L. V., Polyakov, M., and Weinstock, S. F. 2006. Where have all the antibiotic
patients gone? Nat. Biotechnol., 24, 1529–1531.
Kieft, T. L. 1991. Soil microbiology in reclamation of arid and semi-arid lands. In: Skujins, J., ed.,
Semiarid Lands and Deserts: Soil Resource and Reclamation. Marcel Dekker, New York, pp. 209–256.
Kumar, K. S., Haritha, R., Mohan, Y. S. Y. V. J., and Ramana, T. 2012a. Screening of marine
Actinobacteria for antimicrobial compounds. Res. J. Microbiol., 6(4), 385–393.
Kumar, V., Bharti, A., Negi, Y. K., Gusain, O., Pandey, P., and Bisht, G. S. 2012b. Screening of acti-
nomycetes from earthworm castings for their antimicrobial activity and industrial enzymes.
Braz. J. Microbiol., 43(1), 205–214.
Kurapova, A. I., Zenova, G. M., Sudnitsyn, I. I., Kizilova, A. K., Manucharova, N. A., Norovsuren, Z.,
and Zvyagintsev, D. G. 2012. Thermotolerant and thermophilic Actinomycetes from soils of
Mongolia desert steppe zone. Microbiology, 81, 105–116.
Kurosawa, K., Ghiviriga, I., Sambandan, T. G., Lessard, P. A., Barbara, J. E., Rha, C., and Sinskey, A. J.
2008. Rhodostreptomycins, antibiotics biosynthesized following horizontal gene transfer from
Streptomyces padanus to Rhodococcus fascians. JACS Commun., 130, 1126–1127.
Lancini, G., and Lorenzetti, R. 1993. Biotechnology of Antibiotics and Other Bioactive Microbial
Metabolites. Plenum Press, New York, pp. 49–57.
Lange, R. P. Locher, H. H., Wyss, P. S., and Then, R. L. 2007. The targets of currently used antibiotic
agents: Lessons for drug discovery. Curr. Pharm. Des., 13, 3140–3154.
Lazzarini, A., Cavaletti, L., Toppo, G., and Marinelli, F. 2001. Rare genera of actinomycetes as poten-
tial producers of new antibiotics. Antonie van Leeuwenhoek, 78, 399–405.
Levy, S. B. 1998. The challenge of antibiotic resistance. Sci Am., 398, 451–456.
Li, G. P. 1989. Isolation of actinomycetes for antibiotic screening. Chin. J. Antibiotic, 14, 452–465.
Liu, L. L., He, L. S., Xu, Y., Han, Z., Li, Y. X., Zhong, J. L., and Qian, P. Y. 2013. Caspase-3-dependent
apoptosis of citreamicin epsilon-induced HeLa cells is associated with reactive oxygen species
generation. Chem. Res. Toxicol., 26(7), 1055–1063.
Makut, M. D. and Abdalazeez, U. S. 2012. A survey of antibiotic-producing actinomycetes in the
soil environment of Keffi metropolis, Nasarawa State, Nigeria. J. Microbiol. Antimicrobial., 4(5),
74–78.
Marinelli, F. 2009. Antibiotics and Streptomyces: The future of antibiotic discovery. Microbiology
Today, pp. 20–23.
Mendes, T. D., Borges, W. S., Rodrigues, A., Solomon, S. E., Vieira, P. C., Duarte, M. C., and Pagnocca,
F. C. 2013. Anti-Candida properties of urauchimycins isolated from Actinobacteria associ-
ated with Trachymyrmex ants. Biomed. Res. Int. 8. Article ID 835081. Accessed January 20, 2014.
http://www.hindawi.com/journals/bmri/2013/835081/cta/.
Mlambo, C. K., Warren, R. M., Poswa, X., Victor, T. C., Duse, A. G., and Marais, E. 2008. Genotypic
diversity of extensively drug-resistant tuberculosis (XDR-TB) in South Africa. Int. J. Tuberc.
Lung. Dis., 12(1), 99–104.
Nelson, L. M. and Levy, S. B. 2011. The history of the tetracyclines. Ann. NY Acad. Sci., 1241, 17–32.
Nicolaou, K. C., Boddy, C. N. C., Bräse, S., and Winssinger, N. 1999. Review: Chemistry, biology, and
medicine of the glycopeptide antibiotics. Angew. Chem. Int. Ed., 38, 2096–2152.
Nithyanand, P., Thenmozhi, R., Rathna, J., and Pandian, S. K. 2010. Inhibition of S. pyogenes biofilm
formation by coral associated Actinomycetes. Curr. Microbiol., 60, 454–460.
Niyomvong, N., Pathom-aree, W., Thamchaipenet, A., and Duangmal, K. 2012. Actinomycetes from
tropical limesone caves. Chiang Mai J. Sci., 39(3), 373–388.
Osman, M. E., Ahmed, F. A. H., and Abd El All, W. S. M. 2011. Antibiotic production from local
Streptomyces isolates from Egyptian soil at Wady El Natron: Isolation, identification and opti-
mization. Aust. J. Basic Appl. Sci., 5(9), 782–792.
Panaiyadiyan, P. and Chellaia, S. R. 2011. Biodiversity of microorganisms isolated from rhizosphere
soils of Pachamalai hills, Tamilnadu, India. Res. J. Forestry, 5, 27–35.
Payne, D. J., Gwynn, M. N., Holmes, D. J., and Pompliano, D. L. 2007. Drugs for bad bugs: Confronting
the challenge for antibacterial discovery. Nat. Rev. Drug Discov., 6, 29–40.
Peoples, A. J., Zhang, Q., Millett, W. P., Rothfeder, M. T., Pescatore, B. C., Madden, A. A., and Moore, C. M.
2008. Neocitreamicins I and II, novel antibiotics with activity against methicillin-r esistant
Staphylococcus aureus and vancomycin-resistant Enterococci. J. Antibiot. (Tokyo), 61, 457–463.
Piechoski, M., Cundell, D., Bower, A., and Porter, J. 2012. Bioassay and antibiotic activity of Jamaican
Actinomyces isolates. J. Young Investigat. Accessed January 13, 2014. http://www.jyi.org/issue/
bioassay-and-antibiotic-activity-of-jamaican-actinomycetes-isolates/.
Powers, J. H. 2004. Antimicrobial drug development—The past, the present and the future. Clin.
Microbiol. Infect., 10, 23–31.
Prashith Kekuda, T. R., Shobha, K. S., and Onkarappa, R. 2010. Studies on antioxidant and anthel-
mintic activity of two Streptomyces species isolated from Western Ghat soils of Agumbe,
Karnataka. J. Pharm. Res., 3, 26–29.
Raja, A. and Prabakarana, P. 2011. Actinomycetes and drug—An overview. Am. J. Drug Discov. Dev.,
1(2), 75–84.
Rajput, Y., Rai, V., and Biswas, J. 2012. Screening of bacterial isolates from various microhabitat sedi-
ments of Kotumsar Cave: A cogitation on their respective benefits and expected threats for
complete biosphere and tourists. Res. J. Environ. Toxicol., 6, 13–24.
Ray, W. A., Murray, K. T., Hall, K., Arbogast, P. G., and Stein, C. M. 2012. Azithromycin and the risk
of cardiovascular death. N Eng. J. Med., 366(20), 1881–1890.
Schaechter, M., ed. 2010. Desk Encyclopedia of Microbiology. Academic Press, San Diego, CA.
Selvameenal, L., Radhakrishnan, M., and Balagurunathan, R. 2009. Antibiotic pigment from desert
soil actinomycetes; Biological activity, purification and chemical screening. Indian J. Pharm.
Sci., 71(5), 499–504.
Silver, L. 2011. Challenges of antibacterial discovery. Clin. Microbiol. Rev., 24, 71–109.
Singh, A. B., Singh, R. B., and Mishra, S. 2012. Microbial and biochemical aspects of antibiotic pro-
ducing microorganisms from soil samples of certain industrial area of India—An overview.
Open Nutraceut. J., 5, 107–112.
Skujins, J. 1984. Microbial ecology of desert soils. In: Marshal, C. C., ed., Advances in Microbial Biology.
Plenum Press, New York, pp. 49–51.
Solis, P. N., Wright, C. W., Anderson, M. M., Gupta, M. P., and Phillipson, J. D. 1993. A microwell
cytotoxicity assay using Artemia salina. Plant Med., 59, 250–252.
Tabrizi, S. G., Hamedi, J., and Mohammadipanah, F. 2013. Screening of soil actinomycetes against
Salmonella serovar NCTC 5761 and characterization of the prominent active strains. Iran
J. Microbiol., 5, 356–365.
Tomova, I., Lazarkevich, I., Tomova, A., Kambourova, M., and Vasileva-Tonkova, E. 2013. Diversity
and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura
cave, Bulgaria. Int. J. Spelol., 42(1), 65–76.
Vander Meer, R. V. 2012. Ant interactions with soil organisms and associated semiochemicals.
J. Chem. Ecol., 38(6), 728–745.
Varghese, R., Nishamol, S., Suchithra, R., Jyothy, S., and Hatha, A. M. 2012. Distribution and
antibacterial activity of Actinomycetes from shola soils of tropical montane forest in Kerala,
South India. J. Environ., 1, 93–99.
Von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., and Häbich, D. 2006. Antibacterial natural
products in medicinal chemistry—Exodus or revival? Angew. Chem. Int. Ed. Engl., 45, 5072–5129.
Waksman, S. A. and Lechevalier, H. A. 1962. The Actinomycetes, Volume III, Antibiotics of Actinomycetes.
The Williams and Wilkins Company, Baltimore, MD.
Wall Street Journal Market Watch. Vela Minerals acquires 100% interest in Mavis Bank and Port
Antonio properties Jamaica. The Wall Street Journal, June 6, 2013. Accessed January 22, 2014.
www.marketwatch.com/story/vela-minerals-acquires-100-interest-in-mavis-bank-and-port-
antonio-properties-jamaica-2013-06-06.
Wright, G. D. 2010. Q&A: Antibiotic resistance: Where does it come from and what can we do about
it? BMC Biol., 8, 123.
Yucel, S. and Yamac, M. 2010. Selection of Streptomyces isolates from Turkish karstic caves against
antibiotic-resistant organisms. Pak. J. Pharm. Sci., 23, 1–6.
Zgoda, J. R. and Porter, J. R. 2001. A convenient microdilution method for screening natural prod-
ucts against bacteria and fungi. Pharm. Biol., 39, 221–225.
Zvyagintsev, D. G. and Zenova, G. M. 2007. Aktinomitsety zasolennykh i schchelochnykh pochv
(Actinomycetes of Saline and Alkaline Soils). Knizhnyi Som Universitet, Moscow, Russia.
chapter six
Antimicrobial agents from actinomycetes

Chemistry and applications
Contents
6.1 Introduction........................................................................................................................... 99
6.2 Source of antimicrobial agent–producing actinomycetes............................................. 101
6.2.1 Soil actinomycetes�� 101
6.2.2 Endophytic actinomycetes�� 103
6.2.3 Alkaliphilic actinomycetes�� 105
6.2.4 Halophilic actinomycetes�� 107
6.2.5 Marine actinomycetes�� 107
6.3 Future developments.......................................................................................................... 109
Acknowledgments....................................................................................................................... 111
References...................................................................................................................................... 111
6.1 Introduction
The antimicrobial era started after the discovery of penicillin by Alexander Fleming
(1929). Back from a holiday, Fleming noticed that a petri dish containing Staphylococcus
plate culture, which was mistakenly left open, was contaminated by a blue-green mould
(Penicillium), which formed a visible growth. Fleming noticed that there was a halo of
inhibited bacterial growth around the mould. Watching the plate Fleming concluded that
the mould might release a substance that suppressed the growth or killed the bacteria. The
substance was nothing but penicillin (Figure 6.1a), which had an antibacterial effect that
inhibited the growth of Staphylococcus.
Although the discovery was accidental and a lucky one, let us imagine for an instance
that after returning from the holiday, Fleming noticed something “funny” about this
plate and just discarded it as a culture that had been made useless as it was contami-
nated. Perhaps the best known of Pasteur’s obiter is that “in matters of observation, for-
tune favours only the prepared mind” (Kingston, 2008). Fleming discovered the penicillin
because his mind was prepared; in his earlier study, he discovered lysozyme when he
had a heavy cold and mucus from his nose fell on the bacterial plate. After a few days, he
noticed no growth around the mucus. He had hoped that this mucus would have antibac-
terial activity; from this experience, he discovered the penicillin.
99
O CH3
HO H OH
HO O HO
H O
H HO N
R N S O O NH2
CH3
HO HN CH3 N OH NH2
O N CH3
NH2
O
COOH H2N
(a) (b)
Figure 6.1 The two important compounds of antimicrobial era. (a) Structure of penicillin where R
is the variable group. (b) Structure of streptomycin.
This remarkable contribution of Fleming to medicinal science saved many lives dur-
ing World War II. Though penicillin was a very effective antimicrobial agent at that time,
unfortunately, it had one drawback: it has negligible effect on tuberculosis. The great suc-
cess of penicillin made Waksman rush into his laboratory and shout to his colleagues, “see
what these Englishmen have discovered a mould can do. I know the actinomycetes will
do better!” Within a few months, Waksman discovered streptomycin (Figure 6.1b), and
it was announced in a 1944 paper (Schatz et al., 1944; Kingston, 2008). Furthermore, the
Mayo Institute found that “streptomycin” surpassed the activity of penicillin in inhibiting
tuberculosis (Kiple, 1993). Since then, the golden era of actinomycetes started, and today
actinomycetes are well known for producing antimicrobial agents.
Actinomycetes are gram-positive, filamentous bacteria that have a proven capacity to
produce bioactive secondary metabolites. Particularly, it has been estimated that approxi-
mately two-thirds of natural antibiotics have been isolated from actinomycetes, and about
75% of them are produced by members of the genus Streptomyces (Table 6.1) (Franco-
Correa et al., 2010; Wang et al., 2013a). The golden age of antimicrobial agents continues.
Everything seemed to be very well controlled, but because of the widespread and uncon-
trolled use of antimicrobial agents, the golden age came to an end as microorganisms
Table 6.1 Some antimicrobial agents obtained from Streptomyces

Antibiotic/
molecular formula Source Application References
Paromomycin Streptomyces Used to treat intestinal infections such as Davidson
C23H4N5O18S krestomuceticus cryptosporidiosis, amoebiasis, and other et al. (2009)
diseases like leishmaniasis
Carbomycin Streptomyces Used as a mild antibiotic and used to treat Wagner
C42H67NO16 halstedii granuloma inguinale et al. (1953)
Kanamycin Streptomyces Used as an antibacterial agent Garrod
C18H36N4O11 kanamyceticus et al. (1981)
Lincomycin Streptomyces Used against mycoplasma and some Spizek and
C18H34N2O6S lincolnensis species of Plasmodium Rezanka
(2004)
Daptomycin Streptomyces Used to treat skin infections mainly caused Woodworth
C72H101N17O26 roseosporus by gram-positive bacteria et al. (1992)
Tylosin Streptomyces Effective against gram-positive bacteria but Giguere
C46H77NO17 fradiae has limited range on gram-negative bacteria et al. (2007)
Chapter six: Antimicrobial agents from actinomycetes 101
became resistant to them (Deene and Lingappa, 2013). The biggest advantage of microbes
against antibacterial agents is their incredible adaptability to change in the environment,
which is mainly due to their flexible metabolic power (Bérdy, 2012). The increasing emer-
gence of multidrug-resistant bacteria throughout the world and the lack of antibiotics to
combat such pathogenic agents continue to be the major concern of the medical community
(Kitouni et al., 2005). To combat multidrug-resistant bacteria, many different antimicro-
bial agents from actinomycetes isolated from various sources have been reported. In this
chapter, we focus on the chemistry and applications of antimicrobial agents from actino-
mycetes isolated from different sources.
6.2 Source of antimicrobial agent–producing actinomycetes

6.2.1 Soil actinomycetes
Soil is not just a mass of dead debris that resulted simply from the physical and chemical
weathering of rocks, but it teems with life. Every small particle of soil contains numer-
ous types of living organisms so small that they cannot be recognized with the naked
eye. The living organisms comprise numerous types of bacteria, fungi, algae, protozoa,
nematodes, and other invertebrates that vary considerably in their structure, size, mode
of living, and relationship to soil processes (Waksman and Starkey, 1931). Soils contain
by far the greatest diversity of organisms (Bonkowski and Roy, 2005), for instance, it
has been estimated that 1 g of soil contains as many as 1010 –1011 bacteria (Marcel et al.,
2008), of which 107 is actinomycetes (Steffan et al., 1988) mainly belonging to the genus
Streptomyces.
Since the discovery of streptomycin, various scientists started finding new antibacte-
rial agents mainly isolated from soil. Eli Lilly’s research team in 1949 discovered erythro-
mycin, a macrolide antibiotic having the molecular formula C37H67NO13 and a molecular
mass of 733.93 g/mol, mainly obtained from Saccharopolyspora erythraea isolated from soil
samples (McGuire et al., 1952). Erythromycin (Figure 6.2a) is used to treat skin and upper
respiratory tract infections caused by Streptococcus, Staphylococcus, and Haemophilus genera
(Sarbani, 2006).
NH2
O HO O
H3C CH3 HO
NH2 H2N
HO OH H3C O
CH3
H 3C OH CH3 N HO O NH2
H3C HO O OH
H5C2 O O O CH3
O O OCH3 O OH
CH3 R1 NH2
CH3 HO
O OH HO O
CH3 R2
(a) (b)
Figure 6.2 Antimicrobial agent from actinomycetes isolated from soil: (a) structure of e rythromycin
and (b) structure of neomycin.
In the same year, neomycin was discovered by Selman Waksman who was later
awarded the Nobel Prize in 1951 for his comprehensive contribution toward the field of
physiology and medicine (Schatz et al., 1944; Kingston, 2008). Neomycin (Figure 6.2b) is
an amino glycoside antibiotic having the molecular formula C23H46N6O13 and a molecular
mass of 614.644 g/mol. Naturally produced by Streptomyces fradiae, neomycin works by
binding to the bacterial 30S ribosomal subunit inhibiting the translocation of the peptidyl-
tRNA from the A-site to the P-site and also causing misreading of mRNA, leaving the bac-
terium unable to synthesize protein vital to its growth, leading to its death. Streptomyces
was not the only center of attraction, but other actinomycetes were also explored.
Edmund Kornfeld isolated vancomycin in 1953 from Amycolatopsis orientalis
from soil samples collected from the interior jungles of Borneo (Michael and Plotkin,
2003). Vancomycin, a glycopeptide antibiotic (Figure 6.3a), has the molecular formula
C66H75Cl2N9O24 and a molecular mass of 1449.3 g/mol. It is used in the prophylaxis and
treatment of infections caused by gram-positive bacteria like Staphylococcus aureus (Levine,
2006). The minimum inhibitory concentration (MIC) of vancomycin against S. aureus is
found to be ≤2 μg/mL according to the Clinical and Laboratory Standards Institute (CLSI)
guidelines (2012).
Similarly, another aminoglycoside antibiotic, gentamicin, was isolated from
Micromonospora purpurea by Weinstein in 1963. Gentamicin (Figure 6.3b) has the molecular
formula C21H43N5O7 and a molecular mass of 477.596 g/mol (Weinstein et al., 1967), and it
is active against gram-negative bacteria.
OH
HO
OH
O OH
NH2
NH O O HN O
H H H O
N N N
N N
H H O
O O
HO OH
O O
CI CI
O O
HO
HO O
OH
O
H2N
(a) OH
OH
CH3
H3C O H2N
HN O
HO NH2
H3C HO
O O
H2N NH2
(b)
Figure 6.3 Structure of (a) vancomycin and (b) gentamicin.

The antimicrobial agent produced by actinomycetes not only helped us to overcome

human pathogens but is also used as biological control agents for many plant pathogens
and also solved many environmental problems.
Cavity spot disease in the plant caused by the pathogen Pythium coloratum was con-
trolled by the antimicrobial agent produced by Actinoplanes philippinensis isolated from
the soil collected from carrot rhizosphere (El-Tarabily et al., 1997). The antimicrobial
agent from actinomycetes was used to solve many environmental problems like cya-
nobacteria bloom, which posed a serious health threat to animals and humans. Many
cyanobacteria produce cyanotoxins (Sivonen and Jones, 1999). To overcome this toxin
many compounds from actinomycetes were isolated, such as a novel actinomycete,
Streptomyces aurantiogriseus, which showed algicidal activity against the toxic cyano
bacterium Microcystis aeruginosa (Somdee et al., 2013). Though many antimicrobial
agents produced from actinomycetes have been isolated (Figure 6.4a through f), it is
just a “tip of the iceberg” that has been explored (Baltz, 2005).
In the past few decades, researchers have made many attempts to isolate novel anti-
microbial agents not only from the soil but also from the plants generally regarded as
endophytes. Attempts were also made to isolate actinomycetes from extreme conditions
like alkaliphilic actinomycetes (actinomycetes that can grow at high pH), halophilic
actinomycetes (actinomycetes that can grow at high salt concentration), and also actino-
mycetes from the marine ecosystem. In this chapter, we discuss antimicrobial agents from
actinomycetes isolated from the aforementioned environments.
6.2.2 Endophytic actinomycetes
Endophytes are microorganisms that live for the whole or part of their life history inside
plant tissues. As a result of these long-held associations, endophytic microorganisms and
plants have developed good information transfer (Zhao et al., 2011a,b). The antimicrobial
activity of endophytes was reported more than 50 years ago when Smith (1957) succeeded
to isolate Micromonospora from the tomato plant, which had an antagonistic activity. Since
then, many endophytic actinomycete compounds were isolated and have found applica-
tion not only as an antimicrobial agent but also in many fields.
Many endophytic actinomycete compounds were used as biocontrol agents like com-
pounds from Nocardia globerula used to control the Helminthosporium solani pathogen, which
caused the silver scurf disease in potatoes (Elson et al., 1997). Furthermore, compounds
like chitinase produced by the endophyte Actinoplanes missouriensis are used against root
rot disease of lupine caused by Plectosporium tabacinum (El-Tarabily, 2003).
Besides biocontrol agents, various compounds from endophytic actinomycetes helped
to protect plants from fungal pathogens. The compound from Streptomyces sp. isolated
from Rhododendron showed antifungal activity against seven fungal pathogens, namely
Phytophthora cinnamomi Rands, Pythium aphanidermatum (Edson) Fitzpatrick, Rhizoctonia solani
Kühn, Fusarium avenaceum (Corda: Fries) Saccardo, Sclerotinia homoeocarpa Bennett, Botrytis
cinerea Persoon, and Pestalotiopsis sydowiana Bresadola (Shimizu et al., 2000). Similarly, two
white amorphous compounds, 5,7-dimethoxy-4-p-methoxylphenylcoumarin (C18H16O5)
and 5,7-dimethoxy-4-phenylcoumarin (C17H14O5), obtained from Streptomyces aureofaciens
CMUAc130 isolated from the root tissue of Zingiber officinale Rosc. (Zingiberaceae) helped to
suppress the soil fungal pathogens (Taechowisan et al., 2005).
104
OH O
HO O CH3 O
HO HC
H3C CH3
NH2 H2N
O N
HO O NH2 O O HO
H3CO HO
O OH O
O O CH3 CH3
OH
O O
OH
O OH H3C O O
CH3 CH3 HO O HO OH
H2N NH2 H2N O
O HO NH2
HO H3C HO
O O
HO O H3C O H2N NH2
(a) (b) (c)
H3N+
O H O
H N
N N N Me Me
CONH2 O NH H O H O HO
O O HOOC HN
H Me
N N HOOC O AcO OH O
HO N OH OH Me
O HO H H NH Me
H H NH O COOH O OO HO NH
N OH MeO
N H
O N N N Me
H O O
H H O
OH O
O O O CO2H
SCH3
NH2 HO O Me O
(d) (e) (f)
Figure 6.4 Structure of (a) paromomycin, (b) carbomycin, (c) kanamycin, (d) lincomycin, (e) daptomycin, and (f) rifamycin B.
Antimicrobials: Synthetic and natural compounds
O
CH3O
O
N
H
O
CH3O
CH3O
OH O
O
NH2
(a) (b)
Figure 6.5 Structure of (a) geldanamycin and (b) Hsp90–geldanamycin complex.
A macrolide antibiotic obtained from Streptomyces sp. CS isolated from Maytenus

hookeri was not only a good antimicrobial agent but also a good antitumor agent (Lu
and Shen, 2003). Celastramycins A and B isolated from Streptomyces sp. MaB-QuH-8
isolated from the Maytenus aquifolium plant showed excellent antimicrobial activity
(Pullen et al., 2002).
Compounds like “siderophore,” an iron-chelating compound isolated from endo-
phytic actinomycetes, not only help in plant growth but also act as an indirect antimi-
crobial agent (Franco-Correa et al., 2010). Many structures similar to siderophore like
“spoxazomicins A” (molecular formula C16H21N3O3S and molecular weight 335 g/mol),
pyochelin, a siderophore isolated from the orchid endophyte Streptosporangium

oxazolinicum (Inahashi et al., 2011), and geldanamycin, a type of benzoquinone ansa-
mycin antibiotic (C29H40N2O9) isolated from endophytic actinomycetes, also showed
antimicrobial activity (Dhanya and Padmavathy, 2014). Geldanamycin (Figure 6.5a
and b) inhibits the function of heat shock protein 90 (Hsp90) by binding to the unusual
ADP/ATP-binding pocket of the protein. Hsp90 client proteins play important roles in
the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis, and
oncogenesis (Michael and Plotkin, 2003). Similarly, many antimicrobial agents from
endophytic actinomycetes were isolated. Table 6.2 lists some of the endophytic actino-
mycetes producing antimicrobial agents.
6.2.3 Alkaliphilic actinomycetes
Sato’s team was the first to report the production of antibiotics in alkaline medium (pH
of the medium, 10.5) (Sato et al., 1980). Since then, many attempts were made to iso-
late antimicrobial compounds from alkaliphilic actinomycetes like phenazine and its
derivatives.
Phenazine is a water-soluble antibiotic obtained as a yellow-to-brown crystal pow-
der (Figure 6.6a), isolated from the alkaliphile Nocardiopsis dassonvillei OPC-15. It has
the molecular formula C12H8N2, a molecular mass of 180.21 g/mol, and a melting point
of 177°C and is reported to have an antimicrobial activity at pH 10 (Tsujibo et al., 1988;
McDonald et al., 2001).
Similarly, pyrocoll (Figure 6.6b), a constituent of cigarette smoke isolated from the
alkaliphilic Streptomyces sp. AK, has biological activity against various Arthrobacter strains,
filamentous fungi, several pathogenic protozoa, and some human tumor cell lines (Dietera
et al., 2003). Furthermore, an antifungal compound, naphthospironone A, produced by the
Table 6.2 List of some endophytic actinomycetes, origin and their compound applications
Name of endophytic
actinomycetes Origin Compound application References
Microbispora Roots and Antibacterial agent against Araujo et al.
leaves of Bacillus subtilis, S. aureus, (2000)
maize and Micrococcus luteus
Streptomyces sp. Roots of Antifungal activity against Taechowisan
Zingiber Colletotrichum musae and and Lumyong
officinale Fusarium oxysporum (2003)
Streptomyces griseorubiginosus Banana roots Biocontrol agent against wilt Cao et al.
disease of banana, caused (2005)
by the fungal pathogen
Fusarium oxysporum
Actinoplanes philippinensis Cucumber Biocontrol agent against El-Tarabily
damping-off disease caused (2006)
by the pathogen Pythium
aphanidermatum
Microbispora, Streptomyces, and Chinese Antagonists to Lee et al.
Micromonospora cabbage Plasmodiophora brassicae (2008)
Streptomyces, Streptosporangium, From various Antibacterial and antifungal Verma et al.
Microbispora, Streptoverticillium, parts of agents (2009)
Saccharomonospora, and Nocardia Azadirachta
indica
Nocardioides sp. Triticum Used to treat wheat take-all Coombs et al.
aestivum disease caused by (2004)
Gaeumannomyces graminis
var. tritici
O N
N
N O
N
(a) (b)
Figure 6.6 Structure of (a) phenazine and (b) pyrocoll.
Nocardiopsis sp. YIM DT266 isolated from the soil sample (pH 10), is a colorless solid with
the molecular formula C20H18O9, a molecular mass of 403.1023 g/mol, and a melting point
of 173°C. It shows maximum absorption at 227 nm in methanol and has antibacterial, anti-
fungal, and moderate cytotoxic activities (Ding et al., 2010). Similarly, the two pyran-2-one
compounds, namely nocardiopyrones A and B (Figure 6.7a and b), isolated from the novel
alkaliphilic actinomycete species Nocardiopsis alkaliphila YIM-80379 and having the molec-
ular formula C14H20O4 and C12H18O3 and a molecular mass of 275.1255 and 233.1150 g/mol,
respectively, showed antimicrobial activity against Pseudomonas aeruginosa, Enterobacter
aerogenes, Escherichia coli, S. aureus, and Candida albicans (Wang et al., 2013b).
There are only scanty reports on antimicrobial activity produced by alkaliphilic acti-
nomycetes. This may be due to the fact that antibiotics produced were not stable under
higher pH (Horikoshi, 1999). Thus, in future attempts, isolation of stable alkaliphilic anti-
biotics should be made so that they can be applied in many applications.
12
2 O
4
RO 11 O
6
10 8 13
(a) R = Ac
(b) R = H
Figure 6.7 Structure of (a) nocardiopyrone A and (b) nocardiopyrone B.
6.2.4 Halophilic actinomycetes
The actinomycetes growing in saline environments are probably most metabolically
and physiologically different from their type strains that thrive in other environments.
Actinopolyspora halophila is the first halophilic actinomycete isolated as a contaminant of a
culture medium containing 25% NaCl (Johnson et al., 1986). Since then, many compounds
from halophilic actinomycetes were isolated, characterized, and used in many fields as a
biological control agent, and as antibacterial, antifungal, and anticancer agents.
The biological control agents spinosyns A and D, isolated from the halophilic actino-
mycete Saccharopolyspora spinosa, prevent acetylcholine from binding to appropriate recep-
tor sites, which causes paralysis and death in insects, mostly caterpillars, by both contact
and ingestion (Kirst et al., 1992). Himalomycins (Table 6.3) isolated from Streptomyces sp.
have antibacterial activity (Maskey et al., 2003).
The new p-terphenyl 6′-hydroxy-4,2′,3′,4″-tetramethoxy-p-terphenyl obtained as color-
less needles from the halophilic actinomycete Nocardiopsis gilva YIM 90087 is not only a
good antibacterial and antifungal agent but also acts as an antioxidant (Tian et al., 2013).
Furthermore, Table 6.3 presents some of the halophilic actinomycetes and their products.
Due to the diverse application that halophilic actinomycete compounds possess, our
team have made attempts and successfully isolated many novel actinomycetes (listed in
Table 6.4), and furthermore, we believe that our team can isolate many unique antimicro-
bial agents from them in the future.
6.2.5 Marine actinomycetes
More than 70% of our planet’s surface is covered by oceans, and life on Earth is believed
to have originated from the sea. Experts estimate that the biological diversity in marine
ecosystems is much higher than in the tropical rain forests (Haefner, 2003). The early
evidence supporting the existence of marine actinomycetes came from the description
of Rhodococcus marinonascene, the first marine actinomycete species to be characterized
(Helmke and Weyland, 1984). Since then, many researchers have switched over to this
environment to find actinomycetes that produce novel antimicrobial agents mainly due
to the belief that marine environmental conditions are extremely different from terres-
trial conditions. Thus, the marine ecosystem may harbor many unique microorganisms
that produce novel antimicrobial agents and obtain positive results. The compounds iso-
lated from marine actinomycetes have many diverse applications like control of many
multidrug-resistant pathogens, for example, the polyketide antibiotic SBR-22, isolated from
marine streptomycete BT-408, showed antibacterial activity against methicillin-resistant
Table 6.3 List of some antimicrobial agents obtained from halophilic actinomycetes
Halophilic
actinomycetes and
compounds name Structure References
Streptomyces sp. O CH3 Maskey et al.
Himalomycins O (2003)
OH
OH O OH
Actinopolyspora sp. O Huang et al.
YIM90600 (2009)
Erythronolides H and I OH
O
OH 18 O
6 O O
HO 9S O 12R
14R O OH
OH
O OH
Actinopolyspora sp. Zhao et al.

YIM90600 (2011b)
H3CO2C
Actinopolysporins A–C OH
OH
(a)
OH
H3CO2C
OH
(b)
OH
O
(c)
Table 6.4 List of some novel halophilic actinomycetes

Halophilic actinomycetes Salt concentration range References
Streptomonospora alba 5–25 Li et al. (2003b)
Prauserella halophila 5–25 Li et al. (2003a)
Nesterenkonia xinjiangensis 0–25 Li et al. (2004a)
Nocardiopsis salina 3–20 Li et al. (2004b)
Nesterenkonia halotolerans 0–25 Li et al. (2004a)
Nocardiopsis rhodophaea 0–18 Li et al. (2006)
Nocardiopsis chromatogenez 0–18 Li et al. (2006)
Saccharomonospora saliphila 5–20 Syed et al. (2008)
Marinactinospora thermotolerans 0–5 Tian et al. (2009)
Amycolatopsis halophila 1–15 Tang et al. (2010)
Actinopolyspora erythraea 10–25 Tang et al. (2011)
Streptomyces oceani 2.5–12.5 Tian et al. (2012)
S. aureus (Sujatha et al., 2005). Similarly, the compound abyssomicin C, a novel polycyclic
polyketide antibiotic produced by the marine actinomycete Verrucosispora sp., possesses
potent activity against vancomycin-resistant S. aureus (Rath et al., 2005; Riedlinger
et al., 2004).
Another compound, diazepinomicin (for the structure, refer to Table 6.5), isolated
from the Micromonospora strain DPJ12 and having the molecular formula C28H34N2O4,
not only possesses antibacterial activity but also has anti-inflammatory and antitumor
activities (Charan et al., 2004). Furthermore, Pseudonocardia sp. SCSIO 01299, isolated from
marine sediments collected from a depth of 3258 m, produced four compounds: deoxyny-
boquinone and pseudonocardians A–C. These four compounds were extracted from the
biomass using acetone and butanone as a solvent, respectively. The compound deoxyny-
boquinone (Figure 6.8a) was obtained as red needles having a molecular weight of 284.
The compound, having an MIC of 1 µg/mL, showed antibacterial activity against Bacillus
thuringiensis, S. aureus, and Enterococcus faecalis. The compound was tested for in vitro cyto-
toxic activities against three human tumor cell lines, namely SF-268 (human glioma cell
line), MCF-7 (human breast adenocarcinoma cell line), and NCI-H460 (human non–small
cell lung cancer cell line), and the IC50 (µM) values were estimated to be 0.022, 0.015,
and 0.080, respectively. The compound pseudonocardian A (Figure 6.8b) was obtained
as a white solid having the molecular formula C18H18N2O5 with a molecular mass of
343.1305 g/mol. The compound, having an MIC of 4 µg/mL, showed antibacterial activity
against B. thuringiensis, S. aureus, and E. faecalis. The estimated IC50 (µM) against SF-268,
MCF-7, and NCI-H460 were 0.028, 0.027, and 0.209, respectively.
Similarly, pseudonocardian B (Figure 6.8c) was obtained as a white solid having the
molecular formula C19H20N2O5, with a molecular mass of 357.1456. The compound, having
an MIC of 2 µg/mL, showed antibacterial activity against B. thuringiensis, S. aureus, and
E. faecalis. The estimated IC50 (µM) against SF-268, MCF-7, and NCI-H460 were 0.022, 0.021,
and 0.177, respectively. The compound pseudonocardian C (Figure 6.8d) was obtained as
a red-brown powder having the molecular formula C21H24N2O8, with a molecular mass of
433.1609 g/mol. The compound, having an MIC of >128 µg/mL, showed antibacterial activ-
ity against B. thuringiensis, S. aureus, and E. faecalis. The estimated IC50 (µM) against SF-268,
MCF-7, and NCI-H460 were 6.70, 8.02, and 43.28, respectively (Li et al., 2011). Furthermore,
Tables 6.5 and 6.6 list some of the important antimicrobial agents isolated from the marine
ecosystem. In our point of view, the marine ecosystem hinders many novel antimicrobial
agents that can be used against multidrug-resistant pathogens. Thus, in the future, fur-
ther research in finding novel compounds is required in order to compete with harmful
pathogens.
6.3 Future developments

Since antiquity, humans have been fighting with pathogenic microorganisms. Many anti-
microbial agents were discovered, but still we are unable to eradicate them. Since the
discovery of penicillin, many antimicrobial agents were discovered and we think we are
ready for the battle, but we always forget that the microorganisms are also developing
exponentially, though very tiny but still giving equal competition. So, we should not stop
and keep discovering. Actinomycetes are the largest hub for antimicrobial agents. Though
many antimicrobial agents from actinomycetes were reported, it is just the “tip of the ice-
berg” that has been explored. Various unique and virgin areas should be explored, and
different isolation techniques and isolation media should be developed in order to iso-
late novel actinomycetes having an extraordinary antimicrobial agent to triumph against
pathogenic microorganisms.
Table 6.5 List of compounds from marine actinomycetes and their applications
Compound
Compound name/origin applications References
Diazepinomicin from Micromonospora sp. Antibacterial, Charan et al.
HO anticancer, and (2004)
OH anti-inflammatory
H
N
HO N
O
CH3 CH3
H 3C
CH3
Sporolides A and B from Salinispora tropica Antibacterial Buchanan et al.

HO H (2005)
1
O R2
O 13
10΄
R1 H
1΄ H3C OH
O 5
H 4΄
H3CO H
9΄
H O
O OH
O 8΄ 8
HO H
OH
(a) R1 = CI R2 = H
(b) R1 = H R2 = CI
Ammosamide D from Streptomyces variabilis Cytotoxic activity Pan et al.

O NH2 (2012)
N CI
H2N
O
O
O NH
Lajollamycin from Streptomyces nodosus Antibacterial Manam et al.

(2005)
O O
N+ OH O O
O–
N NCH3
H OH
O
Helquinoline from Janibacter limosus Antibacterial Asolkar et al.
CH3 (2004)
O
N CH3
H
O OH
6 4
13 12 3
7 5
B A
O 10
17 16 O N 14
11
N O
O 9 H 1
6 4 O
13
5
12
3
HO
7 O
B A O N N O OH 1΄
N 14
10
11 N1 O HO OH
O 9 18 OH
H 19
O 15 R OH
20
(a) (b) R = CH3 (d)
20 21
(c) R = CH2CH3
Figure 6.8 Structure of (a) deoxynyboquinone, (b) pseudonocardian A, (c) pseudonocardian B, and
(d) pseudonocardian C.
Table 6.6 List of compounds from marine actinomycetes and their applications
Origin Compound name Compound applications References
Streptomyces griseus Frigocyclinone Antibacterial Bruntner et al. (2005)
Streptomyces sp. Glaciapyrroles Antibacterial Macherla et al. (2005)
Streptomyces sp. Resistoflavin Antibacterial and antioxidative Kock et al. (2005)
methyl ether
Verrucosispora sp. Proximicins Antibacterial and anticancer Fiedler et al. (2008)
Streptomyces sp. Caboxamycin Antibacterial and anticancer Hohmann et al. (2009)
Streptomyces sp. 2-Allyloxyphenol Antimicrobial, food preservative, Arumugam et al.
and oral disinfectant (2010)
Salinispora arenicola Arenimycin Antibacterial and anticancer Asolkar et al. (2010)
Streptomyces sp. Tirandamycins Antibacterial Carlson et al. (2009)
Acknowledgments
This research was supported by the Key Project of International Cooperation of Ministry
of Science & Technology (MOST) (No. 2013DFA31980) and the Key Project of Yunnan
Provincial Natural Science Foundation (2013FA004). W.-J. Li was also supported by the
“Hundred Talents Program” of the Chinese Academy of Sciences and Guangdong Province
Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).
References
Araujo, J.M., Silva, A.C., and Azevedo, J.L. 2000. Isolation of endophytic actinomycetes from roots
and leaves of maize (Zea mays L.). Braz. Arch. Biol. Technol. 43, 447–451.
Arumugam, M., Mitra, A., Jaisankar, P., Dasgupta, S., Sen, T., Gachhui, R., and Mukherjee, J. 2010.
Isolation of an unusual metabolite 2-allyloxyphenol from a marine actinobacterium, its bio-
logical activities and applications. Appl. Microbiol. Biotechnol. 86, 109–117.
Asolkar, R.N., Kirkland, T.N., Jensen, P.R., and Fenical, W. 2010. Arenimycin, an antibiotic effective
against rifampin- and methicillin-resistant Staphylococcus aureus from the marine actinomy-
cete Salinispora arenicola. J. Antibiot. 63, 37–39.
Asolkar, R.N., Schroeder, D., Heckmann, R., Lang, S., Wagner-Doebler, I., and Laatsch, H. 2004.
Helquinoline, a new tetrahydroquinoline antibiotic from Janibacter limosus Hel 1+. J. Antibiot.
57, 17–23.
Baltz, R.H. 2005. Antibiotic discovery from actinomycetes: Will a renaissance follow the decline and
fall? SIM News 55, 186–196,
Bérdy, J. 2012. Thoughts and facts about antibiotics: Where we are now and where we are heading.
J. Antibiot. 65, 385–395.
Bonkowski, M. and Roy, J. 2005. Soil microbial diversity and soil functioning affect competition
among grasses in experimental microcosms. Oecologia 143, 232–240.
Bruntner, C., Binder, T., Pathom-aree, W., Goodfellow, M., Bull, A.T., Potterat, O., and Fiedler, H.P.
2005. Frigocyclinone, a novel angucyclinone antibiotic produced by a Streptomyces griseus
strain from Antarctica. J. Antibiot. 58, 346–349.
Buchanan, G.O., Williams, P.G., Feling, R.H., Kauffman, C.A., Jensen, P.R., and Fenical, W. 2005.
Sporolides A and B: Structurally unprecedented halogenated macrolides from the marine acti-
nomycete Salinispora tropica. Org. Lett. 7, 2731–2734.
Cao, L., Qiu, Z., You, J., Tan, H., and Zhou, S. 2005. Isolation and characterization of endophytic
Streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots.
FEMS Microbiol. Lett. 247, 147–152.
Carlson, J.C., Li, S., Burr, D.A., and Sherman, D.H. 2009. Isolation and characterization of tirandamy-
cins from a marine-derived Streptomyces sp. J. Nat. Prod. 72, 2076–2079.
Charan, R.D., Schlingmann, G., Janso, J., Bernan, V., Feng, X., and Carter, G.T. 2004. Diazepinomicin,
a new antimicrobial alkaloid from marine Micromonospora sp. J. Nat. Prod. 67, 1431–1433.
CLSI. 2012. Performing Standards for Antimicrobial Susceptibility Testing: Twenty-second Informational
Supplement. CLSI document M100-S22. Wayne, PA: Clinical and Laboratory Standards Institute.
Coombs, J.T., Michelsen, P.P., and Franco, M.M. 2004. Evaluation of endophytic actinobacteria as
antagonists of Gaeumannomyces graminis var. tritici in wheat. Biol. Control 29, 359–366.
Davidson, R.N., den Boer, M., and Ritmeijer, K. 2009. Paromomycin. Trans. R. Soc. Trop. Med. Hyg.
103, 653–660.
Deene, M. and Lingappa, K. 2013. Antibacterial activity of silver nanoparticles against methicillin-
resistant Staphylococcus aureus synthesized using model Streptomyces sp. pigment by photo-
irradiation method. J. Pharma. Res. 6, 255–260.
Dhanya, N.N. and Padmavathy, S. 2014. Impact of endophytic microorganisms on plants, environ-
ment and humans. Sci. World J., 2011, 1–11.
Dietera, A., Hamm, A., Fiedler, H.P., Goodfellow, M., Müller, W.E., Brun, R., and Bringmann, G. 2003.
Pyrocoll, an antibiotic, antiparasitic and antitumor compound produced by a novel alkaliphi-
lic Streptomyces strain. J. Antibiotics 56, 639–646.
Ding, Z.G., Li, M.G., Zhao, J.Y., Ren, J., Huang, R., Xie, M.J., and Wen, M.L. 2010. Naphthospironone
A: An unprecedented and highly functionalized polycyclic metabolite from an alkaline mine
waste extremophile. Chem. Eur. J. 16, 3902–3905.
Elson, M.K., Schisler, D.A., and Bothast, R.J. 1997. Selection of microorganisms for biological control
of silver scurf (Helminthosporium solani) of potato tubers. Plant Dis. 81, 647–652.
El-Tarabily, K.A. 2003. An endophytic chitinase-producing isolate of Actinoplanes missouriensis, with
potential for biological control of root rot of lupine caused by Plectosporium tabacinum. Aus. J. Bot.
51, 257–266.
El-Tarabily, K.A. 2006. Rhizosphere-competent isolates of streptomycete and non-streptomycete
actinomycetes capable of producing cell-wall degrading enzymes to control Pythium aphanider-
matum damping-off disease of cucumber. Can. J. Bot. 84, 211–222.
El-Tarabily, K.A., Hardy, G.E., Sivasithamparam, K., Hussein, A.M., and Kurtböke, D. 1997. The
potential for the biological control of cavity spot disease of carrots caused by Pythium colora-
tum by streptomycete and non-streptomycete actinomycetes in Western Australia. New Phytol.
137, 495–507.
Fiedler, H.P., Bruntner, C., Riedlinger, J., Bull, A.T., Knutsen, G., Goodfellow, M., and Sussmuth, R.D.
2008. Proximicin A, B and C, novel aminofuran antibiotic and anticancer compounds isolated
from marine strains of the actinomycete Verrucosispora. J. Antibiot. 61, 158–163.
Fleming, A. 1929. On the antibacterial action of cultures of a penicillium, with special reference to
their use in the isolation of B. influenzae. Br. J. Exp. Pathol. 10(3), 226.
Franco-Correa, M., Quintana, A., Duque, C., Suarez, C., Rodríguez, M.X., and Barea, J.M. 2010.
Evaluation of actinomycete strains for key traits related with plant growth promotion and
mycorrhiza helping activities. Appl. Soil Ecol. 45, 209–217.
Garrod, L.P., Lambert, H.P., and O’Grady, F. 1981. Antibiotic and Chemotherapy. Churchill Livingstone,
New York, p. 131.
Giguere, S., Prescott, J.F., Baggot D., Walker R.D., and Dowling, P.M. 2007. Antimicrobial Therapy in
Veterinary Medicine, 4th edn. Blackwell Publishing, Ames, IA.
Haefner, B. 2003. Drugs from the deep: Marine natural products as drug candidates. Drug. Discov.
Today 8, 536–544.
Helmke, E. and Weyland, H. 1984. Rhodococcus marinonascens sp. Nov., an actinomycete from the sea.
Int. J. Syst. Bacteriol. 34, 127–138.
Hohmann, C., Schneider, K., Bruntner, C., Irran, E., Nicholson, G., Bull, A.T., and Fiedler, H.P. 2009.
Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain
Streptomyces sp. NTK 937. J. Antibiot. 62, 99–104.
Horikoshi, K. 1999. Alkaliphiles: Some applications of their products for biotechnology. Microbiol.
Mol. Biol. Rev. 63, 735–750.
Huang, S.X., Zhao, L.X., Tang, S.K., Jiang, C.L., Duan, Y., and Shen, B. 2009. Erythronolides H
and I, new erythromycin congeners from a new halophilic actinomycete Actinopolyspora sp.
YIM90600. Org. Lett. 11, 1353–1356.
Inahashi, Y., Iwatsuki, M., Ishiyama, A., Namatame, M., Nishihara-Tsukashima, A., Matsumoto, A.,
and Shiomi, K. 2011. Spoxazomicins A-C, novel antitrypanosomal alkaloids produced by an
endophytic actinomycete, Streptosporangium oxazolinicum K07–0450T. J. Antibiot. 64, 303–307.
Johnson, K.G., Lanthier, P.H., and Gochnauer, M.B. 1986. Studies of two strains of Actinopolyspora
halophila, an extremely halophilic actinomycete. Arch. Microbiol. 143, 370–378.
Kingston, W. 2008. Irish contributions to the origins of antibiotics. Ir. J. Med. Sci. 177, 87–92.
Kiple, K.F. (ed.). 1993. Cambridge World History of Human Disease. Cambridge University Press,
Cambridge, U.K.
Kirst, H.A., Michel, K.H., Mynderase, J.S., Chio, E.H., Yao, R.C., Nakasukasa, W.M., and Deeter, J.B.
1992. Discovery, isolation, and structure elucidation of a family of structurally unique, fermen-
tation derived tetracyclic macrolides. In: Baker, D.R., Fenyes, J.G., Steffens, J.J. (eds.) Synthesis
and Chemistry of Agrochemicals III. American Chemical Society, Washington, DC, pp. 214–225.
Kitouni, M., Boudemagh, A., Oulmi, L., Reghioua, S., Boughachiche, F., Zerizer, H., and Boiron, P.
2005. Isolation of actinomycetes producing bioactive substances from water, soil and tree bark
samples of the north–east of Algeria. J. Mycol. Med. 15, 45–51.
Kock, I., Maskey, R.P., Biabani, M.A., Helmke, E., and Laatsch, H. 2005. 1-Hydroxy-1-norresistomycin
and resistoflavin methyl ether: New antibiotics from marine-derived Streptomycetes. J. Antibiot.
58, 530–534.
Lee, S.O., Choi, G.J., Choi, Y.H., Jang, K.S., Park, D.J., Kim, C.J., and Kim, J.C. 2008. Isolation and
characterization of endophytic actinomycetes from Chinese cabbage roots as antagonists to
Plasmodiophora brassicae. J. Microbiol. Biotechnol. 18, 1741–1746.
Levine, D.P. 2006. Vancomycin: A history. Clin. Infect. Dis. 42(Suppl. 1), S5–S12.
Li, S., Tian, X., Niu, S., Zhang, W., Chen, Y., Zhang, H., and Zhang, C. 2011. Pseudonocardians A-C,
new diazaanthraquinone derivatives from a deep-sea actinomycete Pseudonocardia sp. SCSIO
01299. Mar. Drugs 9, 1428–1439.
Li, W.J., Chen, H.H., Zhang, Y.Q., Schumann, P., Stackebrandt, E., Xu, L.H., and Jiang, C.L. 2004a.
Nesterenkonia halotolerans sp. nov. and Nesterenkonia xinjiangensis sp. nov., actinobacteria from
saline soils in the west of China. Int. J. Syst. Evol. Microbiol.54, 837–841.
Li, W.J., Kroppenstedt, R.M., Wang, D., Tang, S.K., Lee, J.C., Park, D.J., and Jiang, C.L. 2006. Five novel
species of the genus Nocardiopsis isolated from hypersaline soils and emended description of
Nocardiopsis salina. Int. J. Syst. Evol. Microbiol. 56, 1089–1096.
Li, W.J., Park, D.J., Tang, S.K., Wang, D., Lee, J.C., Xu, L.H., and Jiang, C.L. 2004b. Nocardiopsis salina
sp. nov., a novel halophilic actinomycete isolated from saline soil in China. Int. J. Syst. Evol.
Microbiol. 54, 1805–1809.
Li, W.J., Xu, P., Tang, S.K., Xu, L.H., Kroppenstedt, R.M., Stackebrandt, E., and Jiang, C.L. 2003a.
Prauserella halophila sp. nov. and Prauserella alba sp. nov., moderately halophilic actinomycetes
from saline soil. Int. J. Syst. Evol. Microbiol. 53, 1545–1549.
Li, W.J., Xu, P., Zhang, L.P., Tang, S.K., Cui, X.L., Mao, P.H., and Jiang, C.L. 2003b. Streptomonospora
alba sp. nov., a novel halophilic actinomycete, and emended description of the genus
Streptomonospora. Int. J. Syst. Evol. Microbiol. 53, 1421–1425.
Lu, C.H. and Shen, Y.M. 2003. A new macrolide antibiotics with antitumor activity produced by
Streptomyces sp. CS, a commensal microbe of Maytenus hookeri. J. Antibiot. 56, 415–418.
Macherla, V.R., Liu, J., Bellows, C., Teisan, S., Lam, K.S., and Potts, B.C.M. 2005. Glaciapyrroles A, B
and C, pyrrolosesquiterpenes from a Streptomyces sp. isolated from an Alaskan marine sedi-
ment. J. Nat. Prod. 68, 780–783.
Manam, R.R., Teisan, S., White, D.J., Nicholson, B., Grodberg, J., Neuteboom, S.T.C., Lam, K.S., Mosca,
D.A., Lloyd, G.K., and Potts, B.C. 2005. Lajollamycin, a nitro-tetraene spiro-b-lactone-c-lactam
antibiotic from the marine actinomycete Streptomyces nodosus. J. Nat. Prod. 68, 240–243.
Maskey, R.P., Helmke, E., and Laastsch, H. 2003. Himalomycin A and B: Isolation and structure elu-
cidation of new fridamycin type antibiotics from a marine Streptomyces isolate. J. Antibiot. 56,
942–949.
McDonald, M., Mavrodi, D.V., Thomashow, L.S., and Floss, H.G. 2001. Phenazine biosynthesis in
Pseudomonas fluorescens: Branchpoint from the primary shikimate biosynthetic pathway and
role of phenazine-1,6-dicarboxylic acid. J. Am. Chem. Soc. 123, 9459–9460.
McGuire, J.M., Bunch, R.L., Anderson, R.C., Boaz, H.E., Flynn, E.H., Powell, H.M., and Smith, J.W.
1952. Ilotycin, a new antibiotic. Antibiot. Chemother. 2, 281–283.
Michael, S. and Plotkin, M.J. 2003. The Killers Within: The Deadly Rise of Drug-Resistant Bacteria. New
York: Little Brown & Company. ISBN 978–0–316–73566–7.
Pan, E., Jamison, M., Yousufuddin, M., and MacMillan, J.B. 2012. Ammosamide D, an oxidatively
ring opened ammosamide analog from a marine-derived Streptomyces variabilis. Org. Lett. 14,
2390–2393.
Pullen, C., Schmitz, P., Meurer, K., Bamberg, D.D.V., Lohmann, S., Franca, S.D.C., and Leistner,
E. 2002. New and bioactive compounds from Streptomyces strains residing in the wood of
Celastraceae. Planta 216, 162–167.
Rath, J.P., Kinast, S., and Maier, M.E. 2005. Synthesis of the full functionalized core structure of the
antibiotic abyssomicin C. Org. Lett. 7, 3089–3092.
Riedlinger, J., Reicke, A., Zähner, H.A.N.S., Krismer, B., Bull, A.T., Maldonado, L.A., and Fiedler,
H.P. 2004. Abyssomicins, inhibitors of the para-aminobenzoic acid pathway produced by the
marine Verrucosispora strain AB-18-032. J. Antibiot. 57, 271–279.
Sarbani, P. 2006. A journey across the sequential development of macrolides and ketolides related to
erythromycin. Tetrahedron 62, 3171–3200.
Sato, M., Beppu, T., and Arima, K. 1980. Properties and structure of a novel peptide antibiotic no.
1970. Agric. Biol. Chem. 44, 3037–3040.
Schatz, A., Bugie, E., and Waksman, S.E. 1944. Streptomycin, a substance exhibiting antibiotic activity
against gram-positive and gram negative bacteria. Proc. Soc. Exp. Biol. Med. 55, 65–69.
Shimizu, M., Nakagawa, Y., Yukio, S., Furumai, T., Igarashi, Y., Onaka, H., and Kunoh, H. 2000.
Studies on endophytic actinomycetes (I) Streptomyces sp. Isolated from Rhododendron and its
antifungal activity. J. Gen. Plant Pathol. 66, 360–366.
Sivonen, K. and Jones, G. 1999. Cyanobacterial toxins. In: Chorus, I. and Bartran, J. (eds.) Toxic
Cyanobacteria in Water: A Guide to Their Public Health Consequences. Monitoring and Management.
E&FN Spon., London, U.K., pp. 41–111.
Smith, G.E. 1957. Inhibition of Fusarium oxysporum f.sp. lycopersici by a species of Micromonospora
isolated from tomato. Phytopathology 47, 429–432.
Somdee, T., Sumalai, N., and Somdee, A. 2013. A novel actinomycete Streptomyces aurantiogriseus
with algicidal activity against the toxic cyanobacterium Microcystis aeruginosa. J. Appl. Phycol.
25, 1587–1594.
Spizek, J. and Rezanka, T. 2004. Lincomycin, cultivation of producing strains and biosynthesis. Appl.
Microbiol. Biotechnol. 63, 510–519.
Steffan, R.J., Goskoyr, J., Bej, A.K., and Atlas, R.M. 1988. Recovery of DNA from soil and sediments.
Appl. Environ. Microbiol. 54, 2908–2915.
Sujatha, P., Bapi Raju, K.V.V.S.N., and Ramana, T. 2005. Studies on a new marine streptomycete BT-408
producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus
aureus. Microbiol. Res. 160, 119–126.
Syed, D.G., Tang, S.K., Cai, M., Zhi, X.Y., Agasar, D., Lee, J.C., and Li, W.J. 2008. Saccharomonospora saliph-
ila sp. nov., a halophilic actinomycete from an Indian soil. Int. J. Syst. Evol. Microbiol. 58, 570–573.
Taechowisan, T., Lu, C., Shen, Y., and Lumyong, S. 2005. Secondary metabolites from endophytic
Streptomyces aureofaciens CMUAc130 and their antifungal activity. Microbiology 151, 1691–1695.
Taechowisan, T. and Lumyong, S. 2003. Activity of endophytic actinomycetes from roots of Zingiber
officinale and Alpinia galanga against phytopathogenic fungi, Ann. Microbiol. 53, 291–298.
Tang, S.K., Wang, Y., Guan, T.W., Lee, J.C., Kim, C.J., and Li, W.J. 2010. Amycolatopsis halophila sp. nov.
a halophilic actinomycete isolated from a salt lake. Int. J. Syst. Evol. Microbiol. 60, 1073–1078.
Tang, S.K., Wang, Y., Klenk, H.P., Shi, R., Lou, K., Zhang, Y.J., and Li, W.J. 2011. Actinopolyspora alba
sp. nov. and Actinopolyspora erythraea sp. nov., isolated from a salt field, and reclassification of
Actinopolyspora iraqiensis as a heterotypic synonym of Saccharomonospora halophile. Int. J. Syst.
Evol. Microbiol. 61, 1693–1698.
Tian, S.Z., Pu, X., Luo, G., Zhao, L.X., Xu, L.H., Li, W.J., and Luo Y. 2013. Isolation and characterization
of new p-terphenyls with antifungal, antibacterial, and antioxidant activities from a halophilic
actinomycete Nocardiopsis gilva YIM 90087. J. Agric. Food. Chem. 27, 3006–3012.
Tian, X.P., Tang, S.K., Dong, J.D., Zhang, Y.Q., Xu, L.H., Zhang, S., and Li, W.J. 2009. Marinactinospora
thermotolerans gen. nov., sp. nov., a marine actinomycete isolated from a sediment in the north-
ern South China Sea. Int. J. Syst. Evol. Microbiol. 59, 948–952.
Tian, X.P., Xu, Y., Zhang, J., Li, J., Chen, Z., Kim, C.J., and Zhang, S. 2012. Streptomyces oceani sp. nov., a
new obligate marine actinomycete isolated from a deep-sea sample of seep authigenic carbon-
ate nodule in South China Sea. Antonie Van Leeuwenhoek 102, 335–343.
Tsujibo, H., Sato, T., Inui, M., Yamamoto, H., and Inamori, Y. 1988. Intracellular accumulation of phen-
azine antibiotics production by an alkalophilic actinomycete. Agric. Biol. Chem. 52, 301–306.
Van der Heijden, M.G.A., Bardgett, R.D., and van Straalen, N.M. 2008. The unseen majority: Soil
microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett. 11,
296–310.
Verma, V.C., Gond, S.K., Kumar, A., Mishra, A., Kharwar, R.N., and Gange, A.C. 2009. Endophytic
actinomycetes from Azadirachta indica A. Juss.: Isolation, diversity, and anti-microbial activity.
Microb. Ecol. 57, 749–756.
Wagner, R.L., Hochstein, F.A., Murai, K., Messina, N., and Regna, P.P. 1953. Magnamycin, a new
antibiotic. J. Am. Chem. Soc. 75, 4684–4687.
Waksman, S.A. and Starkey, R.L. 1931. The Soil and the Microbe. An Introduction to the Study of the
Microscopic Population of the Soil and Its Role in Soil Processes and Plant Growth. John Wiley &
Sons, Inc., New York.
Wang, X., Tabudravu, J., Jaspars, M., and Deng, H. 2013a. Tianchimycins, A-B, 16-membered macro-
lides from the rare actinomycete Saccharothrix xinjiangensis. Tetrahedron 69, 6060–6064.
Wang, Z., Fu, P., Liu, P., Wang, P., Hou, J., Li, W., and Zhu, W. 2013b. New pyran-2-ones from alka-
lophilic actinomycete, Nocardiopsis alkaliphila sp. nov. YIM-80379. Chem. Biodivers. 10, 281–287.
Weinstein, M.J., Wagman, G.H, Oden, E.M., and Marquez, J.A. 1967. Biological activity of the antibi-
otic components of the gentamicin complex. J. Bacteriol. 94, 789–790.
Woodworth, J.R., Nyhart, E.H., Brier, G.L., Wolny, J.D., and Black, H.R. 1992. Single-dose pharma-
cokinetics and antibacterial activity of daptomycin, a new lipopeptide antibiotic, in healthy
volunteers. Antimicrob. Agents. Chemother. 36, 318–325.
Zhao, K., Penttinen, P., Guan, T., Xiao, J., Chen, Q., Xu, J., and Strobel, G.A. 2011a. The diversity and
anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in Panxi
Plateau, China. Curr. Microbiol. 62, 182–190.
Zhao, L.X., Huang, S.X., Tang, S.K., Jiang, C.L., Duan, Y., Beutler, J.A., and Shen, B. 2011b.
Actinopolysporins A-C and tubercidin as a Pdcd4 stabilizer from the halophilic actinomycete
Actinopolyspora erythraea YIM 90600. J. Nat. Prod. 74, 1990–1995.
chapter seven
Actinobacteria
A predominant source
of antimicrobial compounds
Ramasamy Vijayakumar, Govindaraj Vaijayanthi,
Annamalai Panneerselvam, and Nooruddin Thajuddin
Contents
7.1 Antibiotics: An introduction............................................................................................. 118
7.2 Origin of antibiotics............................................................................................................ 119
7.3 Types of antibiotics............................................................................................................. 120
7.3.1 Antibacterial compounds...................................................................................... 120
7.3.2 Antifungal compounds.......................................................................................... 120
7.3.3 Antiviral compounds............................................................................................. 120
7.3.4 Antiparasitic compounds...................................................................................... 121
7.3.5 Anticancer compounds.......................................................................................... 121
7.4 Sources of antibiotics.......................................................................................................... 122
7.4.1 Plants........................................................................................................................ 122
7.4.2 Animals.................................................................................................................... 124
7.4.3 Bacteria..................................................................................................................... 124
7.4.4 Fungi......................................................................................................................... 125
7.4.5 Actinobacteria......................................................................................................... 126
7.4.6 Other sources........................................................................................................... 126
7.5 Isolation of actinobacteria.................................................................................................. 127
7.5.1 Selection of samples for the isolation of actinobacteria.................................... 127
7.5.2 Pretreatment of the samples.................................................................................. 128
7.5.2.1 Method 1 (dry/stamp)............................................................................. 128
7.5.2.2 Method 2 (sterile dry/scrape)................................................................. 128
7.5.2.3 Method 3 (dry/dilute).............................................................................. 128
7.5.2.4 Method 4 (dilute/heat)............................................................................ 129
7.5.2.5 Method 5 (dilute/heat/2)........................................................................ 129
7.5.2.6 Method 6 (dry/stamp + dilute/heat)..................................................... 129
7.5.2.7 Method 7 (freeze/dilute)......................................................................... 129
7.5.2.8 Method 8 (freeze/dilute/2)..................................................................... 129
7.5.3 Isolation and culture conditions........................................................................... 129
7.6 Identification of actinobacteria......................................................................................... 131
7.6.1 Phenotypic characterization.................................................................................. 131
7.6.1.1 Microscopic characterization................................................................. 131
7.6.1.2 Colony morphological/cultural characterization................................ 131
117
7.6.1.3 Biochemical characterization................................................................. 131

7.6.1.4 Physiological characterization............................................................... 131
7.6.2 Molecular characterization.................................................................................... 133
7.7 Screening of antibiotic production................................................................................... 133
7.7.1 Primary screening: Cross streak plate technique.............................................. 133
7.7.2 Secondary screening: Well diffusion assay........................................................ 133
7.8 Extraction and separation of antimicrobial compounds.............................................. 134
7.8.1 Thin-layer chromatography.................................................................................. 134
7.8.2 High-performance liquid chromatography........................................................ 134
7.9 Characterization and identification of antibiotics.......................................................... 135
7.9.1 pH stability test....................................................................................................... 135
7.9.2 Temperature stability test...................................................................................... 135
7.9.3 Melting point........................................................................................................... 135
7.9.4 Quality test.............................................................................................................. 135
7.10 Prediction of antibiotic structures.................................................................................... 135
7.10.1 Ultraviolet spectrum.............................................................................................. 135
7.10.2 Fourier transform infrared spectrum.................................................................. 136
7.10.3 Mass spectrum........................................................................................................ 136
7.10.4 Nuclear magnetic resonance spectrum............................................................... 136
7.11 Conclusion........................................................................................................................... 136
References...................................................................................................................................... 137
7.1 Antibiotics: An introduction

An antibiotic is an agent that either kills or inhibits the growth of a microorganism
(Dorlands, 2010). The term antibiotic was first used in 1942 by Selman Waksman and his
collaborators and they described it as any substance produced by a microorganism that is
antagonistic to the growth of other microorganisms (Waksman, 1947). Most of the recent
antibacterials are semisynthetic and are modifications of various natural compounds (Von
Nussbaum et al., 2006). These include the beta-lactam antibiotics, which include the peni-
cillins (produced by fungi in the genus Penicillium), cephalosporins, and carbapenems.
Compounds that are still isolated from living organisms are the aminoglycosides, whereas
other antibacterials, for example, the sulfonamides, quinolones, and oxazolidinones, are
produced solely by chemical synthesis. In accordance with this, many antimicrobial com-
pounds are classified on the basis of chemical/biosynthetic origin into natural, semisyn-
thetic, and synthetic. Another classification system is based on biological activity; in this
classification, antibacterials are divided into two broad groups according to their biologi-
cal effect on microorganisms. Bactericidal agents kill bacteria, and bacteriostatic agents
slow down or stall bacterial growth.
Antibiotics are produced industrially by a process of fermentation, where the source
microorganism is grown in large containers (100,000–150,000 L or more) containing a liq-
uid growth medium. Oxygen concentration, temperature, pH, and nutrient levels must be
optimal and are closely monitored and adjusted if necessary. As antibiotics are secondary
metabolites, the population size must be controlled very carefully to ensure that maximum
yield is obtained before the cells die. Once the process is complete, the antibiotic must be
extracted and purified to a crystalline product. This is simpler to achieve if the antibiotic
is soluble in organic solvent. Otherwise, it must first be removed by ion exchange, adsorp-
tion, or chemical precipitation.
Chapter seven: Actinobacteria 119
Marine organisms represent a promising source for natural antibiotics of the future
due to the incredible diversity of chemical compounds that were isolated particularly
from the marine microorganisms. The oceans cover almost 70% of the earth’s surface
and over 90% of volume of its crust (Aghighi et al., 2005). From the entire microbial flora,
actinobacteria from the marine environment have been traditionally a rich source for
biologically active metabolites. Although heavily studied over the past three decades,
actinobacteria continue to prove themselves as reliable sources of novel antimicrobial
compounds. Among the well-characterized pharmaceutically relevant microorganisms,
actinobacteria remain major sources of novel, therapeutically relevant natural products
(Arasu et al., 2009). The majority of these compounds demonstrate one or more bioactiv-
ities, many of which developed into antibiotics for treatment of a wide range of diseases
in human, veterinary, and agriculture sectors (Atalan et al., 2000).
7.2 Origin of antibiotics

After accidental discovery of penicillin (first antibiotic) by Sir Alexander Fleming from
Penicillium notatum, a fungus in 1929 that inhibited the growth of Staphylococcus aureus, a
dangerous/major microbial killer from the early twentieth century to our days, has been
a fascinating, exciting, continuously changing, and developing adventure. Following the
discovery of penicillin is the second most important antibiotic, streptomycin, which was
discovered by Waksman from Streptomyces griseus, an actinobacterium. Interests toward
the field were generally increasing, although sometimes declining; the interest and the
whole story show some cyclic features with successes and failures and evolved around
changing clinical needs and new enabling technology.
Generally, the microbial natural products appear as the most promising source of the
future antibiotics that society is expecting (Fernando, 2006). Antibiotics are produced by
bacteria, fungi, actinobacteria, algae, lichens, and green plants. Since the isolation of actino-
mycin and streptomycin, the actinobacteria have received tremendous attention (Waksman
and Schatz, 1946). Members of streptomycetes are a rich source of bioactive compounds,
notably antibiotics, enzymes, enzyme inhibitors, and pharmacologically active agents, and
about 75% of the known commercially and medically useful antibiotics are produced by
streptomycetes (Sujatha et al., 2005). Beijerinck (1900) and Eriko (2002) established that acti-
nobacteria occur in great abundance in the soil and have a great role in the management of
microbial stability with the production of antibiotic substances. Actinobacteria live in soil
and decompose organic matter such as cellulose, hemicellulose, pectin, and chitin. In the
drug discovery, these microorganisms are widely recognized for their ability to produce
secondary metabolites with commercially viable antibiotic activity. Actinobacteria were
first recognized as a common and important group of soil microorganisms (Waksman
et al., 1941; Berger et al., 1949). Streptomycin, the first antibiotic for tuberculosis, was derived
from the largest genus of actinobacteria, Streptomyces; erythromycin and tetracycline are
two other common medicines derived originally from these microorganisms.
Most streptomycetes and other actinobacteria produce a diverse array of antibiotics,
including aminoglycosides, macrolides, peptides, polyenes, polyether, and tetracyclines. In
searching for new antibiotics, over 1000 different bacteria (including actinobacteria), fungi, and
algae have been investigated. To prevent exponential emergence of microorganisms becoming
resistant to the clinically available antibiotics already marketed, the periodic replacement of
the existing antibiotics is necessary. Thus, the microbiologists/pharmacologists have regularly
screened/searched the novel antibiotics particularly from microorganisms.
7.3 Types of antibiotics

On the basis of inhibitory action against different types of pathogenic microorganisms, the
antibiotics are grouped as antibacterial, antifungal, antiparasitic, antiviral, anticancer, etc.
They also classified as broad-spectrum and narrow-spectrum antibiotics as they p ossess
inhibitory action on several pathogenic microorganisms as well as single pathogens,
respectively.
7.3.1 Antibacterial compounds

The emergence of multidrug-resistant bacteria is a phenomenon of concern to the clini-
cian and the pharmaceutical industry, as it is the major cause of failure in the treatment
of infectious diseases. The most common resistance mechanism of pathogenic bacteria to
the antibiotics, namely, aminoglycoside, beta-lactam (penicillins and cephalosporins), and
chloramphenicol types, involves the enzyme inactivation of the antibiotic by hydrolysis
or by formation of inactive derivatives. Such resistance determinants most probably were
acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera,
including antibiotic-producing organisms. Certain antibiotics produced by actinobacteria
have a high activity upon bacteria and fungi. Some antibiotics are used by the geneticists
to select a mutant bacteria. Others are used by the biochemists as specific inhibitors of
metabolic reaction, such as chloramphenicol (inhibition of protein synthesis), antimycin A
(inhibition of cytochrome oxidase), and actinomycin (activity upon nucleic acids). Among
the various uses, the treatment of various infectious diseases of human and animals is the
remarkable role of antibiotics (Waksman, 1943).
7.3.2 Antifungal compounds

Antifungal compounds have been overshadowed by antibacterials in research interest
and applications due to the greater impact of bacterial infections on health. Resistance to
antibacterial drugs and the resultant clinical impact are of widespread concern regarding
public health (Parks and Casey, 1996). However, resistance by pathogenic fungal infections
to drug treatment has become more common in the past two decades. Certain antibiotics
produced by actinobacteria have a high activity upon fungi. Some of them like nystatin,
candicidin, candidin, trichomycin, hamycin, and amphotericin are polyenes in nature.
Some of these antibiotics, notably actidione, and some of the polyenes have found practical
applications.
7.3.3 Antiviral compounds

The term “antiviral agents” has been defined in very broad terms as substances other than
a virus or virus-containing vaccine or specific antibody that can produce either a protective
or a therapeutic effect to the clear detectable advantage of the virus-infected host (Swallow,
1977). Unlike the search for antibiotics, which took root from the discovery of penicillin
during the late 1930s, the search for antiviral agents began in the 1950s (Kinchington et al.,
1995) but had a breakthrough in 1964 (Kucera and Hermann, 1965). Early success in this
direction included the use of methisazone for the prophylaxis of small pox and the use of
idoxuridine for the treatment of herpes keratitis. Two major obstacles to the development
and use of effective antiviral chemotherapy are the close relationship that exists between
the multiplying virus and the host cell and that viral diseases can only be diagnosed and
Table 7.1 Chemical class and action of antiviral compounds

Antiviral compound Chemical class Main action
Phytolacca American protein Glycoprotein (amphoteric) Polio and influenza
Antiviral factor Interferon 1 Antiviral
Lycoricidinol N2-containing heterocyclic antibiotic (acidic) Antiviral
Chelerythrine Alkaloid (basic) Antiphage
5–X Pyran derivative Herpes and polio
Fisetin Flavanone (acidic) Antiviral
Source: Data from Abonyi, D.O. et al., Afr. J. Biotechnol., 8(17), 3989, 2009.
recognized after it is too late for effective treatment. In the first case, an effective antiviral
agent must prevent the completion of the viral growth cycle in the infected cell without
being toxic to the surrounding normal cells (Kinchington et al., 1995). One encouraging
development is the discovery that some virus-specific enzymes are synthesized during
multiplication of the viral particles, and this may be a point of attack by a specific inhibi-
tor (Berdy, 1982). Some of the antiviral compounds currently used for therapy are given in
Table 7.1.
7.3.4 Antiparasitic compounds

During the evolution of humans, a broad set of parasites have evolved, which inhabits
with us as a host organism. Usually, a parasite will not kill its host (at least not immedi-
ately), as this would by an evolutionary dead end for a parasite. However, most parasites
are either unpleasant for us (lice and fleas) or weaken our health (most internal parasites).
However, some parasitic infections such as malaria, trypanosomiasis, or chagas can be
deadly if the patients are not treated with adequate therapeutics. Because humans usually
live in close proximity and often without good hygienic conditions, the transmission of
parasites within a human population is often facilitated. It is very likely that humans have
always tried to get rid or minimize the impact of parasites. External parasites (ectopara-
sites) could be reduced or eliminated mechanically. This could be done individually or in
groups. Grooming is a common behavior in primates and monkeys, delousing each other
in sequence. Humans probably did the same. More complicated to treat were internal par-
asites (endoparasites). We know that humans have used medicinal plants for several thou-
sands of years to treat illness and health disorders (Van Wyk and Wink, 2004). Whereas the
availability of effective drugs for the treatment of parasitic infections is scanty, only azole
compounds and very few other drugs are effective for these parasitic infections.
7.3.5 Anticancer compounds

Anticancer, or antineoplastic, drugs are used to treat malignancies, or cancerous growths.
Drug therapy may be used alone or in combination with other treatments such as surgery
or radiation therapy. Cancer is commonly defined as the uncontrolled growth of cells,
with loss of differentiation and commonly, with metastasis, spread of the cancer to other
tissues and organs. In contrast, benign growths remain encapsulated and grow within
a well-defined area. Although benign tumors may be fatal if untreated, due to pressure
on essential organs, as in the case of a benign brain tumor, surgery and radiation are the
preferred methods of treating growths that have a well-defined location. Drug therapy
is used when the tumor has spread, or may spread, to all areas of the body (Canetta and
Eisenhauer Rozencsweig, 1994). Several classes of drugs may be used in cancer treatment,
depending on the nature of the organ involved. Newer methods of antineoplastic drug
therapy have taken different approaches, including angiogenesis—the inhibition of for-
mation of blood vessels feeding the tumor and contributing to tumor growth. Although
these approaches hold promise, they are not yet in common use. Developing new anti-
cancer drugs is the work of ongoing research. In 2003, a new technique was developed to
streamline the search for effective drugs. Researchers pumped more than 23,000 chemical
compounds through a screening technique to identify those that help to fight cancer while
leaving healthy cells unharmed. The system identified nine compounds matching the pro-
file, including one previously unidentified drug for fighting cancer (U.S. Administration of
Food and Drug, 2008). The chemical classes and usage of anticancer compounds are listed
in Table 7.2.
7.4 Sources of antibiotics

7.4.1 Plants
The use and search of drugs and dietary supplements derived from plants have accel-
erated in recent years. Ethnopharmacologists, botanists, microbiologists, and natural
product chemists are combing the earth for phytochemicals and “leads” that could be
developed for the treatment of infectious diseases. While 25%–50% of current pharma-
ceuticals are derived from plants, none are used as antimicrobials. Traditional healers
have long been used plants to prevent or cure infectious conditions; western medicine is
trying to duplicate their successes. Plants are rich in a wide variety of secondary metab-
olites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found
in vitro to have antimicrobial properties. Since many of the bioactive compounds are
currently available as unregulated botanical preparations and their use by the p ublic
is increasing rapidly, clinicians need to consider the consequences of patients self-
medicating with these preparations. Plants have an almost limitless ability to synthesize
aromatic substances, most of which are phenols or their oxygen-substituted derivatives
(Elvin-Lewis and Lewis, 1995). Most are belonging to secondary metabolites, of which at
least 12,000 have been isolated from plants, a number estimated to be less than 10% of the
total metabolites (Berkada, 1978). In many cases, these substances serve as plant defense
mechanisms against predation by microorganisms, insects, and herbivores. Some, such
as terpenoids, give plants their odors, and others (quinones and tannins) are responsible
for plant pigment. Many compounds are responsible for plant flavor (e.g., the terpenoid
capsaicin from chili peppers), and some of the same herbs and spices used by humans to
season food yield useful medicinal compounds (Savoia, 2012). The approximate number
of the known natural products derived from the main types of plant and animal organ-
isms is summarized in Table 7.3.
Mainstream medicine is increasingly receptive to the use of antimicrobial and other
drugs derived from plants, as traditional antibiotics become ineffective and as new, par-
ticularly viral, diseases remain intractable to this type of drug. Another driving factor for
the renewed interest in plant antimicrobials in the past two decades has been the rapid
rate of plant species extinction (Georges and Pandelai, 1949). Unfortunately, there is an
alike feeling among natural products, chemists, and microbiologists that the multitude
of potentially useful phytochemical structures that could be synthesized chemically is at
risk of being lost irretrievably (Rojas et al., 1992). There is a scientific discipline known as
Table 7.2 Chemical class and usage of anticancer compounds

Generic (brand name) Clinical uses
Altretamine (Hexalen) Treatment of advanced ovarian cancer
Asparaginase (Elspar) Commonly used in combination with other drugs; refractory
acute lymphocytic leukemia
Bleomycin (Blenoxane) Lymphomas, Hodgkin’s disease, testicular cancer
Busulfan (Myleran) Chronic granulocytic leukemia
Carboplatin (Paraplatin) Palliation of ovarian cancer
Carmustine Hodgkin’s disease, brain tumors, multiple myeloma, malignant
melanoma
Chlorambucil (Leukeran) Chronic lymphocytic leukemia, non-Hodgkin’s disease
Cisplatin (Platinol) Treatment of the bladder, ovarian, uterine, testicular, and head
and neck cancers
Cladribine (Leustatin) Hairy cell leukemia
Cyclophosphamide (Cytoxan) Hodgkin’s disease, non-Hodgkin’s lymphomas, neuroblastoma;
breast, ovarian, and lung cancers; acute lymphoblastic leukemia
in children; multiple myeloma
Cytarabine (Cytosar-U) Leukemias occurring in adults and children
Dacarbazine (DTIC-Dome) Hodgkin’s disease, malignant melanoma
Diethylstilbestrol (DES) Breast cancer in postmenopausal women, prostate cancer
Ethinyl estradiol (Estinyl) Advanced breast cancer in postmenopausal women, prostate
cancer
Etoposide (VePesid) Acute leukemias, lymphomas, testicular cancer
Mitomycin (Mutamycin) Bladder, breast, colon, lung, pancreas, rectum, and head and neck
cancers, malignant melanoma
Mitotane (Lysodren) Cancer of the adrenal cortex (inoperable)
Mitoxantrone (Novantrone) Acute nonlymphocytic leukemia
Paclitaxel (Taxol) Advanced ovarian cancer
Pentostatin (Nipent) Hairy cell leukemia unresponsive to alpha-interferon
Pipobroman Chronic granulocytic leukemia
Plicamycin (Mithracin) Testicular tumors
Prednisone (Meticorten) Used in combined therapy for palliation of symptoms in
lymphomas, acute leukemia, and Hodgkin’s disease
Procarbazine (Matulane) Hodgkin’s disease
Streptozocin (Zanosar) Islet cell carcinoma of pancreas
Tamoxifen (Nolvadex) Advanced breast cancer in post menopausal
Teniposide (Vumon) Acute lymphocytic leukemia in children
Vinblastine (Velban) Breast cancer, Hodgkin’s disease, metastatic testicular cancer
Vincristine (Oncovin) Acute leukemia, Hodgkin’s disease, lymphomas
Source: Data from Kelecom, A., Ann. Braz. Acad. Sci., 74(1), 151, 2002.
ethnobotany/ethnopharmacology, whose goal is to utilize the impressive array of knowl-

edge assembled by indigenous peoples about the plant and animal products they have
used to maintain health (Silva et al., 1996). Ultimately, the ascendancy of the human immu-
nodeficiency virus has spurred intensive investigation into the plant derivatives that may
be effective, especially for use in underdeveloped nations with little access to expensive
western medicines.
Table 7.3 Approximate number of known natural products

Source All known compounds Bioactives Antibiotics
Natural products Over one million 200,000–250,000 25,000–30,000
Plant kingdom 600,000–700,000 150,000–200,000 ~25,000
Microbes Over 50,000 22,000–23,000 ~17,000
Algae, lichens 3,000–5,000 1,500–2,000 ~1,000
Higher plants 500,000–600,000 ~100,000 10,000–12,000
Animal kingdom 300,000–400,000 50,000–100,000 ~5,000
Protozoa Several hundreds 100–200 ~50
Invertebrates ~100,000 NA ~500
Marine animals 20,000–25,000 7,000–8,000 3,000–4,000
Insects/worms 8,000–10,000 800–1,000 150–200
Vertebrates (mammals, 200,000–250,000 50,000–70,000 ~1,000
fishes, amphibians, etc.)
Source: Data from Berdy, J., J. Antibiot., 58(1), 1, 2005.
7.4.2 Animals
Louis Pasteur, for instance, began to study rabies in animals in 1881, and over a number
of years he developed methods of producing attenuated virus preparations by progres-
sively drying spinal cord of rabbits experimentally infected with the agent. Also through
experimental transmission to mice, in 1900, Walter Reed demonstrated that yellow fever
was caused by a virus and spread by mosquitoes. This discovery eventually enabled Max
Theiler in 1937 to propagate the virus in chick embryo and to produce an attenuated vac-
cine from the 17 D strain that is still in use today. The success of this approach had led
to increased use of animal systems to identify and propagate pathogenic viruses even
with the adoption/perfection of tissue culture technique. In the area of antiviral chemo-
therapeutic research, animal models have been used either primarily as screening tools or
applied in testing the efficacy of the test compound when it had been identified as effec-
tive/potent using any other method (Likar and Japelj, 1977; Sloan et al., 1977).
7.4.3 Bacteria
Among the many challenges facing drug discovery research in recent years, the search
for new antibacterial agents has proved to be among the most unproductive. Many factors
have contributed to this problem, but one of the key areas for improvement is the need
to test compounds that are more appropriate, as it has become obvious that the screen-
ing of randomly assembled, diverse compound libraries has produced extremely low hit
rates (Fernando, 2006). Besides, in vitro screening often delivers nondrug-like and non-
target-specific structures that tend to face serious efficacy issues in in vivo experiments.
To address the major challenge of antibacterial drug discovery, it is critical to access
compound libraries that are capable of delivering excellent chemical starting points for
completely new classes of antibacterials. A large proportion of known antibacterials have
derived from natural products, and these compounds clearly have structures and prop-
erties that have made them a particularly rich source. The discovery of novel antibiot-
ics and other potential molecules of pharmaceutical interest through microbial secondary
metabolite screening is becoming increasingly fruitful. There is wide acceptance that
Table 7.4 Approximate number of bioactive microbial products according to their producers
Other Total Practically used
bioactive bioactive (in human Inactive
Source Antibiotics metabolites metabolites therapy) metabolites
Bacteria 2,900 900 3,800 10 ~ 12 (8 ~ 10) 3,000–5,000
Actinobacteria 8,700 1,400 10,100 100 ~ 120 (70 ~ 75) 5,000–10,000
Fungi 4,900 3,700 8,600 30 ~ 35 (13 ~ 15) 2,000–15,000
Total 16,500 6,000 22,500 140 ~ 160 (~100) 20,000–25,000
Source: Data from Berdy, J., J. Antibiot., 58(1), 1, 2005.
microorganisms are virtually unlimited sources of novel substances with many therapeu-
tic applications. Among them, actinobacteria hold a predominant position due to their
diversity and had proven their ability to provide new and novel substances (Gayathri et al.,
2011). As the best approximate, the total number of additional inactive microbial products is
about 20,000–25,000; therefore, today close to 50,000 microbial metabolites may be known
(Vijayakumar et al., 2013). According to the main types of microbial producers, the num-
bers of compounds, including both antibiotics and other bioactive metabolites, practically
used compounds, and the approximate numbers of the inactive microbial metabolites are
summarized in Table 7.4.
7.4.4 Fungi
Fungi make an extraordinarily important contribution to managing disease in humans
and other animals. At the beginning of the twenty-first century, fungi were involved in
the industrial processing of more than 10 of the 20 most profitable products used in human
medicine. In 1941, penicillin from the fungus Penicillium chrysogenum was first used suc-
cessfully to treat an infection caused by a bacterium. The use of penicillin revolution-
ized the treatment of pathogenic disease. Many formally fatal diseases caused by bacteria
became treatable, and new forms of medical intervention were possible. When penicillin
was first produced, the concentration of active ingredient was approximately 1 µL/mL
of broth solution. Today, improved strains and highly developed fermentation technolo-
gies produce more than 700 µL/mL of active ingredient. The natural penicillins have a
number of disadvantages. They are destroyed in the acid stomach and so cannot be used
orally; they are sensitive to beta-lactamases, which are produced by resistant bacteria, thus
reducing their effectiveness. Also, they only act on gram-positive bacteria. Modifications
to manufacturing conditions have resulted in the development of oral forms. However,
antibiotic resistance among bacteria is becoming an extremely important aspect determin-
ing the long-term use of all antibiotics (Zabriskie and Jackson, 2000). Cephalosporins also
contain the beta-lactam ring. The original fungus found to produce the compounds was
Cephalosporium. As with penicillin, the cephalosporin antibiotics have a number of disad-
vantages. Industrial modification of the active ingredients has reduced these problems.
The only broadly useful antifungal agent from fungi is griseofulvin. The original source
was Penicillium griseofulvum. Griseofulvin is fungistatic, rather than fungicidal. It is used
for the treatment of dermatophytes, as it accumulates in the hair and skin following topical
application (Erturk, 2006).
More recently, several new groups have been developed. Strobilurins target the ubihy-
droquinone oxidation center, and in mammals, the compound from fungi is immediately
excreted. Basidiomycetes, especially from tropical regions, produce an enormous diversity
of these compounds. Sordarins are structurally complex molecules that show a remarkably
narrow range of action against yeasts and yeastlike fungi. The compounds inhibit protein
biosynthesis and so may become important agents against a number of fungal pathogens
of humans. Echinocandins are cyclic peptides with a long fatty acid side chain. They target
cell wall formation. Semisynthetic members of the group of compounds include pneumo-
candins that are in use in humans.
7.4.5 Actinobacteria
A rapid increase in discoveries of new antibiotics occurred from the late 1910s to 1960s,
followed by a gradual fall until 1968 and then again raised in the 1970s. But most of the
major antibiotics had been discovered by the 1960s. The second increase in the isolation
of new antibiotics in recent years may reflect the development of new technique that has
enabled us to isolate and characterize antibiotic of closely related chemicals structures,
which are antibiotics present in the culture as minor components. The recent increase
in the description of new antibiotics might also be attributed to the rediscoveries of old
antibiotics, which were once isolated in crude state and completely described in the early
stage of antibiotic era. Since more than 2000 antibiotics have already been isolated and
described, the chances of finding new antibiotic have now become poor. Under such a situ-
ation, the importance of the preliminary identification of antibiotic producer in the first
step of screening cannot be stressed too much. If one could identify the antibiotic producer
simply by taxonomic studies on the antagonistic actinobacteria under study, it would be
of a great help in the search for new antibiotics produced simply by taxonomic placement
of actinobacteria and their antibiotic production. Krasilnikov (1960) reported that the pro-
duction of specific antibiotics by actinobacteria can be used as one of the criteria for their
classification. On the other hand, Baldacci et al. (1954) claimed that the uses of antibiotic
production in taxonomy present staggering problems since actinobacteria produced sev-
eral hundred antibiotics in various mixtures. Lechavalier et al. (1971) suggested that the
antibiotic production helped in the speciation of actinobacteria.
Generally, the productivity and activity of antimicrobial compounds are not uniform,
which may be varied between the producing organisms where they have isolated and
against test pathogenic microorganisms, respectively. In a study, among the 24 antibac-
terial antagonistic actinobacteria, 20 isolates inhibited the growth of gram-positive bac-
teria, 17 inhibited the growth of gram-negative bacteria, and 13 inhibited the growth of
both gram-positive and gram-negative bacteria. The maximum percentage of antibacterial
compound producing actinobacteria was found in saltpan soil (47.4%), followed by sea-
shore soil (33.33%) and mangrove soil (28.57%) (Vijayakumar, 2006). Correspondingly, out
of 50 isolates, 29 (58%) isolates exhibited antimicrobial activity. Among them, 22 (75.89%)
isolates had activity against gram-positive bacteria, 26 (89.66%) against gram-negative bac-
teria, and 20 (68.97%) against both gram-positive and gram-negative bacteria (Cholarajan,
2014). Several reports have been published by various researchers, and they bring out the
potentials of actinobacteria as producer of commercially valuable antibiotics and other
bioactive compounds. The actinobacteria producing some antibiotics are given in Table 7.5.
7.4.6 Other sources

Numerous compounds isolated in the past few years from lichens proved to be active in
certain physiological and pharmacological terms, and a number of them were confirmed
to be effective antimicrobial agents. These lichen products belong to several distinct
Table 7.5 Some examples for actinobacteria-producing antibiotics

Organism Antibiotic
Micromonospora galeriensis Primycin
Actinoplanes ianthinogenes Purpuromycin
Streptosporangium albidum Sporaviridin
Planomonospora parontospora Sporangiomycin
Actinosporangium griseoroseum Hepcin
Nocardia acidophilus Mycomycin
Micromonospora chalcea Chalacidin
Thermoactinopolyspora coremialis Thermothiocin
Streptomyces venezuelae Chloramphenicol
Streptomyces roseosporus Daptomycin
Streptomyces fradiae Fosfomycin
Streptomyces lincolnensis Lincomycin
Streptomyces fradiae Neomycin
Streptomyces alboniger Puromycin
Streptomyces griseus Streptomycin
Streptomyces rimosus, Streptomyces aureofaciens Tetracycline
Streptomyces avermitilis Avermectin
Source: Data from Kelecom, A., Ann. Braz. Acad. Sci., 74(1), 151, 2002.
chemical classes including polysaccharides, coumarone derivatives, lactone derivatives,

orcinol and orcinol-type despsides and depsidones, and long-chain aliphatic oligocarboxy
hydroxy acids (Esimone, 1997). Most of the lichen products are primarily active against
gram-positive bacteria and mycobacteria, though the extracts of Parmelia caperata, Evernia
prunastri, and Usnea spp. have been shown to be active against gram-negative bacteria
(Rowe et al., 1989). Some lichen polysaccharides and other lichen products such as psoronic
acid had antitumor activity, and usnic acid, the most widely distributed and best-known
lichen antibiotics, has been used in several countries as a topical antibacterial agent for
human skin diseases (Berdy, 1982) and is also reported to exert some antimitotic action,
but at low concentrations, the acid displays a capacity to stimulate cell metabolism in some
biological systems tested (Cardarelli et al., 1997). In Table 7.6, some of the clinically impor-
tant antibiotics are given.
7.5 Isolation of actinobacteria

7.5.1 Selection of samples for the isolation of actinobacteria
Many thousands of actinobacteria have been isolated from the environment until now,
but little information only is available about the geographical or ecological distribution
of these microbes. So, it is generally impossible to predict the sites in which a particular
actinobacterial taxon or strain will occur. Thus, the selection of macro or microenviron-
ments as a source of useful isolates remains largely a matter of chance and hopeful initia-
tive. According to the results of Takahashi et al. (1993), most of the actinobacteria occur
within one meter below the ground. Compared to terrestrial soils, the marine sediments
have proved to be the best source for the isolation of antagonistic actinobacteria by various
workers not only from marine soils and sediments (Vijayakumar et al., 2007, 2012a–c) but
also from salt mining samples (Yang et al., 2008); mangrove environments (Dhanasekaran
Table 7.6 Some clinically important antibiotics

Antibiotic Producer organism Activity Site or mode of action
Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis
Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis
Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules
Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis
Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane
Amphotericin B Streptomyces nodosus Fungi Cell membrane
Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis
Neomycin Streptomyces fradiae Broad spectrum Protein synthesis
Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis
Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis
Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis
Gentamicin Micromonospora purpurea Broad spectrum Protein synthesis
Rifamycin Streptomyces mediterranei Tuberculosis Protein synthesis
Source: Data from Kelecom, A., Ann. Braz. Acad. Sci., 74(1), 151, 2002, modified and reprinted with permission
from Prescott, L.M. et al., Microbiology, 5th edn., Tata McGraw-Hill Company, New Delhi, India, 2002.
et al., 2008, 2009, 2011; Remya and Vijayakumar, 2008), estuaries, sand dunes and indus-
trially polluted coast soil, and salt marsh soil (Al-Zarban et al., 2002; Kathiresan et al.,
2005); coral reefs (Lam, 2006); marine sediments (Olano et al., 2009); salt pan environment
(Vijayakumar et al., 2012c); sea anemone (Chen et al., 2009); marine sponge (Gandhimathi
et al., 2009); beach soil (Ogunmwonyi et al., 2010); endophytic actinobacteria (Ravikumar
et al., 2010); seawater (Reddy et al., 2011); and saltern (Chun et al., 2000).
7.5.2 Pretreatment of the samples

The collected samples were processed and inoculated onto various culture media using
one method, or in some cases (especially for the deeper sediments), as many as eight meth-
ods described in the following are used.
7.5.2.1 Method 1 (dry/stamp)

Sediment was dried overnight in a laminar flow hood and, when clumping occurred,
ground lightly with an alcohol-sterilized mortar and pestle. An autoclaved foam plug
(2 cm in diameter) was pressed onto the sediment and then repeatedly onto the surface of
an agar plate in a clockwise direction creating a serial dilution effect.
7.5.2.2 Method 2 (sterile dry/scrape)

The method was used for small rocks that had been dried overnight in a laminar air flow
hood and then scraped with a spatula generating a powder that was processed as per
Method 1. In some cases, the powder was collected with a wet cotton-tipped applicator,
or the rock was rubbed directly with the applicator that was then used to inoculate the
surface of an agar plate.
7.5.2.3 Method 3 (dry/dilute)

Dried sediment (0.5 g) was diluted with 5 mL of sterile seawater (SSW). The diluted sample
was vortex mixed and allowed to settle for a few minutes, and 50 µL of the resulting solu-
tion was inoculated onto the agar surface and spread with sterile glass rod.
7.5.2.4 Method 4 (dilute/heat)

Dried sediment was volumetrically added to 3 mL of SWW (dilutions 1:3 or 1:6) and heated
to 55°C for 6 min, and 50–75 µL of the resulting suspension was inoculated onto agar
media as per Method 3.
7.5.2.5 Method 5 (dilute/heat/2)

Dried sediment was treated as per Method 4 (dilution 1:6) with the addition of a second
heat treatment at 60°C for 10 min.
7.5.2.6 Method 6 (dry/stamp + dilute/heat)

The surface of an agar medium was inoculated using a sample treated as Method 1. The
dried sediments were then processed using Method 4, and the same agar plate was inocu-
lated a second time with the heat-treated samples.
7.5.2.7 Method 7 (freeze/dilute)

Wet sediment was frozen at −20°C for at least 24 h, thawed, and volumetrically diluted in
SSW (1:3 to 1:120 depending on particle size), and 50 µL of the resulting suspension was
inoculated onto the surface of an agar plate as per Method 3.
7.5.2.8 Method 8 (freeze/dilute/2)

Wet sediment was treated as per Method 7 except that the thawed and diluted samples
were incubated at room temperature for 48 h before inoculation onto the surface on agar
plate (Jensen et al., 2005).
7.5.3 Isolation and culture conditions

First, the isolates were isolated on starch casein agar (SCA) medium (g/L, starch 10,
casein 0.3, KNO3 2, NaCl 2, K2HPO4 2, MgSO4 2, 7H2O 0.05, CaCO3 0.02, FeSO4 7H2O 0.01,
and agar 18); supplemented with griseofulvin and cycloheximide (Himedia, Mumbai)
of 25 and 10 µg/mL density (Vijayakumar et al., 2007). The plates were incubated for
14 days in an incubator of darkness at 28°C ± 1°C. Colonies of morphologically distinct
isolates were purified by using pure culture technology (streak plate method) (Figures 7.1
and 7.2). The studies on colony morphological characteristics of the isolated marine acti-
nobacteria were carried out following the methods recommended by the International
Streptomyces Project (ISP) (Waksman, 1943; Shirling and Gottlieb, 1966). The population
Figure 7.1 Isolated colonies of actinobacteria on a SCA medium.

Figure 7.2 Various colonial morphologies of some actinobacteria on a SCA medium.

of actinobacteria will be varied depending on the samples and their collection location
as well as media with and without antibiotics (inhibit other bacterial and fungal pop-
ulation) used for cultivation. Thus, usage of different culture media for the isolation
of actinobacteria from different sampling environments will give the complete picture
(occurrence) of them (Table 7.7).
7.6 Identification of actinobacteria

7.6.1 Phenotypic characterization
7.6.1.1 Microscopic characterization
Purified isolates of actinobacteria were initially characterized by using morphological
properties followed by the cultural, biochemical, and physiological characteristics by the
methods of ISP. The morphology of the spore-bearing hyphae with the entire spore chain,
the structure and arrangement of the spore chain with the substrate, and the aerial myce-
lium of the actinobacteria were examined and identified using slide culture technique.
After growth, the microscopic morphology including the formation of aerial and substrate
mycelia and sporophore nature was observed by light microscopy. The spore and mycelial
sizes and spore surface nature were observed under scanning electron microscopy.
7.6.1.2 Colony morphological/cultural characterization

Aerial spore mass color and reverse side pigments of the actinobacterial isolates were
recorded in various culture media such as oat meal agar (ISP 3), yeast extract–malt extract
agar (ISP 2), inorganic salt starch agar (ISP 4), glycerol–asparagine agar (ISP 5), tyrosine
agar base (ISP 7), starch–casein agar, and Czapek–Dox agar. The reverse side pigments of
the colony, namely, distinctive (+) and not distinctive (−), were tested using peptone–yeast
extract–iron agar (ISP 6) (Das et al., 2008). The production of melanoid pigments was tested
on tryptone–yeast extract agar (ISP 1) and ISP 7 medium. Colors of aerial spore mass and
reverse side colors were determined with the ISCC-NBS centroid color charts (Kenneth
et al., 1976). Generally, it is difficult to characterize all the isolated actinobacteria with
respect to colony morphology on various culture media and biochemically and physio-
logically. Hence, bioactive actinobacteria alone studied for their phenotypic and genotypic
characteristics will allow us to consume the time duration.
7.6.1.3 Biochemical characterization

The biochemical characteristics are used to differentiate the microorganisms at species/
strain level, in which indole production; methyl red and Voges–Proskauer tests, triple
sugar iron test; citrate utilization; nitrate reduction; production of urease, catalase, and
oxidase; hydrolyses of gelatin, starch, lipid, and casein; degradation of xanthine; fermenta-
tion of carbohydrates; tolerance to pH; and temperature and salt tests are performed and
used to identify the actinobacteria (Holt, 1989). Methodologies for all the described exami-
nations were performed with reference to Cappuccino and Sherman (2005) and Bergey
and Holt (1994). In addition, the analysis of cell wall amino acids and whole cell sugars of
the actinobacteria has also provided valuable information for generic-level identification.
7.6.1.4 Physiological characterization

The ability of the isolates to utilize various carbon and nitrogen sources was studied as per
the methods recommended by ISP. Carbon sources like glucose, mannitol, fructose, xylose,
sucrose, raffinose, inositol, arabinose, and rhamnose were tested on carbon utilization agar
Table 7.7 Different sources and media for isolation of actinobacteria

Source Media References
From soil
Forest soil Starch–casein medium Kuster and Williams (1964)
Humus layer of forest soil Humic acid–vitamin agar Choi and Park (1993)
Starch–casein–nitrate (SCN) Hayakawa and Nonomura
agar (1987)
Hair hydrolysate–vitamin
agar
Bennett’s agar Seong et al. (2001)
Cornfield, cow’s barnyard, and Arginine–glycerol salt Porter et al. (1960)
forest medium
Chitin medium Lingappa and Lockwood
(1961)
Modified Benedict’s medium Porter et al. (1960)
Soybean meal–glucose Tsao et al. (1960)
medium
Gauze’s agar medium Rehacek (1959)
Czapek’s agar medium Waksman (1961)
Egg albumen medium
Glucose–asparagine medium
Glycerol–asparagine agar 2
Lake soil Chitin agar Hsu and Lockwood (1975)
Soil Coal–vitamin agar (CVA) Wakisaka et al. (1982)
Antarctic soil Mineral salt (MS) medium Kosmachev (1954)
Mitidja plain (Algeria) Yeast extract–malt extract Shirling and Gottlieb (1966)
agar
Marine soil SCN agar medium Amorso et al. (1998)
From water
Stream sediments and lake muds Chitin agar media Lingappa and Lockwood
(1961, 1962)
M3 agar medium Jones (1949)
Bennett’s medium
Marine sediments Starch–casein agar Grein and Meyers (1958)
Asparagine agar
Glycerol–glycine agar Lindenbein (1952)
Marine sediments (South China) Actinomycete isolation agar You et al. (2007)
(AIA) medium
From root and stem samples of four
plants
Cinnamomum zeylanicum, Zingiber Starch–yeast–casein–agar, Zin et al. (2007)
spectabile, Elettariopsis curtisii, and AIA, humic acid–vitamin–
Labisia pumila gellan gum, tap water–yeast
extract agar, CVA
Mangrove sediments Asparagine–glucose agar Shirling and Gottlieb (1966)
medium
Source: Data from Sharma, M., Int. J. Curr. Microbiol. App. Sci., 3(2), 801, 2014.
(ISP 9) supplemented with 1% carbon sources (Nonomura, 1974). The ability of the isolate to
utilize various nitrogen sources like leucine, histidine, tryptophan, serine, glutamic acid,
lysine, arginine, methionine, and tyrosine for growth was also tested.
7.6.2 Molecular characterization

Sequence-based identification is becoming an increasingly important tool of identification.
Based on the molecular characterization of phylogenetic relatedness, 16S rRNA genes pro-
vide enough information for the species-level identification of actinobacteria. The genomic
DNA isolation of actinobacteria was done by the method described by Sambrook et al.
(1989) and amplified by polymerase chain reaction (PCR) using master mix (Kit Medox
Mix). PCR conditions, the primers and the methodology for sequencing, were adapted
from Magarvey et al. (2004) and Mincer et al. (2002). The sequencing was carried out in
both sense and antisense directions. The similarity and homology of the 16S rDNA partial
gene sequence were analyzed with the similar existing sequences available in the data
bank of NCBI using BLAST search. The DNA sequences were aligned and phylogenetic
tree was constructed by neighbor joining (NJ tree) method using neighbor tree software.
A bootstrap analysis of 100 replicates was carried out.
7.7 Screening of antibiotic production

7.7.1 Primary screening: Cross streak plate technique
The search for novel metabolites especially from actinobacteria requires a large number
of isolates (over thousands) in order to discover actinobacterial population with novel
compound of pharmaceutical interest. Because of this, the research will be more prom-
ising if diverse and more actinobacteria are sampled and screened. Such attempts need
to be continued both in the sample collection area and from the adjacent places dur-
ing various climatic conditions as to screen more isolates for novel antimicrobial com-
pounds. Antimicrobial producing property of the actinobacteria was initially screened
by cross streak method (Egorov, 1985). The single streak of the actinobacteria was made
on the surface of the modified nutrient agar medium and incubated at 28°C ± 2°C.
After observing a good ribbon-like growth of the actinobacteria on the Petri plates,
the test pathogens were streaked at right angles to the original streak of actinobacteria
and incubated at 27°C. The inhibition zone was measured after 24 and 48 h. By this
cross streak plate method, deserved candidates (antibiotic-producing actinobacteria)
were selected for further studies. The isolates with no activity may also be cultured and
maintained for identification so as to understand the actinobacteria with or without
biological activities.
7.7.2 Secondary screening: Well diffusion assay

The molten sterile nutrient agar was prepared and inoculated with the test organisms.
Wells were made using sterile borer; 50 µL of clear broth (supernatant) was added to
each well. The plates were kept in a refrigerator for about 2 h to allow the diffusion of
the bioactive metabolite. After 2 h, plates were incubated at 37°C in an incubator. The
inhibition zones were measured after 24 h using an antibiotic zone reader (Grove and
Randall, 1955).
7.8 Extraction and separation of antimicrobial compounds

The selected antagonistic actinobacterial isolates were inoculated into liquid starch–
casein medium and incubated at 28°C on a shaker (200–250 rpm) for 7 days. After incu-
bation, the medium was filtered through Whatman No. 1 filter paper and then through
0.45 µm Millipore filter (Millipore Millex-HV Hydrophilic PVDF). The filtrate was trans-
ferred aseptically into a conical flask and stored at 4°C for further assay. To the culture
filtrate, equal volume of various solvents (viz., chloroform, ethyl acetate, petroleum ether,
and methanol) was added separately and centrifuged at 8000 rpm for 10 min at 4°C to
extract the antimicrobial compound (Sambamurthy and Ellaiah, 1974). The supernatant
was collected and maintained in the refrigerator for further use. Extraction of an anti-
microbial compound is a crucial and vital process with as much as organic solvents.
Therefore, it is essential to keep in mind that the solvent selection is crucial step not only
for the extraction of antimicrobial compounds but also for the minimization of the total
production cost of an antibiotic.
7.8.1 Thin-layer chromatography

The crude extract of the selected actinobacteria was applied manually on a preparative
thin-layer chromatography (TLC) glass plate (20 × 20 cm; 1.5 cm thickness) with inor-
ganic fluorescent indicator binder. After air drying, the plate was developed, using the
same mobile phase as used in the analytical TLC, in a presaturated glass chamber. In
each experiment, two plates were used in parallel. One plate from each set of experiment
was sprayed with 2,3,5-triphenyl tetrazolium chloride solution, as described earlier,
and the bands with antimicrobial activity determined by bioautography were scraped
off carefully from the second plate of each set of experiment. The scratched sample
was dissolved in high-performance liquid chromatography (HPLC) grade methanol
and centrifuged at 12,000 rpm for 15 min in order to remove debris of silica gel. The
supernatant was collected, filtered from 0.22 µm filter, and dried under reduced pres-
sure. Further, all the dried samples were passed under nitrogen gas for 5 min and then
dissolved in methanol for further characterization and bioactivity analysis. The entire
purification process was carried out under dark or dim light conditions (Rajauria and
Abu-Ghannam, 2013).
7.8.2 High-performance liquid chromatography

HPLC is a technique used to separate a mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying, quantifying, and purifying the individual
components of the mixture. The chromatographic separation of antibacterial compound
was carried out on a LC-10 AT vp model HPLC using 250 × 4.60 mm Rheodyne column
(C-18). The solvent system used was methanol (HPLC grade) and water (HPLC grade) in
the ratio of 88:12. The operating pressure was 114 kgf, at a flow rate 0.8 mL/min, and the
temperature was set at 30°C. The ultraviolet–visible (UV-Vis) (SPD-10 A vp) detector was
set at 210 nm. The sample was mixed with the solvent in the ratio of 50:50 and filtered
using Millipore filter before injection. About 25 μL of the sample filtrate was injected into
the column. The sample was run for 10 min and the retention time was noted. The elution
time was compared with the standard, and the compound was determined (Swami et al.,
1983; Sethi, 2001).
7.9 Characterization and identification of antibiotics

7.9.1 pH stability test
Five milliliters of actinobacterial culture filtrates were put in vials using 0.1 N HCl or 0.1 N
NaOH adjusted to the pH levels 2, 4, 6, 8, 10, and 12. These antibiotic-containing media
were held for 3–4 h at room temperature (20°C–24°C) and then readjusted to pH 6.0 (Muiru
et al., 2012). The experiment was replicated three times and arranged in a completely ran-
domized design as outlined earlier. The activity of these treated antibiotic culture filtrates
was determined against pathogenic microorganisms by the measurements of the inhibi-
tion zones using the paper disc method as described earlier (Grove and Randall, 1955).
7.9.2 Temperature stability test

The culture filtrates of actinobacteria were adjusted to pH 6.0 using 0.1 N HCl or 0.1 N
NaOH, and 5 mL of each culture filtrate was put in sterile universal bottles and then
subjected to different temperatures in a water bath for 10 min. The temperature range
from 30°C to 121°C was employed for the determination of the stability of antimicrobial
compounds. Autoclaving was done for the temperature 121°C at one bar pressure for
15 min. The stability of the various culture filtrates to these temperatures was determined
by measuring the size of the inhibition zones using the paper disc method as described
earlier. For each temperature, three replicates (plates) were maintained (Muiru et al., 2012).
7.9.3 Melting point

The melting point of the antimicrobial compound was determined on electrically heated
oil bath. The dried antimicrobial compound was taken in the glass capillary tubes. The
tubes were kept in the electrical heat oil bath. Thermometer was monitored regularly to
notice the melting point of the secondary metabolite (Gandhi et al., 1976).
7.9.4 Quality test

Quality assurance is essential to ensure the quality of antimicrobial susceptibility test by
diffusion methods. Routine internal quality control testing with a range of control strains
is a major part of the quality assurance process, as it facilitates monitoring of the per-
formance of the test. Most standardized methods include tables of acceptance zone size
ranges for control strains, and in addition to checking that control zone diameters are
within the published ranges, rules or statistical approaches may be applied to indicate
deviations from acceptable performance (King and Brown, 2001).
7.10 Prediction of antibiotic structures

7.10.1 Ultraviolet spectrum
UV–visible (UV–Vis or UV/Vis) spectrophotometry refers to absorption spectroscopy or
reflectance spectroscopy in the UV–Vis spectral region. This means that it uses light in the
visible and adjacent (near-UV) and near-infrared ranges. The absorption or reflectance in
the visible range directly affects the perceived color of the chemicals involved. In this region
of the electromagnetic spectrum, molecules undergo electronic transitions. This technique
is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions

from the excited state to the ground state, while absorption measures transitions from the
ground state to the excited state. Molecules containing π-electrons or nonbonding elec-
trons (n-electrons) can absorb the energy in the form of UV or visible light to excite these
electrons to higher antibonding molecular orbitals (Douglas et al., 2007). The more easily
excited the electrons (i.e., lower energy gap between the HOMO and the LUMO), the longer
the wavelength of light it can absorb.
7.10.2 Fourier transform infrared spectrum

The powdered antimicrobial compound samples were mixed with potassium bromide
(KBr pellet) and subjected to a pressure of about 5.106 Pa in an evacuated to produce a
clear transparent disc of diameter 13 mm and thickness 1 mm. IR spectra in frequency
region 4000–400 cm−1 were recorded at room temperature on a PerkinElmer fourier trans-
form spectrometer equipped an air-cooled deuterated triglycine sulfate detector. For each
spectrum, 100 scans were coadded at a spectral resolution of 4 cm. The frequencies for
all sharp bands were accurate to 0.01 cm. All the spectral values were expressed in (%)
transmittance.
7.10.3 Mass spectrum

Mass spectroscopy is an important physicochemical tool applied for structural elucida-
tion of compounds from natural product including medicinal plants. The fundamental
principle of mass spectrum (MS) is the use of different physical means for sample ioniza-
tion and separation of the ions generated based on their mass (m) to change (z) ratio (m/z)
(De Rijke et al., 2006), for example, ionization (APCI), electron ionization, fast atom bom-
bardment, and matrix-assisted laser desorption ionization. Mass spectroscopy has high
sensitivity with detection limit of fentogram compared to nuclear magnetic resonance
(NMR) with sensitivity limit of nanogram range and above. The high sensitivity and the
flexibility for hyphenation with other chromatographic techniques made MS a versatile
analytical instrument.
7.10.4 Nuclear magnetic resonance spectrum

One-dimensional (1H and 13C) and 2D NMR spectra (1H, 1HCOSY, HMQC, and HMBC) were
recorded on a varian VNMRS 600 spectrometer with tetramethylsilane as internal stan-
dard. Standard pulse sequence was used for homo- and heteronuclear correlation experi-
ments. 1H NMR spectra were measured at 599 MHz, whereas 13C NMR spectra were run at
150 MHz multiplicities of 13C NMR resonances determined by DEPT experiments. All NMR
experiments were performed at constant temperature (27°C) using software supplied by the
manufacturer, employing deuteriochloroform, deuteriomethanol, and deuteriosulfoxide as
solvent on the basis of solubility of the sample and literature data (Figure 7.3).
7.11 Conclusion
Around 23,000 bioactive secondary metabolites produced by microorganisms have been
reported, and over 10,000 of these compounds are produced by actinobacteria. Their
metabolic potential offers a strong area for research. For novel drug delivery, scientists
still exploit the chemical and biological diversity from diverse actinobacterial group to
3.26
N H 2.0 0.91 0.91

2.21
2.25;2.00 0.91 O
O 3.05
7.59 3.2
0.91 2.21 O 4.41 O
5.80
O 4.41 N
7.42 3.30 4.98;4.96
N
H N H O 5.08 1.54
7.42 N
1.86 8.0 O 8.0
8.17 1.54 0.91 2.21
O 0.91 8.0 H
O 5.08 O N O
7.59 4.41
O 4.41 4.89 O 2.21
0.91
N N
4.22 8.17 7.42
2.65
H 7.42 O H O 0.91
(a) 8.0 (b) 8.0
0.91
0.91
Figure 7.3 Structure of antimicrobial compounds: (a) staurosporine and (b) octa-valinomycin.
maximize the possibility of successful discovery of novel strain. However, further charac-
terization of actinobacteria and their product for utilization in plant biotechnology, envi-
ronmental biotechnology, urban waste management, and some other applications is yet to
be done. The potential numbers of metabolites from actinobacteria may be discovered in
the future.
References
Abonyi, D.O., Adikwu, M.U., Esimone, C.O., and Ibezim, E.C. 2009. Plants as sources of antiviral
agents. Afr. J. Biotechnol. 8(17):3989–3994.
Aghighi, S.G.H., Bonjar, S., and Saadoun, I. 2005. First report of antifungal properties of a new strain
of Streptomyces plicatus (strain 101) against four Iranian phytopathogenic isolate of Verticillium
dahliae, a new horizon in biocontrol agents. Biotechnology 3(1):90–97.
Al-Zarban, S.S., Al-Musallam, A.A., Abbas, I.H., and Fasasi, Y.A. 2002. Noteworthy salt-loving acti-
nomycetes from Kuwait. Kuwait J. Sci. Eng. 29(1):99–109.
Amorso, M.J., Castro, G.R., Carlino, F.J., Romero, N.C., Hill, R.T., and Oliver, G. 1998. Screening
of heavy metal-tolerant actinomycetes isolated from the Salí River. J. Gen. Appl. Microbiol.
44:129–132.
Arasu, M.V., Duraipandiyan, V., Agastian, P., and Ignacimuthu, S. 2009. In vitro antimicrobial activity
of Streptomyces spp. ERI-3 isolated from Western Ghats rock soil (India). J. Mycol. Méd. 19:22–28.
Atalan, E., Manfio, G.P., Ward, A.C., Kroppenstedt, R.M., and Goodfellow, M. 2000. Biosystematic
studies on novel Streptomycetes from soil. Anton van Leeuwhoek 77: 337–353.
Baldacci, E., Spalla, C., and Grein, A. 1954. The classification of the Actinomyces (Streptomyces) spe-
cies. Arch. Microbiol. 20:347–357.
Beijerinck, M.W. 1900. Streptothrix chromogena. Proc. Natl Acad. USA 100(12):4555–4561.
Berdy, J. 1982. Handbook of Antibiotics Compounds, Vol. IX. Antibiotics from Higher Forms of Life: Lichens,
Algae and Animal Organisms. Boca Raton, FL: CRC Press, pp. 39–227.
Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58(1):1–26.
Berger, J., Jampolsky, L.M., and Goldberg, M.W. 1949. Borrelidin, a new antibiotic with antiborrelia
activity and penicillin-enhancement properties. Arch. Biochem. 22:476–478.
Bergey, D.H. and Holt, J.G. 1994. Bergey’s Manual of Determinative Bacteriology, 9th edn. Philadelphia,
PA: Lippincott Williams and Wilkins.
Berkada, B. 1978. Preliminary report on warfarin for the treatment of herpes simplex. J. Irish Coll.
Phys. Surg. 22:56.
Canetta, R.M. and Eisenhauer Rozencsweig, E.M. 1994. Methods for Administration of Taxol for Cancer
Treatment with Reduced Toxicity. New York: Bristol-Myers Squibb Co., Application, p. 38.
Cappuccino, J.G. and Sherman, N. 2005. Microbiology: A Laboratory Manual, 7th edn. San Francisco,
CA: The Benjamin Cummings.
Cardarelli, M., Serino, G., Campanella, L., Ercole, P., Nardone, F.D.C., Alesiani, O., and Rossiello, F. 1997.
Antimitotic effects of usnic acid on different biological systems. Cell. Mol. Life Sci. 53:667–672.
Chen, Y.G., Wang, Y.X., Zhang, Y.Q., Tang, S.K., Liu, Z.X., Xiao, H.D., and Li, W.J. 2009. Nocardiopsis
litoralis sp. nov., a halophilic marine actinomycete isolated from a sea anemone. J. Syst. Evol.
Microbiol. 59:2708–2713.
Choi, J.D. and Park, U.Y. 1993. Identification of the marine microorganisms producing bioactives.1.
Isolation and cultural conditions of the marine actinomycetes no. 101 producing antimicrobial
compounds. Bull. Kor. Fish. Soc. 26(4):305–311.
Cholarajan, A. 2014. Diversity, characterization and antimicrobial compounds from actinobacteria in
terrestrial soil of Thanjavur District, Tamilnadu, India. PhD thesis, Bharathidasan University,
Tiruchirappalli, Tamil Nadu, India.
Chun, J., Bae, K.S., Moon, E.Y., Jung, S.O., Lee, H.K., and Kim, S.J. 2000. Nocardiopsis kunsanensis sp.
nov., a moderately halophilic actinomycete isolated from a saltern. Int. J. Syst. Evol. Microbiol.
50:1909–1913.
Das, S., Lyla, P.S., and Khan, S.A. 2008. Characterization and identification of marine actinomycetes
existing systems, complexities and future directions. Natl. Acad. Sci. Lett. 31:149–160.
De Rijke, E., Out, P., Niessen, W.M.A., Ariese, F., Gooijer, C., and Brinkman, U.A. 2006. Analytical
separation and detection methods for flavonoids. J. Chromatograp. 1112(1–2):31–63.
Dhanasekaran, D., Selvamani, S., Panneerselvam, A., and Thajuddin, N. 2009. Isolation and char-
acterization of actinomycetes in Vellar Estuary, Annagkoil, Tamil Nadu. Afr. J. Biotechnol.
8(17):4159–4162.
Dhanasekaran, D., Thajuddin, N., and Panneerselvam, A. 2008. Distribution and ecobiology of antago-
nistic Streptomycetes from agriculture and coastal soil in Tamilnadu, India. J. Cul. Coll. 6:10–20.
Dhanasekaran, D., Thajuddin, N., and Panneerselvam, A. 2011. Applications of actinobacterial fungi-
cides in agriculture and medicine. In Fungicides for Plant and Animal Diseases, Dhanasekaran, D.,
Thajuddin, N., and Panneerselvam, A., eds. Rijeka, Croatia: InTech, pp. 29–54.
Dorlands Medical Dictionary: Antibacterial. Archived from the original on 2010–11–17. Accessed
October 29, 2010.
Douglas, A., Skoog, F., Holler, J., Stanley, R. Crouch. 2007. Principles of Instrumental Analysis, 6th edn.,
Belmont, CA: Thomson Brooks/Cole, pp. 169–173.
Egorov, N.S. 1985. Antibiotics: A Scientific Approach. Moscow, Russia: Mir Publishers.
Elvin-Lewis, M. and Lewis, W.H. 1995. New concepts and medical and dental ethnobotany.
In Ethnobotany Evolution of a Discipline, Schultes, R. and Von Reis, S., eds. Portland, OR: Discords
Press, pp. 303–310.
Eriko, T. 2002. g-Butyrolactones: Streptomyces signalling molecules regulating antibiotic production
and differentiation. Opin. Microbiol. 9:287–294.
Erturk, O. 2006. Antibacterial and antifungal activity of ethanolic extracts from eleven spice plants.
Biologia. (Bratisl) 61(3):275–278.
Esimone, C.O. 1997. Antimicrobial properties of extracts from the lichen Ramalina farinacea. Masters
Degree Dissertation, Department of Pharmaceutics, University of Nigeria, Nsukka, Nigeria.
Fernando, P. 2006. The history delivery of antibiotics from microbial nature products. Biochem
Pharmacol. 71:981–990.
Food and Drug Administration U.S. Approval statistics of oncology drugs. 2008. http://www.
accessdata.fda.gov/scripts/cder/onctools/statistics.cfm. Accessed November 5, 2002.
Gandhi, N., Sawant, S., and Joshi, J. 1976. Studies on the lipozyme-catalyzed synthesis of butyl lau-
rate. Biotechnol. Bioeng. 46:1–12.
Gandhimathi, R., Seghal Kiran, G., Hema, T.A., Selvin, J., Rajeetha Raviji, T., and Shanmughapriya, S.
2009. Production and characterization of lipopeptide biosurfactant by a sponge-associated
marine actinomycetes Nocardiopsis alba MSA10. Bioprocess Biosyst. Eng. 32:825–835.
Gayathri, V., Madhanraj, P., and Panneerselvam, A. 2011. Diversity, antibacterial activity and
molecular characterization of actinomycetes isolated from saltpan region of Kodiakarai,
Nagapattinam district. Asian J. Pharm. Tech. 1(3):79–81.
Georges, M. and Pandelai, K.M. 1949. Investigations on plant antibiotics. IV. Further search for anti-
biotic substances in Indian medicinal plants. Ind. J. Med. Res. 37:169–181.
Grein, A. and Meyers, S.P. 1958. Growth characteristics and antibiotic production of actinomycetes
isolated from littoral sediments and materials suspended in seawater. J. Bacteriol. 76:457–463.
Grove, D.C. and Randall, W.A. 1955. Assay Methods of Antibiotics: A Laboratory Manual [Antibiotics
Monographs, 02]. New York: Medical Encyclopedia, Inc., p. 80.
Hayakawa, M. and Nonomura, H. 1987. Humic acid-vitamin agar, a new medium for the selective
isolation of soil actinomycetes. J. Ferment. Technol. 65:501–509.
Holler, F.J., Skoog, D.A., and Crouch, S.R. 2007. Principles of Instrumental Analysis, 6th edn. Belmont,
CA: Thomson Brooks/Cole, pp. 169–173.
Holt, J.G. 1989. Streptomycetes and related genera. In Bergey’s Manual of Systematic Bacteriology,
Williams, S.T. and Sharpe, M.E., eds. Baltimore, MD: Williams and Wilkins, p. 747.
Hsu, S.C. and Lockwood, J.L. 1975. Powdered chitin agar as a selective medium for enumeration of
actinomycetes in water and soil. Appl. Microbiol. 29(3):422–426.
Jensen, P.R., Gontang, E., Mafnas, C., Mincer, T.J., and Fenical, W. 2005. Culturable marine actinomy-
cetes diversity from tropical Pacific Ocean sediments. Environ. Microbiol. 7(7):1039–1048.
Jones, K.L. 1949. Fresh isolates of actinomycetes in which the presence of sporogenous aerial mycelia
is a fluctuating characteristic. J. Bacteriol. 57:141–145.
Kathiresan, K., Balagurunathan, R., and Masilamani Selvam, M. 2005. Fungicidal activity of marine
actinomycetes against phytopathogenic fungi. Ind. J. Biotechnol. 4:271–276.
Kelecom, A. 2002. Secondary metabolites from marine microorganisms. Ann. Braz. Acad. Sci.
74(1):151–170.
Kelly, K.L. and Judd, D.B. 1976. National Bureau of Standards, Spec. Publ. 440, NBS/ISCC Centroids.
U.S. Government Printing Office, Washington, DC, pp. 189.
Kinchington, D., Kangro, H., and Jeffries, K.J. 1995. Design and testing of antiviral compounds. In
Medical Virology: A Practical Approach, Desselberger, U., ed. New York: Oxford University Press,
pp. 147–171.
King, A. and Brown, D.F.J. 2001. Quality assurance of antimicrobial susceptibility testing by disc
diffusion. J. Antimicrob. Chemother. 48:71–76.
Kosmachev, A.E. 1954. Thermophilic actinomycetes and their antagonistic properties. PhD thesis,
Institute of Microbiology, Moscow, Russia (in Russian).
Krasilnikov, N.A. 1960. Taxonomic principles of the actinomycetes. J. Bacteriol. 79:65–74.
Kucera, L.S. and Hermann, E.E. 1965. Annual Report in Medicinal Chemistry. London, U.K.: Academic
Press, pp. 129.
Kuster, E. and Williams, S.T. 1964. Production of hydrogen sulphide by Streptomyces and methods for
its detection. Appl. Microbiol. 12:46–52.
Lam, K.S. 2006. Discovery of novel metabolites from marine actinomycetes. Curr. Opin. Microbiol.
9:245–251.
Lechavalier, H.A., Lechavalier, M.P., and Gerber, N.N. 1971. Chemical composition as a criterion in
the classification of actinomycetes. Adv. Appl. Microbiol. 14:47–72.
Likar, M. and Japelj, M. 1977. Animal corneas as tools for the testing of antiviral compounds. Ann.
N.Y. Acad. Sci. 284:182–189.
Lindenbein, W. 1952. Uber einige chemisch interresante Actinomycetenstamme und ihre
Klassifizierung. Arch. Mikrobiol. 17:361–383.
Lingappa, Y. and Lockwood, J.L. 1961. A chitin medium for isolation, growth and maintenance of
actinomycetes. Nature 189:158–159.
Lingappa, Y. and Lockwood, J.L. 1962. Chitin media for selective isolation and culture of actinomy-
cetes. Phytopathology 52:317–323.
Magarvey, N.A., Keller, J.M., Bernan, V., Dworkin, M., and Sherman, D.H. 2004. Isolation and char-
acterization of novel marine-derived actinomycete taxa rich in bioactive metabolite. Appl.
Environ. Microbiol. 70(12):7520–7529.
Mincer, T.J., Jensen, P.R., Kauffman, C.A., and Fenical, W. 2002. Widespread and persistent popula-
tions of a major new marine actinomycete taxon in ocean sediments. Appl. Environ. Microbiol.
68(10):5005–5011.
Muiru, W.M., Mutitu, E.W., and Mukunya, D.M. 2012. Characterization of antibiotic metabolites
from actinomycete isolates. Afr. Crop Sci. Conf. Proc. 8:2103–2107.
Nonomura, H. 1974. Key for classification and identification of 458 species of the Streptomyces
included in ISP. J. Ferment. Technol. 52:78–92.
Ogunmwonyi, I.H., Mazomba, N., Mabinya, L., Ngwenya, E., Green, E., Akinpelu, D.A., and Okoh,
A.I. 2010. Studies on the culturable marine actinomycetes isolated from the Nahoon beach in
the Eastern Cape Province of South Africa. Afr. J. Microbiol. Res. 4(21):2223–2230.
Olano, C., Méndez, C., and Salas, J.A. 2009. Antitumor compounds from marine actinomycetes. Mar.
Drugs. 7:210–248.
Parks, L.W. and Casey, W.M. 1996. Fungal sterols. In Lipids of Pathogenic Fungi, Prasad, R. and
Ghannoum, M., ed. Boca Raton, FL: CRC Press, pp. 63–82.
Porter, J.N., Wilhelm, J.J., and Tresner, H.D. 1960. Method for the preferential isolation of actinomy-
cetes from soils. Appl. Microbiol. 8:174–178.
Prescott, L.M., Harley, J.P., and Klein, D.A. 2002. Microbiology, 5th edn. New Delhi, India: Tata
McGraw-Hill Company.
Rajauria, G. and Abu-Ghannam, N. 2013. Isolation and partial characterization of bioactive fuco-
xanthin from Himanthalia elongata brown seaweed: A TLC-based approach. Int. J. Anal. Chem.
Article ID 802573: 1–6. doi: 10.1155/2013/802573.
Ravikumar, S., Gnanadesigan, M., Thajuddin, N., Deepan Chakkaravarthi, V.S., and Beula Banerjee,
M. 2010. Anticancer property of sponge associated actinomycetes along Palk Strait. J. Pharm.
Res. 3(10):2415–2417.
Reddy, N.G., Ramakrishna, D.P.N., and Raja Gopal, S.V. 2011. A morphological, physiological and
biochemical studies of marine Streptomyces rochei (MTCC 10109) showing antagonistic activity
against selective human pathogenic microorganisms. Asian J. Biol. Sci. 4(1):1–14.
Rehacek, Z. 1959. Isolation of actinomycetes and determination of the number of their spores in soil.
Microbiology 28:220–225.
Remya, M. and Vijayakumar, R. 2008. Isolation and characterization of marine antagonistic actino-
mycetes from West Coast of India. Facta Universitatis: Med. Biol. 15(1):13–19.
Rojas, A., Hernandez, L., Pereda-Miranda, R., and Mata, R. 1992. Screening for antimicrobial
activity of crude drug extracts and pure natural products from Mexican medicinal plants.
J. Ethnopharmacol. 35:275–283.
Rowe, J.G., Saenz, M.T., and Garcia, M.D. 1989. Antibacterial activity of some lichens from Southern
Spain. Ann. Pharm. Francaises. 47:89–94.
Sambamurthy, K. and Ellaiah, P. 1974. A new Streptomycin producing Neomycin (B and C) complex,
S. marinensis (part-1). Hind. Antibiot. Bull. 17:24–28.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Savoia, D. 2012. Plant-derived antimicrobial compounds: Alternatives to antibiotics. Future Microbiol.
7(8):979–990.
Seong, C.N., Choi, J.H., and Baik, K.S. 2001. An improved selective isolation of rare actinomycetes
from forest soil. J. Microbiol. 39(1):17–23.
Sethi, P.D. 2001. High Performance Liquid Chromatography: Quantitative Analysis of Pharmaceutical
Formulations, 1st edn. New Delhi, India: CBS Publishers and Distributors.
Sharma, M. 2014. Actinomycetes: Source, identification and their applications. Int. J. Curr. Microbiol.
App. Sci. 3(2):801–832.
Shirling, E.B. and Gottlieb, D. 1966. Methods for characterization for Streptomyces species. Int. J. Syst.
Bacteriol. 16:313–340.
Silva, O., Duarte, A., Cabrita, J., Pimentel, M., Diniz, A., and Gomes, E. 1996. Antimicrobial activity
of Guinea-Bissau traditional remedies. J. Ethnopharmacol. 59:55–59.
Sloan, B.J., Kielty, J.K., and Miller, F.A. 1977. Effect of a novel adenosine deaminase inhibitor upon
the antiviral activity in vitro and in vivo of vidarabine for DNA virus replication. Ann. N. Y.
Acad. Sci. 284:60–80.
Sujatha, P., Bapi Raju, K.V.V.S.N., and Ramana, T. 2005. Studies on a new marine Streptomycete BT-408
producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus
aureus. Microbiol. Res. 160(2):119–126.
Swallow, D.L. 1977. Progress in medicinal chemistry, Ellis, G.P. and West, G.B. eds. London, U.K.:
Butterworth Group, p. 120.
Swami, M.B., Sastry, M.K., Nigudkar, A.G., and Nanda, R.K. 1983. Correlation of HPLC retention
time with structure and functional group of macrolide polyene. Hind. Antibiot. Bull. 25:81–99.
Takahashi, S., Miyaoka, H., Tanaka, K., Enokita, R., and Okazaki, T. 1993. Milbemycins alpha11,
alpha12, alpha13, alpha14 and alpha15: A new family of milbemycins from Streptomyces hygro-
scopicus sub sp. aureolacrimosus. J. Antibiot. 46(9):1364–1371.
Tsao, P.H., Leben, C., and Keitt, G.W. 1960. An enrichment method for isolating actinomycetes that
produce diffusible antifungal antibiotics. Phytopathology 50:88–89.
Van Wyk, B.E. and Wink, M. 2004. Medicinal Plants of the World: An Illustrated Scientific Guide to
Important Medicinal Plants and Their Uses. Portland, OR: Timber Press.
Vijayakumar, R. 2006. Studies on actinomycetes from Palk Strait region of Tamilnadu coast with
reference to antibiotic production. PhD thesis, Bharathidasan University, Tiruchirappally,
Tamilnadu, India, p. 66.
Vijayakumar, R., Gopika, G., Dhanasekaran, D., and Saravanamuthu, R., 2012a. Marine actino-
mycetes: A potential biocontrol agent against Colletotrichum vulgatum, Thielaviopsis paradoxa,
Trichoderma virida and Fusarium semitectum. Arch. Phytopathol. Pl. Prot. 45(9):1010–1025.
Vijayakumar, R., Muthukumar, C., Thajuddin, N., Panneerselvam, A., and Saravanamuthu, R. 2007.
Studies on the diversity of actinomycetes in the Palk Strait region of Bay of Bengal, India.
Actinomycetologica 21(2):59–65.
Vijayakumar, R., Panneer Selvam, K., Muthukumar, C., Thajuddin, N., Panneerselvam, A., and
Saravanamuthu, R. 2012b. Optimization of antimicrobial production by the marine actino-
mycetes Streptomyces afghaniensis VPTS3–1 isolated from Palk Strait, East Coast of India. Ind.
J. Microbiol. 52(2):230–239.
Vijayakumar, R., Panneer Selvam, K., Muthukumar, C., Thajuddin, N., Panneerselvam, A., and
Saravanamuthu, R. 2012c. Antimicrobial potentiality of a halophilic strain of Streptomyces
sp. VPTSA18 isolated from the saltpan environment of Vedaranyam, India. Ann. Microbiol.
62(3):1039–1047.
Vijayakumar, R., Panneerselvam, A., and Thajuddin, N. 2013. Marine actinobacteria—A potential
source for antifungal compounds. In Marine Pharmacognosy: Trends and Applications, Kim,
S.K., ed. Busan, South Korea: CRC Press, pp. 231–252.
Von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., and Häbich, D. 2006. Medicinal chemistry
of antibacterial natural products—Exodus or revival. Angew. Chem. Int. Ed. 45(31):5072–5129.
Wakisaka, Y., Kawamura, Y., Yasuda, Y. Koizumi, K., and Nishimoto, Y. 1982. A selective isolation
procedure for Micromonospora. J. Antibiot. 35:822–836.
Waksman, S.A. 1943. Production and activity of streptothricin. J. Bacteriol. 45:299–310.
Waksman, S.A. 1947. What is an antibiotic or an antibiotic substance? Mycologia 39(5):565–569.
doi:10.2307/3755196. JSTOR 3755196. PMID 20264541.
Waksman, S.A. 1961. The Actinomycetes: Classification, Identification and Descriptions of Genera and
Species, Vol. II. Baltimore, MD: Williams and Wilkins Co., p. 363.
Waksman, S.A. and Henrici, A.T. 1943. The nomenclature and classification of actinomycetes.
J. Bateriol. 46:337–341.
Waksman, S.A., Horning, E.S., Welsch, M., and Woodruff, H.G. 1941. The distribution of antagonistic
actinomycetes in nature. Soil. Sci. 54:281–296.
Waksman, S.A. and Schatz, A. 1946. Production of antibiotic substances by actinomycetes. Ann.
N. Y. Acad. Sci. 48:73–86.
Yang, R., Zhang, L.P., Guo, L.G., Shi, N., Lu, Z., and Zhang, X. 2008. Nocardiopsis valliformis sp. nov.,
an alkaliphilic actinomycete isolated from alkali lake soil in China. Int. J. Syst. Evol. Microbiol.
58:1542–1546.
You, J., Xue, X., Cao, L., Lu, X., Wang, J., Zhang, L., and Zhou, S. 2007. Inhibition of Vibrio biofilm for-
mation by a marine actinomycete strain A66. Appl. Microbiol. Biotechnol. 76(5):1137–1144.
Zabriskie, T.M. and Jackson, M.D. 2000. Lysine biosynthesis and metabolism in fungi. Nat. Prod. Rep.
17(1):85–97.
Zin, N.M., Sarmin, N.I., Ghadin, N., Basri, D.F., Sidik, N.M., Hess, W.M., and Strobel, G.A. 2007.
Bioactive endophytic streptomycetes from the Malay Peninsula. FEMS Microbiol. Lett. 274:83–88.
chapter eight
Novel antimicrobial and anticancer

drugs from bacteria
Ranjith N. Kumavath
Contents
8.1 Introduction......................................................................................................................... 143
8.1.1 Production of secondary metabolites from aromatic amino acids.................. 143
8.2 Indole terpenoid ethers and esters................................................................................... 144
8.2.1 Rhodethrin............................................................................................................... 145
8.2.2 Rubrivivaxin............................................................................................................ 145
8.2.3 Rhodestrin............................................................................................................... 145
8.2.4 2.4.3,6-Disubstituted indoles A, B, and C............................................................ 146
8.3 Indole terpenoid ethers and esters................................................................................... 146
8.3.1 Indole-3-acetic acid................................................................................................. 146
8.3.2 Indigo........................................................................................................................ 146
8.3.3 Violacein................................................................................................................... 146
8.3.4 Indolmycin............................................................................................................... 147
8.4 Phenols and its derivatives................................................................................................ 147
8.4.1 Alkyl esters of gallic acid....................................................................................... 147
8.5 Polyketides........................................................................................................................... 148
8.5.1 Daryamides A, B, and C........................................................................................ 148
8.6 Piericidin and derivatives.................................................................................................. 148
8.6.1 Piericidins C7 and C8.............................................................................................. 149
8.7 Conclusion........................................................................................................................... 149
Acknowledgment......................................................................................................................... 149
References...................................................................................................................................... 149
8.1 Introduction
8.1.1 Production of secondary metabolites from aromatic amino acids
Microorganisms such as bacteria and fungi are promising sources for structurally diverse
and potent bioactive compounds (Laatsch, 2006; Lebar et al., 2007). Actinomycetes are
mostly responsible for the production of half of the discovered secondary metabolites
(Bull, 2004; Berdy, 2005), such as antibiotics (Strohl, 2004), antitumor agents (Olano et al.,
2009), immunosuppressive agents (Mann, 2001), and enzymes (Pecznska-Czoch and
Mordarski, 1988; Oldfield et al., 1998). The studies on metabolism of aromatic amino acids in
microorganisms show that they evolved secondary metabolic pathways with the capacity
to produce compounds displaying an effective array of pharmacological applications
143
including pigments, toxins, enzyme inhibitors, pesticides, herbicides, antiparasitics, myco-

toxins, antitumor agents, antibiotics, cytotoxicity activities, and growth promoters of ani-
mals and plants. Structurally diverse classes of secondary metabolites were produced by
the marine Bacillus species, such as lipopeptides, polypeptides, macrolactones, fatty acids,
polyketides, lipoamides, and isocoumarins (Hamdache et al., 2011). These compounds
show a wide range of biological activities such as antimicrobial, anticancer, antialgal, and
antiperonosporomycetal (Brauzzi et al., 2011).
These bioactive compounds had a wide range of applications such as chemothera-
peutic agents for the treatment of human and animal diseases (Schwartsmann et al., 2001;
Jha and Zi-rong, 2004). There is a continuing demand for novel bioactive compounds
for the treatment of drug-resistant human and animal pathogens (Nathan, 2004; von
Nussbaum et al., 2006) and the management of destructive pathogens of crops, which
are insensitive to existing chemical pesticides (Islam, 2005; Islam et al., 2011; Islam and
Hossain, 2013). Another application for the bioactive compounds is quorum sensing
(QS), which helps the bacteria for cell-to-cell communication using intermediary sub-
stances, which are excreted from bacterial cells into the environment. The metabolite
concentration reaches the threshold level in the bacteria; results in certain types of gene
expression resulting in bioluminescence, biofilm formation, swarming motility, anti-
biotic biosynthesis, and virulence factor production (Falcao et al., 2004; Antunes et al.,
2010); and is triggered by the metabolite (Kazuhiro et al., 2013). The strong impact of
classical natural compounds with advanced microbial genetics and bioinformatics will
help to overcome supply and sustainability issues from the past and to promote bioac-
tive substances from microbial species.
8.2 Indole terpenoid ethers and esters

The novel indole terpenoids, namely, sphestrin (Sunayana et al., 2005a) and rhodestrin
(Sunayana et al., 2005b), were isolated from the culture supernatants of Rhodobacter
sphaeroides OU5 grown in the presence of 2-aminobenzoate. Indole terpenoid esters/
ethers are recent discoveries of biomolecules produced by a purple nonsulfur bacterium
Rhodobacter sphaeroides OU5 having phytohormonal activity. They were produced in the
presence of a processor like 2-aminobenzoate (Nanda et al., 2000) or aniline (Vijay et al.,
2006). There are a few reports of indole ester biosynthesis in plant systems and micro-
organisms. 4-Chloroindole-3-acetic acid and its esters were chemically synthesized from
2-chloro-6-nitrotoluene as the base material (Katayama, 2000). Esters of indole-3-acetic
acid (IAA) were extracted and purified from the liquid endosperm of immature fruits
of various species of the horse chestrint (Aesculus parviflora, Aesculus baumannii, Aesculus
pavia rubra, and Aesculus pavia humulis). The liquid endosperm contained at least 12 chro-
matographically distinct esters. One of these compounds were purified and characterized
as an ester of IAA and myoinositol (Domagaski et al., 1987). Indole-3-acetyl-myo-inositol
esters have been demonstrated as an endogenous component of etiolated Zea mays shoot
tissue. The amount of indole-3-acetyl-myo-inositol esters in the shoots was determined to
be 74 nmoles/kg fresh weight (Chisnell, 1984).
Indoles are extensively produced by the chemical industry for a variety of appli-
cations including pharmaceuticals, pesticides, and dyes, and they are widely used as
analgesics (Magniez et al., 1995), anti-inflammatory agents (Verma et al., 1994), antihyper-
tensive (Frishman, 1983), anti-HIV compound (Britcher et al., 1995), and phytohormones
(Elsorra et al., 2003). Many indole esters were found as COX-2 selective enzyme inhibitors
(Olgen and Nebioglu, 2002; Olgen et al., 2007); they were also found to have anti–lipid
Chapter eight: Novel antimicrobial and anticancer drugs from bacteria 145
peroxidation (LP) activity and antisuperoxide formation (SOD) (Olgen and Coban, 2003,
Olgen et al., 2007). Indole esters were also found to have more phytohormonal activity
than their corresponding acids in the auxin bioassay (Katayama, 2000).
8.2.1 Rhodethrin
Rhodethrin, which is a terpenoid metabolite, is isolated from the culture superna-
tants of Rhodobacter sphaeroides OU5 when grown on L-tryptophan as a sole source of
nitrogen under photoheterotrophic conditions. The International Union of Pure and
Applied Chemistry (IUPAC) name of the compound is 3-hydroxy-6-(1H-indol-3-yloxy)-
4-methylhexanoic acid. This compound better acts as a phytohormone, along with its
cytotoxic activity against SUP-T1 lymphoma and Colo-125 cancer cell lines at 10 nM
(Ranjith et al., 2007).
OH
O COOH
N
H
8.2.2 Rubrivivaxin
The Rubrivivax benzoatilyticus JA2 bacterial species produces a phenol terpenoid
called rubrivivaxin. The IUPAC name of the compound is 3,4-dihy-droxy-5-carboxy-
3-
methylpentyl ester. The significance of cyclooxygenase-I inhibitory, antimicrobial,
cytotoxic activities against U937 (human leukemic monocyte lymphoma), and Jurkat
(T lymphocyte) cell lines is conferred by rubrivivaxin (Ranjith et al., 2011).
OH
COOH
O O
OH
OH
8.2.3 Rhodestrin
Rhodestrin is a phytohormone isolated from the metabolite of anthranilate pho-
tobiotransformation by Rhodobacter sphaeroides OU5. The IUPAC name of the
compound is 24-hydroxy-2,6,10,14,19 pentamethyl tetrecosa-2,4,6,8,10,12,14,16,18 nonenyl-
2(hydroxymethyl)-1H-indole-3-carboxylate. This molecule shows antitumor and antimi-
crobial activities (Sunayana et al., 2005).
OH
O
O
N OH
H m/z 595 [MH]+
8.2.4 2.4.3,6-Disubstituted indoles A, B, and C

The rich sources of compounds for the development of pharmaceutical agents were found
in the marine microorganisms (Fenical, 1993). One of them is marine Streptomyces sp. strain
BL-49–58–005 that was identified for the production of the 3,6-disubstituted indole com-
pounds, which are cytotoxic in nature. These compounds are in three forms based upon
the functional group substituted at the first position of the indole ring of the pyrimidine
structure (Sanchez Lopez et al., 2003).
CH2OH CH = NOH C N
CH3 CH3
CH3 N
N N
CH3 H CH3 H CH3 H
3,6-Disubstituted indole A 3,6-Disubstituted indole B 3,6-Disubstituted indole C
8.3 Indole terpenoid ethers and esters

8.3.1 Indole-3-acetic acid
Light/horseradish peroxidase (HRP) activation has been suggested as a new photody-
namic cancer therapy by forming free radicals (Dong et al., 2006) such as indolyl and
peroxyl radicals (Folkes et al., 2002), which can cause LP. The combination of IAA and HRP
shows cytotoxic to mammalian cells including G361 human melanoma cells (Domagalski
et al., 1987; Dong et al., 2006) and human pancreatic cancer BXPC-3 cells (Chen et al., 2005).
8.3.2 Indigo
The blue dye indigo has been known since prehistoric times and is still one of the most eco-
nomical important textile dyes; the first report of microbial indigo production was recorded
in 1928 (Gray, 1928). It is biosynthesized in bacteria via the oxidation of indole by a naphtha-
lene dioxygenase and subsequent oxidation and dimerization (Ensley et al., 1983). The desire
to achieve a competitive alternative to the chemical production of indigo rejuvenated interest
in microbial indigo production (Murdock et al., 1993) since many microorganisms expressing
both monooxygenase (Allen et al., 1997) and dioxygenase (Murdock et al., 1993) during the
growth of aromatic hydrocarbons have been shown to transform indole to indigo, and the
production of these has focused on the naphthalene dioxygenase from Pseudomonas putida
PpG7 expressed in Escherichia coli. Some of the genes of indigo biosynthetic pathway have
been cloned and used to construct “engineering bacteria” with this kind of bacteria; more
efficient fermentation systems for indigo production have been exploited (Han et al., 2008).
O H
N
N
H O
8.3.3 Violacein
Chromobacterium violaceum was first reported as an isolate from wet rice paste; one of
the characteristics of this microorganism is the ability to produce a purple (deep violet)
pigment known as violacein under aerobic conditions. The biological role of violacein in
Chromobacterium violaceum, as well as its biosynthesis pathway, was well reported (DeMoss
and Evans, 1959), in addition to the role of tryptophan and other indole derivatives
(DeMoss and Evans, 1960; Hoshino et al., 1987; Hoshino and Ogasawara, 1990; Duran et al.,
1994; Momen and Hoshino, 2000). Tryptophan appears to be the only precursor m olecule
in violacein biosynthesis; its production is apparently essential for pigment production in
Chromobacterium violaceum (Vasconcelos et al., 2003; Regina and Creczynski-Pasa, 2004).
The IUPAC name and molecular mass of violacein are (3-[1,2-dihydro-5-(5-hydroxy-
1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ylidene]-1,3-dihydro-2H-indol-2-one) and [m/z 343.34],
respectively. Violacein has attracted interest owing to its important multiple biological
activities and pharmacological potentials such as antibiotic, bactericide (Duran et al., 1983),
trypanocide (Duran et al., 1994), antitumoral (Melo et al., 2003; Ferreira et al., 2004; Saraiva
et al., 2004), antiviral (Kodach et al., 2006), and genotoxic (Andrighetti et al., 2003) proper-
ties, as well as its antioxidant efficiency against oxygen- and nitrogen-reactive species as a
scavenger of hydroxyl, superoxide, and nitric oxide radicals (Konzen et al., 2006). In addi-
tion, it is capable of inducing apoptosis in cancer cell cultures (Duran and Menck, 2001)
and effective against a panel of neoplastic cell lines, including leukemia lineage cancer
diseases (Melo et al., 2003).
H
N O
HO
N N
O
H H
8.3.4 Indolmycin
Indolmycin is a secondary metabolite produced by Streptomyces griseus ATCC 1248 (for-
mally Streptomyces albus BA 3972A), which was isolated from a sample of African soil.
Indolmycin completely inhibits bacterial tryptophanyl-tRNA synthetase enzyme (Makoto
et al., 2002), and it exhibits antimicrobial activity against gram-positive and gram-negative
bacteria. Recently, researchers have shown that indolmycin is active against Mycobacteria
and Helicobacter pylori, which is known as a major causative agent of chronic active gastritis.
O
N
H3C
CH3
O N
H H
NH
8.4 Phenols and its derivatives

8.4.1 Alkyl esters of gallic acid
The chief source for obtaining gallic acid is through the hydrolysis of plant-based prod-
ucts like tannins (Inoue et al., 1995). Microbial production of gallic acid was also reported
using tannic acid as substrate (Kar et al., 1999). The esters iso-amyl-(iAG), n-amyl-(nAG),
iso-butyl-(iBG), n-butyl-(nBG), and isopropyl gallate (iPG) were chemically synthesized
from gallic acid (Christian et al., 2007). Gallic acid (3,4,5-trihydroxybenzoic acid) is an
industrially important phenol and finds its applications in various fields (Kar et al., 1999).
O
HO
OH
HO
OH
Alkyl esters of gallate are an important group of biogenic molecules reported from
plants (Yang et al., 2003), bee propolis (Ahn et al., 2004), and yeasts like Candida (Stevenson
et al., 2007). These molecules are of biotechnological significance since they are known to
have antioxidant (Chen and Ho, 1997), anticancer (Samaha et al., 1997; Li et al., 2003), anti-
HIV (Burke et al., 1995), and antifungal/microbial (Tawata et al., 1996) activities. Alkyl
esters of gallic acid have antiviral, antibacterial, and antifungal properties (Fujita and
Kubo, 2002) specifically against gram-positive bacteria (Kubo et al., 2004).
8.5 Polyketides
Polyketides are the products of repetitive condensations of the small carboxylic acid units
similar to fatty acid synthesis. These are synthesized by the polyketide synthase, which
is a mono- or bifunctional enzyme (Staunton and Weissman, 2001). These compounds are
preferentially isolated from the marine Streptomyces sp. JP95 strains (Li and Piel, 2002).
These compounds show functional and structural variabilities; these molecules may be of
simple to complex structures.
8.5.1 Daryamides A, B, and C

Daryamides are the class of polyketides that act as antibiotic and antitumor agents, iso-
lated from marine-derived actinobacteria (Ratnakar et al., 2006). These compounds are of
different types based upon the complex structures designated: A = 1, B = 2, and C = 3.
O H O H O H
1
N N N
7΄
5 3΄ 5΄
3 O O O
HO
HO 7 HO HO
9 O O O
H 2N H2N H2N
1 2 3
8.6 Piericidin and derivatives

Piericidin are the group of molecules found in the marine actinomycete and Streptomyces sp.
These molecules mostly act as anti-QS, antimicrobial (Kazuhiro et al., 2013), and antitu-
mor agents. Among these compounds, piericidin C7 and C8 are mostly distributed in the
marine microorganisms (Yoichi et al., 2007; Selvakumar, 2010).
8.6.1 Piericidins C7 and C8

The marine Actinomycete sp. produces these antitumor compounds against retinoblas-
toma, and these molecules act as antimicrobial agents for the screening against marine
microorganisms. These molecules are selectively cytotoxic against rat glial cells (Yoichi
et al., 2007).
CH3 R CH3 CH3 CH3 CH3
CH3
H3CO N
O
OH
H3CO CH3
Piericidin C7 R = H
OH Piericidin C8 R = C H3
CH3 R CH3 CH3 CH3
H3CO N CH3
OH
H3CO CH3
Piericidin A1 R = H
OH Piericidin A2 R = C H3
8.7 Conclusion
Microbes are the large repositories of bioactive compounds with different structural and
functional forms, namely, sugars, acids, esters, ethers, terpenoids, peptides, proteins, and
nucleopeptides, which are meant for the immense applications in the treatment of the wide
range of disease types, such as anticancer, anti-inflammatory, antimicrobial, antiviral, and
antifungal. As long as the microorganisms are treated with the classical antibiotics, they
are supposed to be developing the nature of resistance to the specific disease types. Now,
it is time to prepare the new drugs and to make modifications in the structural character-
istic of the target-based drug design for the specific disease. Hence, the utilization of these
vast resources is poorly understood in microorganisms. However, the microbial screening
provides an opportunity to manipulate new-generation drugs for efficient antimicrobial
and anticancer approaches.
Acknowledgment
The authors would like to thank SERB, Govt. of India.
References
Ahn, M.R., Kumazawa, S., Hamasaka, T., Bang, K.S., and Nakayama, T. 2004. Antioxidant activ-
ity and constituents of propolis collected in various areas of Korea. J Agric Food Chem. 52:
7286–7292.
Allen, C.C.R., Boyd, D.R., Larkin, M.J., Reid, K.A., Sharma, N.D., and Wilson, K. 1997. Metabolism
of naphthalene, 1-naphthol, indene, and indole by Rhodococcus sp. strain NCIMB12038. Appl
Environ Microbial. 63: 151–155.
Andrighetti, F.C.R., Antonio, R.V., Creczynski, P.T.B., Barandi, C.R.M., and Simoes, C.M.O. 2003. The
antiviral and cytotoxic activities of violacein using three different methods. MemInst Oswaldo
Cruz. 98: 834–848.
Antunes, L.C., Ferreira, R.B., Buckner, M.M., and Finlay, B.B. 2010. Quorum sensing in bacterial viru-
lence. Microbiology. 156(8): 2271–2282.
Baruzzi, F., Quintieri, L., Morea, M., and Caputo, L. 2011. Antimicrobial compounds produced by
Bacillus spp. and applications in food. In: Science Against Microbial Pathogens: Communicating
Current Research and Technological Advances, Vilas, A.M., Ed. Formatex, Badajoz, Spain, pp.
1102–1111.
Bassler, B.L. and Losick, R. 2006. Bacterially speaking. Cell. 125(2): 237–246.
Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiotics (Tokyo). 58: 1–26.
Britcher, S.F., Lymna, W.C., Young, S.D., Grey, V.E., and Tran, L.D. 1995. 3-Substituted heterocyclic
indoles as inhibitors of HIV reverse transcriptase. Briton UK Pat Appl GB2. 282: 808.
Bull, A.T. 2004. Microbial Diversity and Bioprospecting. ASM Press, Washington, DC.
Chen, H., Li, Y.L., Tu, S.S., Lei, N., Ling, Y., Xiao, Y.H., Jing, S.H., Li, P.S., Yu, L., and Lu, S.S. 2005.
Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination
with horseradish peroxidase. World J Gastroenterol. 11(29): 4519–4523.
Chen, J.H. and Ho, C.T. 1997. Antioxidant activities of caffeic acid and its related hydroxycinnamic
acid compounds. J Agric Food Chem. 45: 2374–2378.
Chisnell, J.R. 1984. Myo-inositol esters of indole-3-acetic acid are endogenous component of Zea
mays L. shoot tissue. Plant Physiol. 74: 278–283.
Christian, F., Mario, P., Gianni, C., Sergio, M., Enrique, R., Jorge, M., Antonio, M., Juan, D.M., and
Jorge, F. 2007. Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated
rat hepatocytes. Comp Biochem Physiol A. 146: 520–527.
DeMoss, R.D. and Evans, N.R. 1959. Physiological aspects of violacein biosynthesis in nonproliferat-
ing cells. J Bacteriol. 78: 583–586.
DeMoss, R.D. and Evans, N.R. 1960. Incorporation of C14-labeled substrates into violacein. J Bacteriol.
79: 729–735.
Domagalski, W., Schulze, A., and Bandurski, R.S. 1987. Isolation and characterization of esters of
indole-3-acetic acid from the liquid endosperm of the horse chestnut (Aesculus species). Plant
Physiol. 84: 1107–1113.
Dong, S.K., So, Y.K., Yun, M.J., Sang, E.J., Myo, K.K., Sun, B.K., Jung, I.N., and Kyoung, C.P. 2006.
Light-activated indole-3-acetic acid induces apoptosis in G361 human melanoma cells. Biol
Pharm Bull. 29(12): 2404–2409.
Duran, N., Antonio, R.V., Haun, M., and Pilli, R.A. 1994. Biosynthesis of a trypanocide by
Chromobacterium violaceum. World J Microbiol Biotechnol. 10: 686–690.
Duran, N., Erazo, S., and Campos, V. 1983. Bacterial chemistry-II: Antimicrobial photoproduct from
pigment of Chromobacterium violaceum. An Acad Bras Cienc. 55: 231–234.
Duran, N. and Menck, C.F. 2001. Chromobacterium violaceum: A review of pharmacological and
industrial perspective. Critical Rev Microbiol. 27: 201–222.
Elsorra, E.I., Domingo, J.I., Manuel, T., and Rainer, B. 2003. Tryptophan dependent production of
indole-3-acetic acid (IAA) affect level of plant growth promotion by Bacillus amyloliquefaciens
FZB42. Mol Plant Microbe Interact. 20(6): 619–626.
Ensley, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M.J., Wackett, L.P., and Gibson, D.T. 1983. Expression
of naphthalene oxidation genes in Escherichia coli results in biosynthesis of indigo. Science. 222:
167–168.
Falcao, J.P., Sharp, F., and Perandio, S.V. 2004. Cell-to-cell signaling in intestinal pathogens. CIIM.
5(1): 9–17.
Fenical, W. 1993. Chemical studies of marine bacteria: Developing a new resource. Chem. Rev.
93:1673–1683.
Ferreira, C.V., Bos, C.L., Versteeg, H.H., Justo, G.Z., Duran, N., and Peppelenbosch, M.P. 2004.
Molecular mechanism of violacein-mediated human leukemia cell death. Blood. 104: 1459–1464.
Fineran, P.C., Slater, H., Everson, L., Hughes, K., and Salmond, G.P. 2005. Biosynthesis of Tripyrrole
and Beta-Lactam secondary metabolites in Serratia: Integration of quorum sensing with mul-
tiple new regulatory components in the control of prodigiosin and carbapenem antibiotic pro-
duction. Mol. Microbiol. 56(6): 1495–1517.
Folkes, L.K., Greco, O., Dachs, G.U., Stratford, M.R., and Wardman, P. 2002. 5-Floroindole-3-acetic
acid: A prodrug activated by a peroxidase with potential for use in target therapy. Biochem
Pharmacol. 63: 265–272.
Frishman, W.H. 1983. Pindolol: A new β-adrenoceptor antagonist with partial activity. N Engl J Med.
308: 940–994.
Fujita, K. and Kubo, I. 2002. Antifungal activity of octyl gallate. Int J Food Microbial. 79: 193–201.
Gray, P.M.M. 1928. Indigo formation by aromatic hydrocarbon-degrading bacteria. Proc Royal Soc
London Ser B. 102: 2263–2279.
Hamdache, A., Lamarti, A., Aleu, J., and Collado, I.G. 2011. Non-peptide metabolites from the genus
Bacillus. J Nat Products. 74: 893–899.
Han, X., Wang, W., and Xiao, X. 2008. Microbial biosynthesis and biotransformation of indigo and
indigo like pigments. Chin J Biotech. 24(6): 921–926.
Hoshino, T., Kondo, T., Uchiyama, T., and Ogasawara, N. 1987. Biosynthesis of violacein: A novel
rearrangement in tryptophan metabolism with a 1,2-shift of the indole ring. Agri Biol Chem.
51: 965–970.
Hoshino, T. and Ogasawara, N. 1990. Biosynthesis of violacein: Evidence for the intermediary of
5-hydroxy-L-tryptophan and the structure of a new pigment, oxyviolacein, produced by the
metabolism of 5-hydroxytryptophan. Agri Biol Chem. 64: 2339–2345.
Inoue, M., Suzuki, R., Sakaguchi, N., Li, Z., Takeda, T., Ogihara, Y., Jiang, B.Y., and Chen, Y. 1995.
Selective induction of cell death in cancer cells by gallic acid. Bio Pharma Bull. 18: 1526–1530.
Islam, M.T. 2011. Potentials for biological control of plant disease by Lysobacter spp., with special ref-
erence to strain SB-K88. In: Bacteria in Agrobiology: Plant Growth Responses, Maheshwari, D.K.,
ed. Springer-Verlag, Berlin/Heidelberg, Germany, pp. 355–364.
Islam, M.T. and Hossain, M.M. 2013. Biological control of Peronosporomycete phytopathogens by
antagonistic bacteria. In: Bacteria in Agrobiology: Plant Disease Management, Maheshwari, D.K.,
ed. Springer-Verlag, Berlin/Heidelberg, Germany, pp. 167–218.
Islam, M.T., von Tiedemann, A., and Laatsch, H. 2011. Protein kinase C is likely to be involved in
zoosporogenesis and maintenance of flagellar motility in the peronosporomycete zoospores.
MPMI. 24: 938–947.
Jha, R.K. and Zi-rong, X. 2004. Biomedical compounds from marine organisms. Marine Drugs 2:
123–146.
Kar, B., Banerjee, R., and Bhattacharya, B.C. 1999. Microbial production of gallic acid by modified
solid-state fermentation. J Ind Micro Biotechnol. 23: 173–177.
Katayama, M. 2000. Synthesis and biological activities of 4-chloroindole-3-acetic acid and its esters.
Biosci Biotechnol Biochem. 64: 808–815.
Kazuhiro, O., Atsushi, F., Tomoe, Y., Kanako, S., Ryo, I., YojiroAnzai, P., and Fumio, K. 2013. Novel
quorum-sensing Inhibitors against Chromobacterium violaceum CV026, from Streptomyces sp.
TOHO-Y209 and TOHO-O348. Open J Med Chem. 3: 93–99.
Kodach, L.L., Bos, C.L., Duran, N., Peppelenbosch, M.P., Ferreira, C.V., and Hardwich, J.C. 2006.
Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and inhib-
its Akt-mediated signal transduction in human colorectal cancer cells. Carcinogenesis. 27:
508–516.
Konzen, M., De Marco, D., Clarissa, A.S. C., Tiago, O.V., Regina, V.A., and Tania, B.C.P. 2006.
Antioxidant properties of violacein: Possible relation on its biological function. Bioorg Med
Chem. 14(24): 8307–8313.
Kubo, I., Fujita, K., Nihei, K., and Nihei, A. 2004. Antibacterial activity of alkyl gallates against
Bacillus subtilis. J Agric Food Chem. 52: 1072–1076.
Laatsch, H. 2006. Marine bacterial metabolites. In: Frontiers in Marine Biotechnology, Proksch, P. and
Muller, W.E.G., eds. Horizon Bioscience, Norfolk, U.K., pp. 225–288.
Lebar, M.D., Heimbegner, J.L., and Baker, B.J. 2007. Cold-water marine natural products. Nat Prod
Rep. 24: 774–797.
Li, A. and Piel, J. 2002. A gene cluster from a marine Streptomyces encoding the biosynthesis of the
aromatic spiroketal polyketide Griseorhodin A. Chem. Biol. 9: 1017–1026.
Makoto, K., Ali, K., Arnold, D., Sakamoto, K., Yokoyama, S., and Soll, D. 2002. Indolmycin resistance
of Streptomyces coelicolor A3 by induced expression of one of its two tryptophanyl-tRNA syn-
thetases. J Biol Chem. 277(26): 23882–23887.
Mann, J. 2001. Natural products as immunosuppressive agents. Nat Prod Rep. 18: 417–430.
McLean, R.J.C., Whiteley, M., Stickler, D.J., and Fuqua, W.C. 1997. Evidence of autoinducer activity in
naturally occurring biofilms. FEMS Microbiol Lett. 154(2): 259–263.
Nanda Devi, P., Sasikala, C., and Ramana, C.V. 2000. Light-dependent transformation of anthranilate
to indole by Rhodobacter sphaeroides OU5. J Ind Microbiol Biotechnol. 24: 219–221.
Nathan, C. 2004. Antibiotics at the crossroads. Nature. 431: 899–902.
Nealson, K.H. 2006. Autoinduction of bacterial luciferase occurrence, mechanism and significance.
Arch Microbiol. 112(1): 73–79.
Olano, C., Mendez, C., and Salas, J.A. 2009. Antitumour compounds from marine actinomycetes.
Marine Drugs. 7: 210–248.
Oldfield, C., Wood, N.T., Gilbert, S.C., Murray, F.D., and Faure, F.R. 1998. Desulphurisation of ben-
zothiophene and dibenzothiophene by actinomycete organisms belonging to the genus
Rhodococcus, and related taxa. Antonie Van Leeuwenhoek. 74: 119–132.
Olgen, S. and Nebioglu, D. 2002. Synthesis and biological evolution of N-substituted indole esters as
inhibitors of cyclo-oxygenase-2 (COX-2). Farmaco. 57: 677–683.
Olgen, S., Zuhal, K., Ada, A., and Coban, T. 2007. Synthesis and anti-oxidant properties of novel N-H
and N-substituted propanamide derivatives. Archv der Pharmazi. 340(3): 140–146.
Pecznska-Czoch, W. and Mordarski, M. 1988. Actinomycete enzymes. In: Actinomycetes in
Biotechnology. Academic Press, London, U.K., pp. 219–283.
Ranjith, N.K., Ramana, C.V., and Sasikala, C. 2007. Rhodethrin: A novel indole terpenoid ether pro-
duced by Rhodobacter sphaeroides has cytotoxic and phytohormonal activities. Biotechnol Lett.
29: 1399–1402, doi: 10.1007/s10529–007–9413–7.
Ranjith, N.K., Ramana, C.V., and Sasikala, C. 2011. Rubrivivaxin, a new cytotoxic and cyclooxygen-
ase-I inhibitory metabolite from Rubrivivax benzoatilyticus JA2. World J Microbiol Biotechnol. 27:
11–16, doi: 10.1007/s11274–010–0420–9.
Ratnakar, N.A., Paul, R.J., Christopher, A.K., and William, F. 2006. Daryamides A-C, weakly cyto-
toxic polyketides from a marine-derived actinomycete of the genus Streptomyces strain CNQ-
085. J Nat Prod. 69: 1756–1759.
Regina, V.A. and Creczynski-Pasa, T.B. 2004. Genetic analysis of violacein biosynthesis by
Chromobacterium violaceum. Genet. Mol. Res. 3(1): 85–91.
Samaha, H.S., Kelloff, G.J., Steele, V., Rao, C.V., and Reddy, B.S. 1997. Modulation of apoptosis
by sulindac, curcumin, phenylethyl 3-methylcaffeate and 6-phenylhexyl isothiocyanate:
Apoptotic index as a biomarker in colon cancer chemoprevention and promotion. Cancer Res.
57: 1301–1305.
Sanchez Lopez, J.M., Martinez Insua, M., Perez Baz, J., Fernandez Puentes, J.L., and Canedo
Hernandez, L.M. 2003. New cytotoxic indolic metabolites from a marine Streptomyces. J Nat
Products. 66: 863–864.
Saraiva, V.S., Marshall, J.C., Cools-Lartigue, J., and Burnier Jr., M.N. 2004. Cytotoxic effects of viola-
cein in human uveal melanoma cell lines. Melanoma Res. 14: 421–424.
Schwartsmann, G., da Rocha, A.B., Berlinck, R.G.S., and Jimeno, J. 2001. Marine organisms as a
source of new anticancer agents. Lancet Oncol. 2: 221–225.
Selvakumar, D. 2010. Marine Streptomyces as a novel source of bioactive substances. World J Microbiol
Biotechnol. 26: 2123–2139.
Slater, H., Crow, M., Everson, L., and Salmond, G.P. 2003. Phosphate availability regulates biosyn-
thesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-
dependent and -independent path-ways. Mol. Microbiol. 47(2): 303–320.
Staunton, J. and Weissman, K.J. 2001. Polyketide biosynthesis: A millennium review. Nat Prod Rep.
18: 380–416.
Stevenson, D.V., Parkar, S.G., Zhang, J., Stanley, R.A., Jenson, D.J., and Cooney, J.M. 2007. Combinatorial
enzymatic synthesis for functional testing of phenolic acid ester catalyzed by Candida Antarctica
lipase B (Novozym 435®). Enzyme Microbial Technol. 40: 1078–1086.
Strohl, W.R. 2004. Antimicrobials. In: Microbial Diversity and Bioprospecting, Bull, A.T. ed. ASM Press,
Washington, DC, pp. 336–355.
Sunayana, M.R., Sasikala, C., and Ramana, V.C. 2005a. Production of novel indole ester from
2-amonobenzoate by Rhodobacter sphaeroides OU5. J Ind Microbiol Biotech. 32: 41–45.
Sunayana, M.R., Sasikala, C., and Ramana, V.C. 2005b. Rhodestrin: A novel indole terpenoid phyto-
hormones from Rhodobacter sphaeroides OU5. Biotech Lett. 27: 1897–1900.
Tawata, S., Taira, S., Kobamoto, N., Nhu, J., Ishihara, M., and Toyama, S. 1996. Synthesis and antifun-
gal activity of cinnamic acid ester. Biosci Biotechnol Biochem. 60: 909–910.
Vasconcelos, A.T.R., Almeida, D.F., Hungria, M. et al. 2003. The complete genome of Chromobacterium
violaceum reveals remarkable and exploitable bacterial adaptability. Proc Natl Acad Sci USA.
100: 11660–11665.
Verma, M., Tripathi, M., Saxena, A.K., and Shanker, K. 1994. Anti-inflammatory activity of novel
indole derivatives. Eur J Med Chem. 29: 941–946.
Vijay, S., Sunayana, M.R., Ranjith, N.K., Sasikala, C., and Ramana, V.C. 2006. Light dependent trans-
formation of aniline to indole esters by a purple bacterium Rhodobacter sphaeroides OU5. Curr
Microbiol. 52: 413–415.
Von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., and Habich, D. 2006. Antibacterial natural
products in medicinal chemistry—Exodus or revival. Angew Chem Int Ed. 45: 5072–5129.
Yang, G., Song, L., Li, K., and Hu, C. 2003. Studies on chemical constituents of Polygonum
o rientale. ZhongguoYaoxue Zazhi. 38: 338–350.
chapter nine
Bacteriocin
A natural alternative to synthetic
antibacterial antibiotics
S. Latha and Dharumadurai Dhanasekaran
Contents
9.1 Introduction......................................................................................................................... 155
9.2 Bacteriocins: A class of antimicrobial peptides.............................................................. 156
9.3 Bacteriocins versus conventional antibiotics.................................................................. 157
9.4 Classification of bacteriocins............................................................................................. 158
9.4.1 Bacteriocins from gram-negative bacteria.......................................................... 158
9.4.2 Bacteriocins from gram-positive bacteria........................................................... 161
9.4.2.1 Lactic acid bacterial bacteriocin............................................................. 161
9.4.2.2 Actinobacterial bacteriocin..................................................................... 162
9.4.3 Bacteriocins from archaea..................................................................................... 164
9.5 Potential applications of bacteriocin................................................................................ 164
9.5.1 Bacteriocins in food biopreservation................................................................... 165
9.5.2 Bacteriocins in human health............................................................................... 166
9.5.3 Bacteriocins in livestock production.................................................................... 166
9.5.4 Bacteriocins in aquaculture................................................................................... 167
9.5.5 Bacteriocins in agricultural settings.................................................................... 168
9.6 Conclusion........................................................................................................................... 169
Acknowledgment......................................................................................................................... 169
References...................................................................................................................................... 169
9.1 Introduction
Antimicrobials are unarguably one of the most important medical discoveries of the
twentieth century and function as a vital medicine for the treatment of bacterial infec-
tions in both humans and animals. Moreover, they have played a major role in the growth
and development of food-producing animals (poultry, goat, sheep, beef, and dairy cattle)
mainly by improving their efficiency in growth rate, feed utilization, mortality reduction,
etc. However, the continuous use of antibiotics led to the emergence of microbial resis-
tance, dissemination of resistant bacteria, and resistance genes to pathogenic bacteria in
both humans and animals. A major issue is that antimicrobial resistance (AMR) not only
occurs among disease-causing organisms but has also become an issue for other resident
organisms in the host. In addition, consumers are also becoming increasingly concerned
about the accumulation of drug residues in meat products of food animals (Thacker, 2013).
155
This alarming spread of resistance to classic antimicrobial agents has driven the search for
new antimicrobials that are broadly effective and less likely to induce AMR.
Bacteriophages (phages), bacterial cell wall hydrolases (BCWHs), and antimicrobial
peptides (AMPs) are some of the promising antimicrobial alternatives (Parisien et al.,
2008). Bacteriophages are highly specific and can be active against a single strain of bac-
teria. Therefore, using bacteriophage against infecting strains was suggested to control
undesirable bacterial species in mucosal systems. This approach was first developed in
the last century and showed much potential; however, it also aroused much controversy
and concern (Gillor et al., 2008). BCWHs are enzymes that degrade peptidoglycan (major
bacterial cell wall component) due to their lytic enzymatic activities, that is, attacking spe-
cific sites in the peptidoglycan network, leading to hydrolysis and consequently bacterioly-
sis (Masschalck and Michiels, 2003). Though they are well established, safe, and efficient
against antibiotic-resistant bacteria, the main obstacle to use them for clinical application
is the high production costs and not being effective on most gram-negative bacteria due to
the presence of outer membrane (Parisien et al., 2008).
AMP is another major group of prospective novel substitute to antibiotics based on their
effectiveness, safety, and enormous diversity. The bactericidal mechanism of AMP includes
formation of ion channels or pores across the cytoplasmic membrane; inhibition of cell
wall biosynthesis, ribonuclease, or deoxyribonuclease (DNase) activities; and induction of
cytoplasmic membrane perturbations by depolarization and perforation of the membrane
(Damasko et al., 2005; Parisien et al., 2008). AMPs are a large family of naturally occur-
ring peptides from various sources, having diverse structures and functionalities. Based
on their origins, they were classified into three types, namely, eukaryotic AMPs, phage-
encoded AMPs, and bacteriocins. This review summarizes the natural AMPs, b acteriocins,
by highlighting their potential as alternative to conventional antibiotics.
9.2 Bacteriocins: A class of antimicrobial peptides

Bacteriocins are proteinaceous compounds (natural AMPs) produced by a bacterium,
which are active against other bacteria. They are generally ribosomally synthesized,
although some are extensively posttranslationally modified. Bacteriocins usually have low
molecular weight (rarely over 10 kDa) and can be easily degraded by proteolytic enzymes
especially by the proteases of the mammalian gastrointestinal tract, which makes them
safe for human consumption. In general, they are cationic, amphipathic molecules as
they contain an excess of lysyl and arginyl residues. They are usually unstructured when
they are incorporated in aqueous solutions; however, they form a helical structure when
exposed to structure-promoting solvents such as trifluoroethanol and anionic phospholip-
ids membranes (Zacharof and Lovitt, 2012).
Bacteriocins are produced by all major lineages of bacteria, archaea, and constitute
a heterogeneous group of peptides with respect to the size, structure, mode of action,
antimicrobial potency, immunity mechanisms, and target cell receptors (Dobson et al.,
2012). Bacteriocin-producing bacteria (BPB) belong to different systematic groups and
occupy various ecological niches such as soil, dairy and meat products, fermented plant
products, and gastrointestinal tract of warm-blooded animals. Bacteriocin provides
them with a competitive advantage in their environment, eliminating competitors to
gain resources. They are mainly known for their bactericidal and bacteriostatic activities
against various human and animal pathogens (Hammami et al., 2007). The inhibitory
spectrum of some bacteriocins also includes food spoilage and food-borne pathogenic
microorganisms.
Chapter nine: Bacteriocin 157
Bacteriocin genes are either chromosomally or plasmid encoded with resulting toxins
employing a variety of killing mechanisms, including cytoplasmic membrane pore formation,
cell wall interference, and nuclease activity (Gillor et al., 2005; Borges et al., 2014). Additional
roles have been proposed for some bacteriocins produced by gram-positive bacteria, such as
chemical mediators in quorum sensing and communication molecules in bacterial consortia.
9.3 Bacteriocins versus conventional antibiotics

All organisms produce AMPs that represent part of the natural and innate immune sys-
tems that protect them against invading organisms. In the microbial world, the AMPs are
an important part of the defense system of bacteria, and they are referred to as bacteriocins.
They are often confused with the antibiotics that would limit their use in food and medical
applications from a legal standpoint. However, bacteriocins are clearly distinguishable from
clinical antibiotics, and also they are safe and more effective in the control of target pathogens.
An important criterion of being a bacteriocin is that they are ribosomally synthesized,
while antibiotics are made by multienzyme complexes (Nes et al., 2007). Besides, they are
produced during the primary phase of growth, whereas antibiotics are usually secondary
metabolites. The major difference between them is that bacteriocins restrict their activity
to strains of species related to the producing species and particularly to strains of the same
species; antibiotics, on the other hand, have a wider activity spectrum, and even if their
activity is restricted, this does not show any preferential effect on closely related strains
(Zacharof and Lovitt, 2012). Moreover, bacteriocins are more potent against their target
bacteria, while higher concentrations of traditional a ntibiotics are needed to kill the target
bacteria (Table 9.1). In contrast to the currently used antibiotics, bacteriocins are often con-
sidered more natural because they are thought to have been present in many of the foods
eaten since ancient times (Cleveland et al., 2001).
Table 9.1 Distinctive features of bacteriocins and conventional antibiotics

S. no. Characteristics Bacteriocins Conventional antibiotics
1. Example Penicillin Defensin
2. Chemical composition Proteinaceous Complex ring structure
3. Synthesis Ribosomal Secondary metabolite
4. Molecular Small amphipathic Small compounds <2000 Da, easy to
characteristics peptides < 10 kDa, synthesize with lower production
feasible to synthesize with cost
higher production cost
5. Activity Narrow spectrum Varying spectrum
6. Application Food Clinical
7. Interaction Sometimes docking Specific target
requirements molecules
8. Host cell immunity Yes No
9. Mechanism of target Usually adaptation Usually a genetically transferable
cell resistance or affecting cell membrane determinant affecting different sites
tolerance composition depending on the mode of action
10. Mode of action Mostly pore formation, Cell membrane or intracellular
but in a few cases targets
possibly cell wall
biosynthesis
11. Toxicity side effects Not known Yes
9.4 Classification of bacteriocins

It has been hypothesized that 99% of bacteria produce at least one bacteriocin, which provides
the basis for an optimistic view that it is only a matter of directing enough research resources
to this area to identify more bacteriocins for different bacteria (Klaenhammer, 1988). Due to
the extensive focus on bacteriocins, a number of classification schemes have been proposed,
which are largely applicable to gram-positive and gram-negative bacteriocins. Methods of
classification include producing strain (bacteria, archaea), method of production (ribosomal,
postribosomal modifications, nonribosomal), mechanism of killing (pore forming, DNase,
nuclease, murein production inhibition, etc.), genetics (large plasmids, small plasmids,
chromosomal), molecular weight and chemistry (large protein, polypeptide, with/without
sugar moiety, containing atypical amino acids like lanthionine), and resistance mechanisms
(Blinkova et al., 2003). According to their origin, bacteriocins are classified as gram-negative
bacteriocin, archaebacterial bacteriocin, and gram-positive bacteriocin (Figure 9.1).
9.4.1 Bacteriocins from gram-negative bacteria

Most bacteriocins produced by gram-negative bacteria are relatively large and range in
size from <10 to 20 kDa (roughly 400 to 700 amino acids). They differ from gram-positive
bacteriocins in two fundamental ways: (1) usually released through cell lysis and (2) depen-
dent on host regulatory pathways, like SOS regulation. Colicins are the most studied gram-
negative bacteriocins and were first discovered by Gratia (2000). They are plasmid-encoded
high-molecular-weight proteins (over 20 kDa) that are active against Escherichia coli strains
and other closely related bacteria, such as Salmonella. Over 30 types of colicins have been
identified, based on killing activity and immunity specificity (Gillor et al., 2005).
Colicins produced by E. coli can be pore-forming toxins or nuclease-type toxins. Their
operons are on plasmids and consist of colicin toxin gene, lysis gene, and immunity gene.
Target specificity is governed by a receptor domain on the colicin protein that binds a spe-
cific cell surface receptor found only on certain strains of Escherichia genus or other entero-
bacteria. Microcins (Figure 9.2a), which are also produced by E. coli, have been treated as a
Bacteriocin
Gram-positive Archaebacterial Gram-negative

bacteriocin bacteriocin bacteriocin
(colicins, pyocins,
(LAB bacteriocin) (halocin) microcins)
Class I Class II Class III Class IV

(non- (Helveticins J, (leuconocin S,
(lantibiotics) lantibiotics) lactacin) lactocin 27)
Two- Class IIa Class IIb Class IIc

Type A Type B component Pediocin-like Two-peptide One-peptide
lantibiotics lantibiotics lantibiotics
bacteriocins bacteriocins bacteriocins
(pediocin PA-1) (lactococcin G) (lacticin Q)
Figure 9.1 Classification of bacteriocin based on producers.

Val H
O S
Gly N Gly
N N H S O
Ile Gly O Gly
N Gly
Gly Gly Glu N N
Gly Gln O Gly
Gly Gly H S Ile
Gly Ser Gln
Gly Gly N His
S N Gly
Gly Gly Gly O Ser
H N N
O O
O N Gly N Gly
H N N
(a) O H O
15
5 Leu
Dha Ala Met
S
lle Leu S Gly Gly
Abu Ala His
H2N lle Dhb Ala Ala Abu Ala Lys Abu Ala Asn Met Lys Ala Abu Ala Ser lle His Val Dha Lys COOH
Pro Gly
S S 20 25 S 30
10
(b)
Gly
Gly Gly
Pro Gly 10
5
S Leu Val
S
H2N Ala Abu Phe Abu Ala Abu Ala lle
1 S Leu Glu
NH
15 Abu Dha
CH
(c) S CH
Asn
Gly Asn
Trp Gly
Tyr Ala
S Asp Trp S
Ala Ala Dhb Asn Dhb Phe D-Ala Leu Ala Ala Abu Leu Abu Lys
His Ala
S
lle Leu S Glu Trp
2-ob D-Ala D-Ala Ala Ala

Met
lle
Dhb Ala
S S S
Ala Pro
Pro Tyr
lle Ala Ala Abu Ala Abu Ala
Pro
Ala Thr Lys Arg Ala
Thr Thr
Dhb
Asn
(d)
Figure 9.2 Covalent structure of peptide bacteriocins: (a) microcin B17, (b) nisin, (c) mersacidin,
(d) lacticin 3147. (Continued)
S S S S
Lys Tyr Tyr Gly Gln Gly Val Thr Cys Cys Ser Val Asp Trp Gly Lys Ala Thr Thr Cys Cys
Lys
Gly Ser Ile His
Lys His Asn
Ile
Asn Gly
Asn Gln
Gly His
Ala Met Ala Trp Ala Thr Gly Gly

(e)
S
Ala Abu Ala
Ile Asp
Leu Glu
10 Asp S His 20
Abu Dha
15 Leu
Gly
Ser Ala
S
Ala Abu
Ala
Lys Lys Thr Lys Lys Asn Ala Trp
Val
S
(f ) 5
25
Leu
Ala
Val 7
D-Ala Pro Thr His
Lys
Pro His
Leu Ala Ala
Val Val Ala
Dhb 19 Gly Ala
Ala Leu D-Ala Asp Ala
O Ala
Glu Val Ala Tyr
O Val 11 S
Phe
D-Ala Met Leu Tyr S Lys
(g)
OH
Val Phe Abu

Asp Ala
Gly Phe
Asn S
S Pro
Abu Ala
Gly
S Arg Gln Ala
Lys Ala Phe
Ala
N
H
(h)
Figure 9.2 (Continued) Covalent structure of peptide bacteriocins: (e) pediocin PA-1, (f) plantaricin
C, (g) l actocin S, and (h) cinnamycin.
separate class because of their significantly smaller molecular weight. Only nine microcins
have been identified so far (Moreno et al., 2002), and unlike colicins, few have been charac-
terized at the level of protein structure or mode of action. The killing spectrum of micro-
cins is broad compared to that of colicins, but they are primarily directed against genera of
Enterobacteriaceae. Microcins kill their target cells by forming pores or by disrupting the
cell membrane potential (Destoumieux-Garzón et al., 2002).
Pyocins are the nuclease proteins produced by Pseudomonas aeruginosa, having a

sequence similar to that of colicin. The colicin and related bacteriocins have typical
domain structure where there are generally a receptor recognition domain, a translocation
domain, and a toxin domain, all of which have sequence similar to their counterparts in
other gram-negative species. Most of the enteric bacteria have the ability to produce bacte-
riocins. Recent surveys of Hafnia alvei, Citrobacter freundii, Klebsiella oxytoca, K. pneumoniae,
and Enterobacter cloacae reveal levels of bacteriocin production ranging from 3% to 26%
of environmental enteric isolates (Riley et al., 2003). Molecular investigations reveal that
enteric bacteriocins employ similar killing mechanisms, although they often utilize novel
receptor recognition and translocation functions, ensuring their narrow killing specifici-
ties (Riley et al., 2001; Wertz and Riley, 2004; Gillor et al., 2005).
9.4.2 Bacteriocins from gram-positive bacteria

Bacteriocins from gram-positive bacteria are even more diverse than those of gram-
negative bacteria. The majority of gram-positive bacteriocins are relatively small consisting
of 30–70 amino acids and seem to possess a broader range of susceptible organisms. They
are generally secreted by the producer cell and thus do not require death of the producer
cell to release mature bacteriocin.
9.4.2.1 Lactic acid bacterial bacteriocin

The best studied members of the gram-positive group of molecules are derived from
lactic acid bacteria (LAB), and they are prolific in their use in cultured food products
as natural preservatives (Lüders et al., 2003). The uses of LAB and their metabolic prod-
ucts are generally considered as safe (GRAS, Grade One). Many bacteriocin-producing
LAB (Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc) and
Bifidobacterium have been isolated from different food matrices, such as fermented dairy
products, vegetables, fruits, meat, and fish, and also from the human and animal gastro-
intestinal tracts (Todorov et al., 2010).
Bacteriocins produced by LAB are small, ribosomally synthesized AMPs or proteins
that possess activity towards closely related gram-positive bacteria, whereas producer cells
are immune to their own bacteriocin(s). The application of the produced antimicrobial com-
pounds as a natural barrier against pathogens and food spoilage caused by bacterial agents
has been proven to be efficient (Leroy, 2007). LAB bacteriocins are categorized into differ-
ent classes based on their biochemical and genetic properties such as mode of action, heat
tolerance, biological activity, presence of modified amino acids, and secretion mechanism.
Class I peptides are the lantibiotics, which are small, posttranslationally modified pep-
tides that contain unusual amino acids such as lanthionine, β-methyl lanthionine, dehy-
droalanine, dehydrobutyrine, and several dehydrated amino acids. They target a broad
range of gram-positive bacteria and are subdivided into three groups on the basis of their
structure and mode of action. Type A lantibiotics (2–5 kDa), such as nisin (Figure 9.2b), are
small, elongated, screw-shaped proteins that contain positively charged molecules, which
kill via the formation of pores, leading to the dissipation of membrane potential and the
efflux of small metabolites from the sensitive cells (Nagao et al., 2006). Type B lantibiotics
(about 2 kDa), such as mersacidin (Figure 9.2c), kill by interfering with cellular enzymatic
reactions, for example, cell wall synthesis (Pag and Sahl, 2002). Another subgroup is com-
posed of two-component lantibiotics, such as lacticin 3147 (LtnA1, LtnA2) consisting of
two lantibiotic peptides (Figure 9.2d) that synergistically display antimicrobial activity
(Wiedemann et al., 2006; Gillor et al., 2008).
Class II includes unmodified, small, heat-stable, non-lanthionine-containing pep-

tides (molecular masses of <10 kDa) that share a conserved N-terminal sequence and
pore formers and have anti-Listeria activity. The majority of bacteriocins in this group kill
the target bacteria by inducing membrane permeabilization and subsequent leakage of
molecules (Oppegård et al., 2007). These bacteriocins are organized into three subgroups,
namely, class IIa pediocin-like bacteriocins (Figure 9.2e), class IIb two-peptide bacterio-
cins (Figure 9.2f), and class IIc others, that is, non-pediocin-like one peptide-bacteriocins
(Borges et al., 2014). Class III peptides are large, thermosensitive proteins (molecular
masses of >10 kDa) such as bacteriolysin, helveticins J, and lactacin B (Dobson et al., 2007).
The best studied bacteriolysin is lysostaphin, a 27 kDa peptide that hydrolyses several
Staphylococcus sp. cell walls, principally S. aureus. Class IV bacteriocins are a group of
complex proteins, associated with other lipid or carbohydrate moieties, which appear to
be required for activity. They are relatively hydrophobic and heat stable (Mahrous et al.,
2013). Little is known about the structure and function of class IV bacteriocins. Examples
include leuconocin S, lactocin 27, and lactocin S (Figure 9.2g) (Choi et al., 1999; Vermeiren
et al., 2006).
9.4.2.2 Actinobacterial bacteriocin

Actinobacteria are a group of gram-positive bacteria that constitute a significant compo-
nent of the microbial population in most soils. Traditionally, actinobacteria have been a
rich source of biotechnological products like antibiotics, industrial enzymes, and other
bioactive molecules. Followed by LAB, actinobacteria is known to be a potential source for
different kinds of bacteriocins.
Among human intestinal microbiota, Bifidobacterium is one of the most familiar genus
that constitutes up to 25% of the total population in the intestinal tract in adults and 95%
in newborns. The positive effect of Bifidobacterium in human is the production of antimi-
crobial compounds other than organic acids, such as bacteriocins. Although some reports
have been suggested that the production of organic acids (via the heterofermentative
pathway) is partially responsible for the inhibitory activity of bifidobacteria (Ibrahim and
Salameh, 2001; Bruno and Shah, 2002), it is well accepted that some genera of this group
also produce the bacteriocins (Cheikhyoussef et al., 2010).
To date, some bacteriocins such as bifidin I (Cheikhyoussef et al., 2010), bifidocin B
(Yildirim et al., 1999), and bacteriocin-like inhibitory substances (BLISs) (Cheikhyoussef
et al., 2009) have been reported to be produced by bifidobacteria. Zouhir et al. (2011) reported
that Bifidobacterium sp. RBL 68 and RBL 85 isolated from newborn feces were found to pro-
duce two BLISs with inhibitory activities against a wide range of gram-positive and gram-
negative bacteria that makes them potentially useful as antimicrobial agents in foods. A
list of known Bifidobacterium-associated bacteriocins and putative bacteriocins as well as
their main characteristics is represented in Table 9.2.
Apart from Bifidobacterium, other actinobacterial genera such as Streptomyces and
Actinomyces also have the ability to produce bacteriocin and inhibit bacterial pathogens.
A strain of A. odontolyticus, originally isolated from human dental plaque, produced a non-
dialyzable, trypsin-sensitive substance called odontolyticin that was bactericidal for certain
strains of bifidobacteria at 42°C but not at 37°C (Franker et al., 1977). Bacteriocin produc-
tion within the genus Streptomyces has been previously reported, with bactericidal spectra
described as species specific (Zhang et al., 2003) or genus specific (Roelants and Naudts,
1964). S. scopuliridis sp. nov., RB72T isolated from woodland bluff soil in northern Alabama,
United States, produces a broad-spectrum bacteriocin with inhibitory activity against
gram-positive and gram-negative bacteria (Farris et al., 2011).
Table 9.2 Bifidobacterium-associated bacteriocins and their inhibitory spectrum

S. no. Bacteriocin Species and strain Inhibitory spectrum References
1. Bifidin B. bifidum NCDC Gram-positive and gram- Anand et al.
1452 negative bacteria (1985)
2. Bifidocin B B. bifidum Bacillus cereus, E. faecalis, Yildirim et al.
NCFB 1454 S. faecalis, P. acidilactici, (1999)
L. monocytogenes
3. Bifilong B. longum Gram-positive and gram- Kang et al.
negative bacteria (1989)
4. Bifilact Bb-46 B. longum Bb-46 S. aureus, S. typhimurium, Saleh et al.
B. cereus, E. coli (2004)
5. Bifilact Bb-12 B. lactis Bb-12 S. aureus, S. typhimurium, Saleh et al.
B. cereus, E. coli (2004)
6. Thermophilicin B. thermophilum Listeria sp., L. acidophilus Von Ah (2006)
B67 RBL67
7. Bifidin I B. infantis LAB strains, Staphylococcus, Cheikhyoussef
BCRC 14602 Streptococcus, Salmonella, et al.
Shigella, Bacillus, E. coli (2009, 2010)
8. Lantibiotic B. longum DJO10A S. thermophilus ST403, Lee et al.
(bisin) S. epidermidis, B. subtilis, Serratia (2011)
marcescens, E. coli DH5a,
Clostridium perfringens
In the same way, bacteriocin of Streptomyces sp. JD9 (KF878075) isolated from the
feces of indigenous chicken (Latha and Dhanasekaran, 2013) exhibited inhibitory activity
against human and animal bacterial pathogens, namely, E. coli MTCC 9537, S. enterica
MTCC 3224, S. flexneri MTCC 9543, K. pneumoniae MTCC 109, E. faecalis MTCC 439, and
S. aureus MTCC 96 and E. coli AP1, S. typhimurium AP2, Pasteurella multocida AP3, and
S. aureus AP4, respectively (Figures 9.3 and 9.4).
Cinnamycin (Figure 9.2h) is one of the peptide antibiotics closely related to type B
lantibiotics duramycin, duramycin B, duramycin C, and ancovenin. These compounds
are derived from 19-aa propeptides and have lanthionine residues in similar positions.
They are all produced by actinobacteria, particularly the duramycins and cinnamycins that
are exclusively produced by streptomycete strains. Despite their antimicrobial activities,
these compounds also have other potentially useful pharmaceutical properties, including
JD6 JD6 JD2 JD2

JD2 JD6
JD8 JD8 JD9 JD10

JD9 JD9
S. typhimurium AP2 P. multocida AP3 S. aureus MTCC 96
Figure 9.3 Determination of bacteriocin activity by spot agar test.

JD6 JD2
JD6 JD2 JD6 JD2
JD9 JD10
JD9 JD10 JD9 JD10
E. faecalis MTCC 439 E. coli MTCC 9537 K. pneumoniae MTCC 109
Figure 9.4 Determination of bacteriocin activity by well diffusion assay.
inhibition of angiotensin-converting enzyme, phospholipase A2 and prostaglandin, and

leukotriene biosynthesis (Widdick et al., 2003).
9.4.3 Bacteriocins from archaea

The archaea produce their own distinct family of bacteriocin-like antimicrobials, known as
archaeocins. The only well-characterized member of archaeocin is the halocin, produced by
the Halobacteriaceae family (Platas et al., 2002; Sun et al., 2005). Halocin production is recog-
nized as a nearly universal feature of halobacteria. However, only the halocins like H1, H4,
H6, S8, and R1 have been purified and described in detail (Parisien et al., 2008). The limited
number of known halocins exhibits substantial diversity in size, ranging from proteins as
large as 35 kDa (e.g., halocin H4) to peptides as small as 3.6 kDa (e.g., halocin S8). The first
discovered halocin is S8, a short hydrophobic peptide of 36 amino acids, which is processed
from a much larger proprotein of 34 kDa (Price and Shand, 2000). It is encoded on a mega-
plasmid and extremely hardy; it can be desalted, boiled, subjected to organic solvents, and
stored at 4°C for extended periods without losing activity. Similar characteristics are also
found in microhalocins, and they are usually quite resistant to acids and bases (Li et al., 2003).
Archaeocins are generally produced during the stationary phase of the cell growth. When
resources are limited, producing cells lyse sensitive cells and enrich the nutrient content of
the local environment. As stable proteins, they may remain in the environment long enough
to reduce competition during subsequent phases of nutrient flux (Riley and Wertz, 2002).
9.5 Potential applications of bacteriocin

Bacteriocins have been the focus of an extensive number of studies for the past 60 years
due to their important role in nature and, more recently, their potential use as probiotics
and therapeutics. The antimicrobial activity of this group of natural substances against
food-borne pathogens, as well as spoilage bacteria, has raised considerable interest for
their application in food preservation. The application of bacteriocins reduces the use of
chemical preservatives and/or the intensity of heat and other physical treatments, satisfy-
ing the demands of consumers for foods that are fresh-tasting, ready to eat, and lightly
preserved. In addition, bacteriocins are of interest in medicine because they are made
by nonpathogenic bacteria that normally colonize the human body. The other most impor-
tant contributions of bacteriocins include animal health improvement and biocontrol of
aquatic and plant bacterial pathogens (Figure 9.5).
Livestock
production
Food
Agriculture preservation
Bacteriocin
Aquaculture Human
health
Figure 9.5 Applications of bacteriocin in diverse field.
9.5.1 Bacteriocins in food biopreservation

Nowadays, bacteriocins have been widely utilized in the field of food preservation. The
uses of bacteriocins in food industry especially on dairy, egg, vegetable, and meat prod-
ucts have been extensively investigated. They can be applied in a purified or crude form
and the product previously fermented with a bacteriocin-producing strain as an ingredi-
ent during food processing or incorporated as a starter culture. Furthermore, bacterio-
cins could be combined with other antimicrobial compounds such as sodium acetate and
sodium lactate resulting in enhanced inactivation of bacteria. They can also be used to
improve the food quality and sensory properties, for example, increasing the rate of prote-
olysis or in the prevention of gas blowing defect in cheese. Another application of bacterio-
cins is bioactive packaging, a process that can protect the food from external contaminants
(Ross et al., 2002; Deegan et al., 2006; Leroy, 2007; Zacharof and Lovitt, 2012).
The potential use of lantibiotics in food, human, and animal health applications has
been well documented. There are several features making them attractive for such applica-
tions such as a relatively broad killing spectrum, an autoregulation system, and stability
and cost-effective production processes. Moreover, several lantibiotics are produced by
food-grade bacteria that have been safely consumed by humans for centuries. The best
studied lantibiotic is undoubtedly nisin, produced by L. lactis. It is thus far the only bac-
teriocin that has been approved by Food and Drug Administration as a food preserva-
tive, and it is being used for this purpose in more than 50 countries. Maisnier-Patin et al.
(1992) demonstrated the potential of using nisin-producing starters for the inhibition of
L. monocytogenes in camembert cheese. Daeschel et al. (1991) and Radler (1990a,b) indicated
the possibility of using nisin for combating contamination in wines as most strains of
Leuconostoc and Pediococcus are nisin sensitive.
Bacteriocin-producing strains of LAB have also been shown to improve the flavor and
quality of meat products when used as starter adjuncts. For example, L. lactis DPC4275,
a transconjugant strain producing lacticin 3147, was used as a starter for the manufac-
ture of salami and was compared to salami manufactured with a conventional starter
(L. sake and S. carnosus) in terms of pH development, water activity (aw) value, weight loss,
color development, and sensory characteristics. Salami produced with L. lactis DPC4275
exhibited pH values below 5.1 and an aw value of 0.9 that is favorable for preservation and
hygienic stability. In addition, these salamis had good sensory and colorimetric qualities
(Coffey et al., 1998). Improvements in the flavor of fermented meat were also observed by
Vogel et al. (1993) when curvacin A–producing L. curvatus LTH1174 was used as a starter
culture in fermented sausages.
9.5.2 Bacteriocins in human health

Bacteriocins produced by several food-grade bacteria have been safely consumed by
humans for centuries. Since the mode of action of bacteriocins is remarkably different
from conventional antibiotics, they considered as a novel source for the control of microbial
pathogens. Besides being used as food preservative, nisin (nontoxic bacteriocin) was also
used as an antibacterial wipe for the udder prior to milking, and it has been suggested as
a contraceptive agent (Gillor et al., 2005). It has a potential in treating peptic ulcer disease
by inhibiting Helicobacter pylori growth and colonization (Delves-Broughton et al., 1996).
Nisin was additionally used to inhibit the growth of multidrug-resistant pathogens
such as Staphylococcus and Streptococcus sp. Bower and his colleagues (2002) treated cath-
eters and tracheotomy tubes with nisin, which had a protective effect against infection by
gram-positive bacteria, albeit for only a short period (5–12 h), and produced no systematic
or adverse effects. It also has the ability to control respiratory tract infections caused by
Staphylococcus sp. De Kwaadsteniet et al. (2009) reported that nisin F inhibited the growth
of S. aureus in the respiratory tract of rats when administered intranasally.
Bacteriocins of LAB are usually not active against gram-negative bacteria, and only
a few works referred to activity against Salmonella sp. Bacteriocin AS-48, produced by
E. faecalis, inhibited the growth of S. choleraesuis at pH 4.0 (Abriouel et al., 1998). Similarly,
a bacteriocin-like substance produced by a strain of L. plantarum inhibited the growth of
Salmonella sp. isolated from mango (Ragazzo-Sanchez et al., 2009). Bacteriocins appear
to be capable of displacing or suppressing the growth of other bacteria and perhaps
provide an advantage to microorganisms in fermenting ecosystems, including the
female genital tract. As bacteriocins do not induce vaginal irritation, they are suitable
for human use.
The inhibitory activity of bacteriocin SB83 produced by P. pentosaceus SB83 against
L. monocytogenes (serotypes 1/2a, 1/2b, and 4b) revealed that the isolate can be used as a
potential vaginal probiotic (Borges et al., 2014). The bacteriocin, subtilosin has proven anti-
microbial activity against a wide variety of human pathogens, including L. monocytogenes,
Gardnerella vaginalis, S. agalactiae, and Micrococcus luteus. Its activity against G. vaginalis,
combined with its lack of effect on probiotic vaginal Lactobacillus isolates, indicates that
subtilosin could target the vaginal pathogen while leaving the healthy vaginal microflora
intact (Sutyak et al., 2008).
9.5.3 Bacteriocins in livestock production

Antibiotic therapy has been a valuable tool used in animal research, as growth promoters
or therapeutic agents, and their efficacy and cost-effectiveness contribute to their popular-
ity. However, in recent years, bacteriocins and probiotic microbes replaced the antibiotic
growth promoters and reduced the antibiotic-associated problems such as occurrence of

antibiotic residues in the environment and veterinary products as well as an increase in
the frequency of resistance among bacterial species (Ochoa-Zarzosa et al., 2008).
Bacteriocins produced by different gram-positive bacteria have been tested both
in vitro and in vivo for the improved livestock production. The peptides that have been
tested differ in their physicochemical characteristics and spectrum of activity, but prelimi-
nary studies indicated that they might be a potential and effective alternative to classical
antibiotics used in animal husbandry. However, due to rapid degradation of the bacterio-
cins in the digestive tract of mammals, feeding or applying bacteriocins alone to livestock
is less common. On the other hand, a variety of probiotic bacteria have been tested to con-
trol animal pathogenic bacteria and to improve the production of livestock. The pathogen
inhibition mechanism of probiotic microorganisms is primarily mediated by the produc-
tion of bacteriocins. Accordingly, the method of feeding BPB is generally preferred as an
alternative antimicrobial strategy in animal husbandry.
In poultry, the use of BPB has been mainly targeted for the control of Salmonella sp.
Administration of bacteriocin-producing E. faecium strain J96 after hatching decreased the
poultry pathogen S. pullorum population and increased the survival rate of young broiler
chicks (Audisio et al., 2000). Microcins produced by E. coli hold promise in reducing the
abundance of S. typhimurium in adult chickens (Portrait et al., 1999; Gillor et al., 2004). BLIS
produced by B. amyloliquefaciens CECT 5940 was also used as a probiotic in poultry systems
that showed the reduction of pathogenic bacteria, such as C. perfringens, E. coli, and Yersinia
sp. (Diaz, 2007).
In cattle, the cow’s rumen serves as a major reservoir for E. coli O157:H7, a pathogen that
is exceedingly difficult to control using antibiotics (Hussein and Bollinger, 2005; Hussein,
2007). In fact, studies have shown that antibiotic treatment increases the amount of Shiga
toxin released by this pathogen, resulting in higher levels of bacterial virulence. Recently,
there have been reports that administration of colicin-producing bacteria into the rumen
of cows can reduce the level of enteric pathogens in the animal. In the same way, the
two-peptide lantibiotic, lacticin 3147, produced by L. lactis was found to be active against
mastitis-causing bacteria Streptococci and Staphylococci in dairy cattle. Significant increase
in cure rates of infections caused by S. agalactiae, S. aureus, and other mastitis pathogens
(90.1%, 50%, and 65.2%) was observed when cows were treated with nisin Z, via intrama-
mmary administration (Wu et al., 2007). The application of BPB for improvements in pro-
ductivity has not been limited to poultry and cattle as several researchers have explored
the use of probiotic strains capable of producing bacteriocins to increase the growth rate
of swine (Rodriguez et al., 2003).
9.5.4 Bacteriocins in aquaculture

Bacteriocinogenic bacterial strains emerged as an excellent antibiotic substitute for dis-
ease control of aquatic animals. Administration of probiotic bacteria was reported to
competitively exclude pathogenic bacteria through the production of inhibitory com-
pounds, improvement of water quality, enhancement of immune response, and nutrition
of host species through the production of supplemental digestive enzymes (Verschuere
et al., 2000). Bacteriocins produced by marine bacteria have generated a great deal of
excitement due to their potential to serve as probiotics and antibiotics in the seafood
industry (Gálvez et al., 2008; Garcia et al., 2010). A recent antimicrobial screening of 258
bacterial strains isolated from water and sediment in the Yucatan Peninsula revealed
that 46 strains belonging to the genera Aeromonas, Bacillus, Burkholderia, Photobacterium,
Pseudomonas, Serratia, and Stenotrophomonas possessed antimicrobial activity. Approxi

mately 50% of this antimicrobial activity was due to bacteriocins or BLISs (De la Rosa-
Garcia et al., 2007).
The first bacteriocin isolated from marine microorganism was detected in Vibrio
harveyi (formerly Beneckea harveyi). McCall and Sizemore (1979) screened a total of 795
Vibrio sp. isolated from Galveston Island, Texas, for bacteriocin production. About 5% of
the Vibrio sp. possessed a high-molecular-weight bacteriocin-like killing agent named as
harveyicin. Piscicocins V1a and V1b, divercin V41, piscicocin CS526, divergicin M35, car-
nocin U149, and carnobacteriocin B2 are some of the bacteriocins isolated from marine
Carnobacterium species (Stoffels et al., 1992; Bhugaloo-Vial et al., 1996; Metivier et al., 1998;
Duffes et al., 1999; Tahiri et al., 2004; Suzuki et al., 2005). These bacteriocins share similar
characteristics with class II bacteriocins of gram-positive bacteria.
A. media strain A199 was found to produce several BLISs and was shown to con-
trol infection by V. tubiashii in pacific oyster larvae (Gibson et al., 1998) and reduce
saprolegniosis-related mortality in eels (Lategan et al., 2004). Irianto and Austin (2002)
reported that cultures of A. hydrophila and V. fluvialis were effective at controlling infec-
tions by A. salmonicida in rainbow trout. In addition, Ruiz-Ponte et al. (1999) found that
BLIS-producing Roseobacter sp. strain BS107 inhibits the pathogenic effect of Vibrio sp.
resulting in enhanced survival of scallop larvae (Gillor et al., 2008).
9.5.5 Bacteriocins in agricultural settings

Bacteria occupying plant niches exhibit diverse lifestyles ranging from epiphytic coloniza-
tion of phyllospheres or rhizospheres to infection of plant tissues as endophytes, symbi-
onts, or pathogens. A number of studies indicate that bacteriocin production plays a major
role in the competitive colonization of the plant environment by phytobacteria. Owing to
their highly selective killing spectrum and potent cytotoxicity, bacteriocins show poten-
tial as targeted next-generation antibiotics for medical and agricultural uses. One possible
application of bacteriocinogenic strains in agriculture includes their use in biological con-
trol of soilborne or phyllosphere-inhabiting bacterial pathogens.
A number of studies have shown that bacteriocins can be used as an effective agent in
the treatment of bacterial diseases in plants. In this way, heterologous production of the
peptide bacteriocin trifolitoxin by an avirulent Agrobacterium strain effectively enhances
the biological control of A. vitis crown gall (Herlache and Triplett, 2002). Expression of
the trifolitoxin genes from Rhizobium leguminosarum bv. trifolii T24 in R. etli CE3 increases
bean nodulation competitiveness of the recombinant strain in the presence of indigenous
rhizobia under agricultural conditions (Robleto et al., 1998; Parret et al., 2005). Dipping
plants in a suspension of a bacteriocin-producing avirulent strain of Ralstonia solanacearum
prevented tobacco wilt infection (Chen and Echandi, 1984). The incidence and severity of
bacterial blight infection that causes leaf streak in rice were reduced by treatment with a
nonpathogenic bacteriocin-producing strain of Xanthomonas campestris pv. oryzae (Sakthivel
and Mew, 1991).
Lavermicocca et al. (2002) reported the use of pyocin, an uncharacterized bacteriocin
from P. syringae pv. ciccarone in the prevention of olive knot disease. The results showed
60%–80% reduction in knot formation when stem wounds were pretreated with crude
bacteriocin before infection with the causal agent of the disease, P. syringae pv. savastanoi.
Additionally, a 350- to 400-fold decrease in the epiphytic numbers of the pathogen was
observed when unwounded olive plants were treated with a crude preparation of the bac-
teriocin (Grinter et al., 2012).
Glycinicin A, the best described bacteriocin produced by X. campestris pv. glycines, is a

heterodimer of two polypeptides. It was found to be active against most tested Xanthomonas
phytopathogenic bacterial strains (Heu et al., 2001; Gillor et al., 2005). A bacteriocin-based
strategy to control fire blight of apple (devastating necrotic disease) was proposed by Jabrane
et al. (1996), based on the inhibitory activity of S. plymuthica J7 culture supernatant against
several strains of γ-proteobacterial species, including Erwinia amylovora. The antagonistic
activity was exerted by a phage-tail-like bacteriocin named serracin P (Jabrane et al., 2002).
9.6 Conclusion
Bacteriocins produced by various bacteria have the potential to cover a broad field of appli-
cations, including food industry and medical sector. In the food industry, bacteriocin-
producing starter or cocultures have been successfully applied in pilot-scale experiments
yielding food quality and food safety advantages. With respect to medical applications,
bacteriocins are antagonistic to many important clinical and veterinary pathogens. In par-
ticular, bacteriocins of probiotic microbes play a major role during the in vivo interactions
occurring in the human and animal gastrointestinal tracts, hence contributing to gut health.
They have the ability to target a relatively narrow range of bacteria without affecting much
of the natural microbiota, which is an important advantage compared to other antibiotics.
Although they do not target many pathogens like antibiotics, they have the potential to
perform a very specific role. However, future studies should turn these bacteriocins into
practical clinical substitutes to antibiotics and prove their anticipated efficacy, safety, and
affordability.
Acknowledgment
The author is grateful to acknowledge the Department of Science and Technology (DST),
New Delhi, India, for the award of INSPIRE fellowship (DST Award Letter No. IF110317/
DST/INSPIRE Fellowship/2011/Dt. 29.06.2011).
References
Abriouel, H., Valdivia, E., Gálvez, A., and Maqueda, M. 1998. Response of Salmonella choleraesuis LT2
spheroplasts and permeabilized cells to the bacteriocin AS-48. Appl. Environ. Microbiol. 64(11):
4623–4626.
Anand, S. K., Srinivasan, R. A., and Rao, L. K. 1985. Antibacterial activity associated with
Bifidobacterium bifidum-II. Cult. Dairy Prod. J. 20: 21–23.
Audisio, M. C., Oliver, G., and Apella, M. C. 2000. Protective effect of Enterococcus faecium J96, a poten-
tial probiotic strain, on chicks infected with Salmonella pullorum. J. Food Prot. 63(10): 1333–1337.
Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Boyaval, P., and Marion, D.
1996. Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacterio-
cins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific
inhibitory activity. Appl. Environ. Microbiol. 62(12): 4410–4416.
Blinkova, L. P., Butova, L. G., Sergeev, V. V., Elkina, S. I., Al’tshuler, M. L., and Kalina, N. G. 2003.
Effectiveness of the oral administration of tomicide in experimental infection. Zh. Mikrobiol.
Epidemiol. Immunobiol. 1: 74–77.
Borges, S., Barbosa, J., Silva, J., and Teixeira, P. 2014. Characterization of a bacteriocin of Pediococcus
pentosaceus SB83 and its potential for vaginal application. Antiinfect. Agents 12(1): 68–74.
Bower, C. K., Parker, J. E., Higgins, A. Z., Oest, M. E., Wilson, J. T., Valentine, B. A., Bothwell, M. K.,
and McGuire, J. 2002. Protein antimicrobial barriers to bacterial adhesion: In vitro and in vivo
evaluation of nisin-treated implantable materials. Colloids Surf. B. 25(1): 81–90.
Bruno, F. and Shah, N. P. 2002. Inhibition of pathogenic and putrefactive microorganisms by 541
Bifidobacterium sp. Milchwissenschaft 57(11/12): 617–621.
Cheikhyoussef, A., Cheikhyoussef, N., Chen, H., Zhao, J., Tang, J., Zhang, H., and Chen, W. 2010.
Bifidin I–A new bacteriocin produced by Bifidobacterium infantis BCRC 14602: Purification and
partial amino acid sequence. Food Control 21(5): 746–753.
Cheikhyoussef, A., Pogori, N., Chen, H., Tian, F., Chen, W., and Tang, J. 2009. Antimicrobial activity
550 and partial characterization of bacteriocin-like inhibitory substances (BLIS) produced by
Bifidobacterium infantis BCRC 14602. Food Control 20: 553–559.
Chen, W. Y. and Echandi, E. 1984. Effects of avirulent bacteriocin‐producing strains of Pseudomonas
solanacearum on the control of bacterial wilt of tobacco. Plant Pathol. 33(2): 245–253.
Choi, H. J., Lee, H. S., Her, S., Oh, D. H., and Yoon, S. S. 1999. Partial characterization and cloning of
leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented
vegetable kimchi. J. Appl. Microbiol. 86(2): 175–181.
Cleveland, J., Montville, T. J., Nes, I. F., and Chikindas, M. L. 2001. Bacteriocins: Safe, natural antimi-
crobials for food preservation. Int. J. Food Microbiol. 71(1): 1–20.
Coffey, A., Ryan, M., Ross, R. P., Hill, C., Arendt, E., and Schwarz, G. 1998. Use of a broad host range
bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami
manufacture. Int. J. Food Microbiol. 43(3): 231–235.
Daeschel, M. A., Jung, D. S., and Watson, B. T. 1991. Controlling wine malolactic fermentation with
nisin and nisin-resistant strains of Leuconostoc oenos. Appl. Environ. Microbiol. 57(2): 601–603.
Damasko, C., Konietzny, A., Kaspar, H., Appel, B., Dersch, P., and Strauch, E. 2005. Studies of the
efficacy of enterocoliticin, a phage‐tail like bacteriocin, as antimicrobial agent against Yersinia
enterocolitica Serotype O3 in a cell culture system and in mice. J. Vet. Med. B. 52(4): 171–179.
Deegan, L. H., Cotter, P. D., Hill, C., and Ross, P. 2006. Bacteriocins: Biological tools for bio-
preservation and shelf-life extension. Int. Dairy J. 16(9): 1058–1071.
De Kwaadsteniet, M., Doeschate, K. T., and Dicks, L. M. T. 2009. Nisin F in the treatment of respira-
tory tract infections caused by Staphylococcus aureus. Lett. Appl. Microbiol. 48(1): 65–70.
De la Rosa-Garcia, S. C., Muñoz‐García, A. A., Barahona‐Pérez, L. F., and Gamboa‐Angulo, M. M.
2007. Antimicrobial properties of moderately halotolerant bacteria from cenotes of the Yucatan
Peninsula. Lett. Appl. Microbiol. 45(3): 289–294.
Delves-Broughton, J., Blackburn, P., Evans, R. J., and Hugenholtz, J. 1996. Applications of the bacte-
riocin, nisin. Antonie van Leeuwenhoek 69(2): 193–202.
Destoumieux-Garzón, D., Peduzzi, J., and Rebuffat, S. 2002. Focus on modified microcins: Structural
features and mechanisms of action. Biochimie 84(5): 511–519.
Diaz, D. 2007. Effect of Bacillus amyloliquefaciens CECT-5940 spores on broiler performance and digest-
ibility. Accessed November 2007. http://en.engormix.com/MA-poultry-industry/articles/
effect-bacillus-amyloliquefaciens-cect5940-t795/p0.htm.
Dobson, A., Cotter, P. D., Ross, R. P., and Hill, C. 2012. Bacteriocin production: A probiotic trait?. Appl.
Environ. Microbiol. 78(1): 1–6.
Dobson, A. E., Sanozky‐Dawes, R. B., and Klaenhammer, T. R. 2007. Identification of an operon and
inducing peptide involved in the production of lactacin B by Lactobacillus acidophilus. J. Appl.
Microbiol. 103(5): 1766–1778.
Duffes, F., Corre, C., Leroi, F., Dousset, X., and Boyaval, P. 1999. Inhibition of Listeria monocytogenes
by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed,
refrigerated cold-smoked salmon. J. Food Protect. 62(12): 1394–1403.
Farris, M. H., Duffy, C., Findlay, R. H., and Olson, J. B. 2011. Streptomyces scopuliridis sp. nov., a
bacteriocin-producing soil streptomycete. Int. J. Syst. Evol. Microbiol. 61(9): 2112–2116.
Franker, C. K., Herbert, C. A., and Ueda, S. 1977. Bacteriocin from Actinomyces odontolyticus with
temperature-dependent killing properties. Antimicrob. Agents Chemother. 12(3): 410–417.
Gálvez, A., López, R. L., Abriouel, H., Valdivia, E., and Omar, N. B. 2008. Application of bacteriocins
in the control of foodborne pathogenic and spoilage bacteria. Crit. Rev. Biotechnol. 28(2): 125–152.
Garcia, P., Rodriguez, L., Rodriguez, A., and Martinez, B. 2010. Food biopreservation: Promising strat-
egies using bacteriocins, bacteriophages and endolysins. Trends Food Sci. Tech. 21(8): 373–382.
Gibson, L. F., Woodworth, J., and George, A. M. 1998. Probiotic activity of Aeromonas media on the
Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii. Aquaculture 169(1): 111–120.
Gillor, O., Etzion, A., and Riley, M. A. 2008. The dual role of bacteriocins as anti-and probiotics. Appl.
Microbiol. Biotechnol. 81(4): 591–606.
Gillor, O., Kirkup, B. C., and Riley, M. A. 2004. Colicins and microcins: The next generation antimi-
crobials. Adv. Appl. Microbiol. 54: 129–146.
Gillor, O., Nigro, L. M., and Riley, M. A. 2005. Genetically engineered bacteriocins and their poten-
tial as the next generation of antimicrobials. Curr. Pharm. Des. 11(8): 1067–1075.
Gratia, J. P. 2000. Andre Gratia: A forerunner in microbial and viral genetics. Genetics 156(2): 471–476.
Grinter, R., Milner, J., and Walker, D. 2012. Bacteriocins active against plant pathogenic bacteria.
Biochem. Soc. Trans. 40(6): 1498.
Hammami, R., Zouhir, A., Hamida, J. B., and Fliss, I. 2007. BACTIBASE: A new web-accessible data-
base for bacteriocin characterization. BMC Microbiol. 7(1): 89.
Herlache, T. C. and Triplett, E. W. 2002. Expression of a crown gall biological control phenotype in an
avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance
genes. BMC Biotechnol. 2(1): 2.
Heu, S., Oh, J., Kang, Y., Ryu, S., Cho, S. K., Cho, Y., and Cho, M. 2001. gly gene cloning and expres-
sion and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv.
glycines 8ra. Appl. Environ. Microbiol. 67(9): 4105–4110.
Hussein, H. S. 2007. Prevalence and pathogenicity of shiga toxin-producing Escherichia coli in beef
cattle and their products. J. Anim. Sci. 85(Suppl 13): E63–E72.
Hussein, H. S. and Bollinger, L. M. 2005. Prevalence of shiga toxin–producing Escherichia coli in beef
cattle. J. Food Protect. 68(10): 2224–2241.
Ibrahim, S. A. and Salameh, M. M. 2001. Simple and rapid method for screening antimicrobial activ-
ities of Bifidobacterium species of human isolates. J. Rapid Meth. Aut. Mic. 9(1): 53–62.
Irianto, A. and Austin, B. 2002. Use of probiotics to control furunculosis in rainbow trout,
Oncorhynchus mykiss (Walbaum). J. Fish Dis. 25(6): 333–342.
Jabrane, A., Ledoux, L., Thonart, P., Deckers, T., and Lepoivre, P. 1996. The efficacy in vitro and
in vivo of bacteriocin against Erwinia amylovora: Comparison of biological and chemical con-
trol of fire blight. Acta Hortic. 411: 355–359.
Jabrane, A., Sabri, A., Compère, P., Jacques, P., Vandenberghe, I., Van Beeumen, J., and Thonart, P. 2002.
Characterization of serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia
amylovora, the fire blight pathogen. Appl. Environ. Microbiol. 68(11): 5704–5710.
Kang, K. H., Shin, H. J., Park, Y. H., and Lee, T. S. 1989. Studies on the antibacterial substances
produced by lactic acid bacteria: Purification and some properties of antibacterial substance
“Bifilong” produced by B. longum. Kor. J. Dairy Sci. 11: 204–216.
Klaenhammer, T. R. 1988. Bacteriocins of lactic acid bacteria. Biochimie 70(3): 337–349.
Lategan, M. J., Torpy, F. R., and Gibson, L. F. 2004. Control of saprolegniosis in the eel Anguilla aus-
tralis Richardson, by Aeromonas media strain A199. Aquaculture 240(1): 19–27.
Latha, S. and Dhanasekaran, D. 2013. Antibacterial and extracellular enzyme activities of gut acti-
nobacteria isolated from Gallus gallus domesticus and Capra hircus. J. Chem. Pharm. Res. 5(11):
379–385.
Lavermicocca, P., Lonigro, S. L., Valerio, F., Evidente, A., and Visconti, A. 2002. Reduction of olive
knot disease by a bacteriocin from Pseudomonas syringae pv. ciccarone. Appl. Environ. Microbiol.
68(3): 1403–1407.
Lee, J. H., Li, X., and O’Sullivan, D. J. 2011. Transcription analysis of a lantibiotic gene cluster from
Bifidobacterium longum DJO10A. Appl. Environ. Microbiol. 77(17): 5879–5887.
Leroy, L. D. V. F. 2007. Bacteriocins from lactic acid bacteria: Production, purification, and food appli-
cations. J. Mol. Microbiol. Biotechnol. 13: 194–199.
Li, Y., Xiang, H., Liu, J., Zhou, M., and Tan, H. 2003. Purification and biological characterization of halo-
cin C8, a novel peptide antibiotic from Halobacterium strain AS7092. Extremophiles 7(5): 401–407.
Lüders, T., Birkemo, G. A., Fimland, G., Nissen-Meyer, J., and Nes, I. F. 2003. Strong synergy between
a eukaryotic antimicrobial peptide and bacteriocins from lactic acid bacteria. Appl. Environ.
Microbiol. 69(3): 1797–1799.
Mahrous, H., Mohamed, A., El-Mongy, M. A., El-Batal, A. I., and Hamza, H. A. 2013. Study bacterio-
cin production and optimization using new isolates of Lactobacillus spp. isolated from some
dairy products under different culture conditions. Food Nutri. Sci. 4(3): 342–356.
Maisnier-Patin, S., Deschamps, N., Tatini, S. R., and Richard, J. 1992. Inhibition of Listeria monocyto-
genes in Camembert cheese made with a nisin-producing starter. Le Lait 72(3): 249–263.
Masschalck, B. and Michiels, C. W. 2003. Antimicrobial properties of lysozyme in relation to food-
borne vegetative bacteria. Crit. Rev. Microbiol. 29(3): 191–214.
McCall, J. O. and Sizemore, R. K. 1979. Description of a bacteriocinogenic plasmid in Beneckea har-
veyi. Appl. Environ. Microbiol. 38(5): 974–979.
Metivier, A., Pilet, M. F., Dousset, X., Sorokine, O., Anglade, P., Zagorec, M., Piard, J., Marlon, D.,
Cenatiempo, Y., and Fremaux, C. 1998. Divercin V41, a new bacteriocin with two disulphide
bonds produced by Carnobacterium divergens V41: Primary structure and genomic organiza-
tion. Microbiology 144(10): 2837–2844.
Moreno, F., Gónzalez-Pastor, J. E., Baquero, M. R., and Bravo, D. 2002. The regulation of microcin B,
C and J operons. Biochimie 84(5): 521–529.
Nagao, J. I., Asaduzzaman, S. M., Aso, Y., Okuda, K. I., Nakayama, J., and Sonomoto, K. 2006.
Lantibiotics: Insight and foresight for new paradigm. J. Biosci. Bioeng. 102(3): 139–149.
Nes, I. F., Yoon, S., and Diep, D. B. 2007. Ribosomally synthesized antimicrobial peptides (bacterio-
cins) in lactic acid bacteria: A review. Food Sci. Biotechnol. 16(5): 675.
Ochoa-Zarzosa, A., Loeza-Lara, P. D., Torres-Rodríguez, F., Loeza-Ángeles, H., Mascot-Chiquito, N.,
Sánchez-Baca, S., and López-Meza, J. E. 2008. Antimicrobial susceptibility and invasive abil-
ity of Staphylococcus aureus isolates from mastitis from dairy backyard systems. Antonie van
Leeuwenhoek, 94(2): 199–206.
Oppegård, C., Rogne, P., Emanuelsen, L., Kristiansen, P. E., Fimland, G., and Nissen-Meyer, J. 2007.
The two-peptide class II bacteriocins: Structure, production, and mode of action. J. Mol.
Pag, U. and Sahl, H. G. 2002. Multiple activities in lantibiotics-models for the design of novel antibi-
otics? Curr. Pharm. Des. 8(9): 815–833.
Parisien, A., Allain, B., Zhang, J., Mandeville, R., and Lan, C. Q. 2008. Novel alternatives to anti-
biotics: Bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. J. Appl.
Microbiol. 104(1): 1–13.
Parret, A. H., Temmerman, K., and De Mot, R. 2005. Novel lectin-like bacteriocins of biocontrol
strain Pseudomonas fluorescens Pf-5. Appl. Environ. Microbiol. 71(9): 5197–5207.
Platas, G., Meseguer, I., and Amils, R. 2002. Purification and biological characterization of halocin
H1 from Haloferax mediterranei M2a. Int. Microbiol. 5(1): 15–19.
Portrait, V., Gendron-Gaillard, S., Cottenceau, G., and Pons, A. M. 1999. Inhibition of pathogenic
Salmonella enteritidis growth mediated by Escherichia coli microcin J25 producing strains. Can.
J. Microbiol. 45(12): 988–994.
Price, L. B. and Shand, R. F. 2000. Halocin S8: A 36-amino-acid microhalocin from the haloarchaeal
strain S8a. J. Bacteriol. 182(17): 4951–4958.
Radler, F. 1990a. Possible use of nisin in winemaking. I. Action of nisin against lactic acid bacteria
and wine yeasts in solid and liquid media. Am. J. Enol. Vitic. 41(1): 1–6.
Radler, F. 1990b. Possible use of nisin in winemaking. II. Experiments to control lactic acid bacteria
in the production of wine. Am. J. Enol. Vitic. 41(1): 7–11.
Ragazzo-Sanchez, J. A., Sanchez-Prado, L., Gutiérrez-Martínez, P., Luna-Solano, G., Gomez-Gil, B.,
and Calderon-Santoyo, M. 2009. Inhibition of Salmonella spp. isolated from mango using bacte-
riocin-like produced by lactobacilli. Cyta J. Food 7(3): 181–187.
Riley, M. A., Goldstone, C. M., Wertz, J. E., and Gordon, D. 2003. A phylogenetic approach to assess-
ing the targets of microbial warfare. J. Evol. Biol. 16(4): 690–697.
Riley, M. A., Pinou, T., Wertz, J. E., Tan, Y., and Valletta, C. M. 2001. Molecular characterization of the
klebicin B plasmid of Klebsiella pneumoniae. Plasmid 45(3): 209–221.
Riley, M. A. and Wertz, J. E. 2002. Bacteriocins: Evolution, ecology, and application. Annu. Rev.
Microbiol. 56(1): 117–137.
Robleto, E. A., Kmiecik, K., Oplinger, E. S., Nienhuis, J., and Triplett, E. W. 1998. Trifolitoxin produc-
tion increases nodulation competitiveness of Rhizobium etli CE3 under agricultural conditions.
Appl. Environ. Microbiol. 64(7): 2630–2633.
Rodriguez, E., Arques, J. L., Rodriguez, R., Nunez, M., and Medina, M. 2003. Reuterin production
by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett. Appl. Microbiol.
37(3): 259–263.
Roelants, P. and Naudts, F. 1964. Properties of a bacteriocin-like substance produced by Streptomyces
virginiae. Antonie van Leeuwenhoek 30(1): 45–53.
Ross, R. P., Morgan, S., and Hill, C. 2002. Preservation and fermentation: Past, present and future.
Int. J. Food Microbiol. 79(1): 3–16.
Ruiz-Ponte, C., Samain, J. F., Sanchez, J. L., and Nicolas, J. L. 1999. The benefit of a Roseobacter species
on the survival of scallop larvae. Mar. Biotechnol. 1(1): 52–59.
Sakthivel, N. and Mew, T. W. 1991. Efficacy of bacteriocinogenic strains of Xanthomonas oryzae pv.
oryzae on the incidence of bacterial blight disease of rice (Oryza sativa L.). Can. J. Microbiol.
37(10): 764–768.
Saleh, F. A., Kholif, A. M., El-Sayed, E. M., Abdou, S. M., and El-Shibiny, S. 2004. Isolation and char-
acterization of bacteriocins produced by Bifidobacterium lactis BB-12 and Bifidobacterium longum
BB-46. 9th Egyptian Conference for Dairy Science and Technology, Cairo, Egypt, pp. 9–11.
Stoffels, G., Nes, I. F., and Guomundsdóttir, A. 1992. Isolation and properties of a bacteriocin‐pro-
ducing Carnobacterium piscicola isolated from fish. J. Appl. Bacteriol. 73(4): 309–316.
Sun, C., Li, Y., Mei, S., Lu, Q., Zhou, L., and Xiang, H. 2005. A single gene directs both production and
immunity of halocin C8 in a haloarchaeal strain AS7092. Mol. Microbiol. 57(2): 537–549.
Sutyak, K. E., Wirawan, R. E., Aroutcheva, A. A., and Chikindas, M. L. 2008. Isolation of the Bacillus
subtilis antimicrobial peptide subtilosin from the dairy product‐derived Bacillus amyloliquefa-
ciens. J. Appl. Microbiol. 104(4): 1067–1074.
Suzuki, M., Yamamoto, T., Kawai, Y., Inoue, N., and Yamazaki, K. 2005. Mode of action of piscicocin
CS526 produced by Carnobacterium piscicola CS526. J. Appl. Microbiol. 98(5): 1146–1151.
Tahiri, I., Desbiens, M., Benech, R., Kheadr, E., Lacroix, C., Thibault, S., Ouellet, D., and Fliss, I. 2004.
Purification, characterization and amino acid sequencing of divergicin M35: A novel class IIa
bacteriocin produced by Carnobacterium divergens M35. Int. J. Food Microbiol. 97(2): 123–136.
Thacker, P. A. 2013. Alternatives to antibiotics as growth promoters for use in swine production: A
review. J. Anim. Sci. Biotechnol. 4(35): 1–12.
Todorov, S. D., Wachsman, M., Tomé, E., Dousset, X., Destro, M. T., Dicks, L. M. T., Franco, B. D.,
Vaz-Velho, M., and Drider, D. 2010. Characterisation of an antiviral pediocin-like bacteriocin
produced by Enterococcus faecium. Food Microbiol. 27(7): 869–879.
Vermeiren, L., Devlieghere, F., Vandekinderen, I., and Debevere, J. 2006. The interaction of the
non-bacteriocinogenic Lactobacillus sakei 10A and lactocin S producing Lactobacillus sakei 148
towards Listeria monocytogenes on a model cooked ham. Food Microbiol. 23(6): 511–518.
Verschuere, L., Rombaut, G., Sorgeloos, P., and Verstraete, W. 2000. Probiotic bacteria as biological
control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64(4): 655–671.
Vogel, R. F., Pohle, B. S., Tichaczek, P. S., and Hammes, W. P. 1993. The competitive advantage of
Lactobacillus curvatus LTH 1174 in sausage fermentations is caused by formation of curvacin A.
Syst. Appl. Microbiol. 16(3): 457–462.
Von Ah, U. 2006. Identification of Bifidobacterium thermophilum RBL67 isolated from baby faeces
and partial purification of its bacteriocin, Doctoral dissertation, Technische Wissenschaften,
Eidgenössische Technische Hochschule ETH Zürich, Nr. 16927.
Wertz, J. E. and Riley, M. A. 2004. Chimeric nature of two plasmids of Hafnia alvei encoding the bac-
teriocins alveicins A and B. J. Bacteriol. 186(6): 1598–1605.
Widdick, D. A., Dodd, H. M., Barraille, P., White, J., Stein, T. H., Chater, K. F., Gasson, M. J., and
Bibb, M. J. 2003. Cloning and engineering of the cinnamycin biosynthetic gene cluster from
Streptomyces cinnamoneus cinnamoneus DSM 40005. Proc. Natl. Acad. Sci. 100(7): 4316–4321.
Wiedemann, I., Böttiger, T., Bonelli, R. R., Wiese, A., Hagge, S. O., Gutsmann, T., Seydel, U. et al. 2006.
The mode of action of the lantibiotic lacticin 3147–a complex mechanism involving specific
interaction of two peptides and the cell wall precursor lipid II. Mol. Microbiol. 61(2): 285–296.
Wu, J., Hu, S., and Cao, L. 2007. Therapeutic effect of nisin Z on subclinical mastitis in lactating cows.
Antimicrob. Agents Chemother. 51(9): 3131–3135.
Yildirim, Z., Winters, D. K., and Johnson, M. G. 1999. Purification, amino acid sequence and mode
of action of bifidocin B produced by Bifidobacterium bifidum NCFB 1454. J. Appl. Microbiol. 86(1):
45–54.
Zacharof, M. P. and Lovitt, R. W. 2012. Bacteriocins produced by lactic acid bacteria: A review article.
APCBEE Procedia 2: 50–56.
Zhang, X., Clark, C. A., and Pettis, G. S. 2003. Interstrain inhibition in the sweet potato pathogen
Streptomyces ipomoeae: Purification and characterization of a highly specific bacteriocin and
cloning of its structural gene. Appl. Environ. Microbiol. 69(4): 2201–2208.
Zouhir, A., Kheadr, E., Fliss, I., and Hamida, J. B. 2011. Partial purification and characterization of
two bacteriocin-like inhibitory substances produced by bifidobacteria. Afr. J. Microbiol. Res.
5(4): 411–418.
chapter ten
Protease inhibitors from marine organisms

L. Karthik and A. Vishnu Kirthi
Contents
10.1 Introduction......................................................................................................................... 175
10.1.1 Protease enzymes................................................................................................... 175
10.1.2 Classification of proteases..................................................................................... 176
10.2 Protease inhibitors.............................................................................................................. 176
10.3 Marine microorganisms as a source of metabolites...................................................... 178
10.3.1 Marine microorganism source of protease inhibitor........................................ 178
10.3.2 Marine animal source of protease inhibitor....................................................... 179
10.3.3 Classification of protease inhibitors..................................................................... 180
10.3.3.1 Cysteine protease inhibitors................................................................... 180
10.3.3.2 Serine protease inhibitors....................................................................... 181
10.3.3.3 Threonine protease inhibitors................................................................ 182
10.3.3.4 Aspartic protease inhibitors................................................................... 183
10.4 Conclusion........................................................................................................................... 184
References...................................................................................................................................... 184
10.1 Introduction
10.1.1 Protease enzymes
Proteases are essential constituents found in prokaryotes, fungi, plants, and animals.
Proteases are enzymes that are involved in the breakdown of proteins in a process called
proteolysis. These enzymes are involved in a multitude of physiological reactions ranging
from simple digestion of food proteins to highly regulated cascades. Proteases determine
the lifetime of other proteins by playing an important physiological role like hormones,
antibodies, or other enzymes. This is one of the fastest switching on and switching off regu-
latory mechanisms in the physiology of organisms. Several bacteria secrete proteases to
hydrolyze the protein peptide bond into simple monomer units. Some of these bacterial
proteases also act as an exotoxin, which will destroy the extracellular structures. Proteases
play a critical role in many complex physiological and pathological processes such as pro-
tein catabolism, blood coagulation, cell growth and migration, tissue arrangement, mor-
phogenesis in development, inflammation, tumor growth and metastasis, activation of
zymogens, release of hormones and pharmacologically active peptides from precursor
proteins, and transport of secretory proteins across membranes (Chambers et al., 2001).
In general, extracellular proteases catalyze the hydrolysis of large proteins to smaller mol-
ecules for subsequent absorption by the cell, whereas intracellular proteases play a criti-
cal role in the regulation of metabolism. Since proteases are physiologically necessary for
living organisms, they are ubiquitous, being found in a wide diversity of sources such
175
as plants, animals, and microorganisms. Besides being necessary from the physiological
point of view, proteases are potentially hazardous to their proteinaceous environment and
the respective cell or organism must precisely control their activity. When uncontrolled,
proteases can be responsible for serious diseases. The control of proteases is generally
achieved by regulated expression/secretion and/or activation of proproteases, by degrada-
tion of mature enzymes, and by the inhibition of their proteolytic activity (Fitzpatrick and
O’Kennedy, 2004).
Moreover, protease enzymes are used for a long time in various forms of clinical thera-
pies. Their use in medicine is gaining more and more attention as several clinical studies
are indicating their benefits in oncology, inflammatory conditions, blood rheology con-
trol, and immune regulation. The pathogenesis of many parasites include their involve-
ment in invasion of a host by parasite migration through host tissue barriers, degradation
of hemoglobin and blood proteins, immune invasions, and activation of inflammation.
Thus, proteases play a foremost role in pathogenesis. However, uncontrolled action causes
deleterious effects in the body. Protease enzyme that is located inside the cancer cell is
capable of breaking through other healthy cell walls and membranes, thereby spreading
and growing into other cell organs and areas of the body, thus causing metastasis (Glinsky,
1993; Kim et al., 1998; Bellail et al., 2004).
10.1.2 Classification of proteases

Proteases are currently classified into six groups:
1. Serine proteases
2. Threonine proteases
3. Cysteine proteases
4. Aspartate proteases
5. Metalloproteases
6. Glutamic acid proteases
During catalysis, serine, aspartate, threonine, and cysteine groups as well as metal ions
will play a major role. All these types of enzymes are present in bacteria. Among these,
glutamic proteases are found only in fungi. Serine, cysteine, and metalloproteases are
widely spread in many pathogenic bacteria, where they play critical functions related to
evasion of host immune defenses, acquisition of nutrient for growth and proliferation,
facilitation of dissemination, or tissue damage during infection (Drag and Salvesen, 2010).
10.2 Protease inhibitors

Protease inhibitors are usually proteins with domains that enter or block the protease
active site to prevent substrate access. Activation of proteases causes alteration of a number
of specific proteins leading to subcellular remodeling and cardiac dysfunction. Most of the
protease inhibitors have been isolated from terrestrial animals, plants, fungi, and actino-
mycetes. The presence of protease inhibitors in microorganisms came into existence from
the studies on antibiotics as they act as inhibitors of the enzymes, which are involved in
the growth and multiplication of microorganisms. Proteolytic enzymes outside of micro-
bial cells hydrolyze organic nitrogen compounds in the medium, so they are thought to be
harmful to cells. The production of inhibitors of the proteolytic enzymes by microorgan-
isms is probably a mechanism to provide cellular protection. In contrast to the inhibitors
Chapter ten: Protease inhibitors from marine organisms 177
of proteolytic enzymes obtained from animals and plants, the inhibitors from microorgan-
isms are of smaller molecules in nature. Specific inhibitors of microbial origin have been
used as useful tools in biochemical analysis of biological functions and diseases (Fear
et al., 2007). Natural protease inhibitors include the family lipocalin proteins, which play
a major role in cell regulation and differentiation. The status of protease inhibitor is sum-
marized in Table 10.1.
Table 10.1 Protease used in structure-based drug design
Peptidase Biological function Disease
Cysteine peptidases
Cathepsin B Antigen processing Acute pancreatitis, cancer
Cathepsins L and S Lysosomal proteolysis Inflammation
FP-2 Hemoglobin degradation Malaria
Caspase 1 Maturation of interleukin 1-β Ameliorate inflammation,
endotoxic shock
Caspases 3 and 7 Executioner caspases in apoptosis Neuronal and cardiac
ischemic injuries
Calpains 1 and 2 Degradation of cytoskeletal Stroke, neural injuries
proteins
Picornain cysteine peptidases Processing of viral proprotein Virus infection
Serine peptidases
Thrombin Proteolysis of fibrinogen Thrombosis
Factor Xa Conversion of prothrombin to Thrombosis
thrombin
Factor VIIa Activation of factors IX and X Thrombosis
Urokinase Activation of plasminogen Cancer
Flavivirus peptidases Processing of polyprotein Viral infection
DPP-4 Processing of hormone precursors Type 2 diabetes mellitus
20S Proteasome Ubiquitin-dependent protein Cancer
degradation
Aspartic peptidases
HIV peptidase Processing of viral proprotein HIV infection
Renin Processing of angiotensinogen Blood pressure
Memapsin 2 β-Secretase activity Alzheimer’s disease
Plasmepsin Hemoglobin degradation Malaria
Metallopeptidases
Angiotensin-converting enzyme Conversion of angiotensin Hypertension
Botulinum neurotoxin Cleavage of SNAP proteins Clostridium and tetanus
infection
Anthrax lethal factor Cleavage of MAPKK Bacillus anthracis infection
Matrix metallopeptidase 1 Degradation of connective tissue Tissue damage in tumor
invasion
FtsH Elimination of misfolded proteins Neurological diseases
Carboxypeptidases B and U Cleavage of tissue plasminogen Blood coagulation
activator
PSMA Liberates glutamate from Marker for prostate
Ac-Asp-Glu in the brain cancer
Source: Mittl, P.R. and Grütter, M.G., Curr. Opin. Struct. Biol., 16(6), 769, 2006.
10.3 Marine microorganisms as a source of metabolites

The biological and chemical diversity of the marine environment has been the source of
unique chemical compounds with the potential for industrial development as pharmaceu-
ticals, cosmetics, nutritional supplements, molecular probes, enzymes, fine chemicals, and
agrochemicals (Ireland et al., 1994). The oceans represent a virtual untapped resource for
the discovery of even more novel compounds with useful activity. Even though the com-
mercial success stories in biotechnology are familiar, such stories in marine biotechnology
are far less familiar and far fewer (Zilinskas and Hill, 1995). In the past two decades, there
has been a continuous effort to learn more about the still largely unexplored realm of
marine-related products.
Besides microorganisms like bacteria, fungi, and actinobacteria, many other marine
organisms such as fishes, prawns, crabs, snakes, plants, and algae have also been studied
to tap the arsenal of the marine world. Properties like high salt tolerance, hyperthermosta-
bility, barophilicity, cold adaptivity, and ease in large-scale cultivation are the key interests
of scientists. These properties may not be expected in terrestrial sources as marine organ-
isms thrive in habitats such as hydrothermal vents, oceanic caves, and some areas where
high pressure and the absence of light are obvious (Debashish et al., 2005).
Marine bioactive compounds are organic compounds produced by both prokaryotes
and eukaryotes. These compounds generally help the host organism to protect themselves
and to maintain homeostasis in their environment. So far, less than 1.0% of the total marine
organisms producing bioactive metabolites are revealed. Based on the review of literature,
the seawater has bactericidal properties and it endorsed the production of antibiotics by
planktonic algae and bacteria, respectively (Lipton, 2003).
10.3.1 Marine microorganism source of protease inhibitor

Imada et al. (1985) isolated the first protease inhibitor–producing marine bacteria from
coastal seawater at the Aburatsubo Inlet of Sagami Bay in Kanagawa Prefecture, Japan.
Based on the amino acid sequences, it is identified as a marinostatin. It is capable to inhibit
the serine protease (Imada et al., 1986). Monastatin was the second protease inhibitor that
had an inhibitory activity against thiol protease (Imada et al., 1985). Leupeptin was the
third protease inhibitor that had an inhibitory activity against thiol and serine proteases
(Hamato et al., 1992). These all are produced by Alteromonas sp. metalloprotease inhibitor
from Spirulina platensis capable to inhibit the melanoma, carcinoma, and fibrosarcoma cell
lines (Mishima et al., 1998). The antioxidants extracted from Chlorella vulgaris have proven
inhibiting MMP-mediated cancer proliferation and progression (Wang et al., 2010).
Enzyme inhibitors are the third important product of marine actinobacteria. Although
it is used for the study of enzyme structures and reaction mechanisms, their use in pharma-
cology was started late (Bode and Huber, 1993). These selective inhibitors can be used as a
powerful tool for inactivating target proteases in the pathogenic processes of human diseases
such as malaria, emphysema, arthritis, pancreatitis, thrombosis, high blood pressure, muscu-
lar dystrophy, cancer, and AIDS (Demuth, 1990). Enzyme inhibitors from marine microorgan-
isms were less studied. However, Streptomyces isolated from terrestrial environment is a one
of the potential producers of enzyme inhibitors (Umezawa et al., 1972). Some of the second-
ary metabolite from actinobacteria was acting as an enzyme inhibitor such as streptomycin
(Martin, 1989), actinomycin (Katz and Weissbach, 1962), chloramphenicol (Jones and Westlake,
1974), and candicidin (Liras et al., 1977). The isolation of novel enzyme inhibitor from terrestrial
sources is rare; hence, marine actinobacteria will provide new potential inhibitors.
10.3.2 Marine animal source of protease inhibitor

In general, marine animals, particularly marine invertebrates, are the promising sources
of novel bioactive compounds. This is an adaptation strategy to thrive in the extreme
environmental conditions of the sea and as a defense strategy to escape from predators
by the marine invertebrates especially soft-bodied animals like sponges (Muller et al.,
2004). Focusing on sponges, a conceptual progress occurred with the study of Thakur
et al. (2003), who suggested that marine animals and their symbiotic microorganisms
(bacteria and fungi) produce an array of bioactive compounds against foreign attackers.
Pharmaceutical interest in sponges was aroused in the early 1950s by the discovery of
a number of unknown nucleosides: spongothymidine and spongouridine in the marine
sponge Cryptotethia crypta (Bergmann and Burke, 1956, Bergmann et al., 1957).
Marine organisms are rich sources of structurally diverse bioactive compounds with
various biological activities. The importance of marine organisms as a source of novel
bioactive substances is growing rapidly. With marine species comprising approximately
one-half of the total global biodiversity, the sea offers an enormous resource for novel com-
pounds. Proteases are enzymes that catalyze the hydrolysis of the peptide bonds forming
the primary structure of proteins (Dixon and Webb, 1979). Evolved over millions of years,
they are a goldmine of genetic diversity and novel secondary metabolites. They are com-
mon to organisms, from microorganism to plants and animals. In higher animals, pro-
teases are involved in several processes including digestion, proenzyme activation, and
defense mechanisms such as blood clotting and complement activation. Due to several
reasons, namely, inaccessibility of their habitats and very low yield of bioactive metabo-
lites, systematic chemical and pharmacological investigations of these organisms were not
popular until a few decades ago. However, the recent advances in underwater exploration,
natural product chemistry, genome mining, and bioassays have led to a great surge in the
search for novel biomolecules from this rather underexploited habitat. A cursory review
of the literature indicates that more than 70% of marine metabolites are obtained from
marine sponges, corals, and microorganisms. The contribution from other organisms like
molluscs, ascidians, and algae adds up to only 30% (Blunt et al., 2007). More than 90% of
marine bacteria are gram-negative psychrophiles, and nearly half of them require high
Na+ concentration for growth (Macleod, 1968). Their microbial growth and the products
of their metabolic activities differ considerably from those of terrestrial microorganisms.
Maeda and Taga (1976) reported that the activity of deoxyribonuclease isolated from a
marine Vibrio sp. was activated strongly by Mg2+ and stabilized by Ca2+. Kobori and Taga
(1980) isolated a marine phosphatase-producing bacterium. At 1000 atm hydrostatic pres-
sure, the enzyme activity of this microbe was more than three times higher than that at
1 atm. This implies that enzymes of marine microorganisms may also differ considerably
from those of terrestrial organisms. Therefore, inhibitors of these enzymes can be expected
to show different characteristics than those of terrestrial inhibitors. Enzyme inhibitors
have received increasing attention as useful tools, not only for the study of enzyme struc-
tures and reaction mechanisms but also for potential utilization in pharmacology (Bode
and Huber, 1993). Specific and selective protease inhibitors are potentially powerful tools
for inactivating target proteases in the pathogenic processes of human diseases such as
emphysema, arthritis, pancreatitis, thrombosis, high blood pressure, muscular dystrophy,
cancer, and AIDS.
Protease inhibitors can be further classified into five groups (serine, threonine, cyste-
ine, aspartyl, and metalloprotease inhibitors) according to the mechanism employed at the
active site of proteases they inhibit. Some protease inhibitors interfere with more than one
type of protease. For example, the serine family of protease inhibitors (serpins) is gener-
ally thought of as active against serine proteases, yet contains several important inhibitors
of cysteine proteases as well. Proteolytic inhibition by protease inhibitors can occur via
two mechanisms: irreversible trapping reactions and reversible tight binding reactions
(Rawlings et al., 2004). Inhibitors that bind through a trapping mechanism change confor-
mation after cleaving an internal peptide bond and trap the enzyme molecule covalently;
neither the inhibitor nor the protease can participate in further reactions. In tight binding
reactions, the inhibitor binds directly to the active site of the protease; these reactions are
reversible, and the inhibitor can dissociate from the enzyme in either the virgin state or
after modification by the protease (Fear et al., 2007).
10.3.3 Classification of protease inhibitors

Protease inhibitors are classifieds based on the type of protease they inhibit.
10.3.3.1 Cysteine protease inhibitors

Nature continues to be one of the most significant sources of pharmacologically active
compounds in the quest for drugs. Penicillium species are widely distributed in nature
and often found living on foods and in indoor environments. They are the source of sev-
eral β-lactam antibiotics, most significantly penicillin. Other secondary metabolites of
Penicillium chrysogenum include various different penicillins, roquefortine C, meleagrin,
chrysogine, xanthocillins, secalonic acids, sorrentanone, sorbicillin, and PR toxin (de Hoog
et al., 2000). Several undescribed, marine-derived Penicillium sp. have been recently iso-
lated from a variety of substrates such as mollusks, sponges, algae, and sands. These
Penicillium species are important producers of new metabolites such as sculezonones
A and B (Komatsu et al., 2000).
Serine and cysteine protease inhibitors named marinostatins and monastatins,
respectively, were isolated from a marine bacterial strain collected from a neritic Japanese
marine environment. The isolated strain, Alteromonas sp. (Imada et al., 1985), is a gram-
negative rod-shaped microorganism with DNA of low G + C contents. It requires seawa-
ter for growth and produces both serine and cysteine protease inhibitors (Imada et al.,
1985). Marine microorganisms have become an important source of pharmacologically
active metabolites. Published reviews show the importance of these organisms as poten-
tial sources of pharmaceutical leads. More specifically, fungi from the marine environ-
ment have shown great potential as suggested by the diversity of secondary metabolites.
Equistatin, a protease inhibitor isolated from the hydrophilic extract of the whole body of
Actinia equine (Lenarcic et al., 1998), is an acidic protein composed of three thyroglobulin
type 1 domains (Galea et al., 2000). It was further shown that the N-terminal domain alone
acts as a Cys protease inhibitor (Ki of 0.6 nM for papain) and the second domain acts as
an aspartic protease inhibitor (Ki of 0.3 for cathepsin D). The function of the third domain
remains unknown (Štrukelj et al., 2000). Sea anemones contain a variety of polypeptide
toxins, presumably in specialized stinging organelles (nematocysts). The sea anemone
polypeptide toxins are considered to serve as chemical weapons to capture prey animals
and as defensive substances against predators. A number of protease inhibitors have
already been isolated from Actinia equine (Ishida et al., 1997), Anemonia sulcata (Wunderer
et al., 1981; Schweitz et al., 1995), Anthopleura aff. xanthogrammica (Minagawa et al., 1997,
1998), Radianthus macrodactylus (Zykova, 1985), Stichodactyla helianthus (Antuch et al., 1993),
and Stichodactyla haddoni (Honma et al., 2008), although their physiological function is not
fully understood yet. Interestingly, three protease inhibitors (AsKC 1–3 or kalicludines 1–3)
from A. sulcata (Schweitz et al., 1995) and one inhibitor (SHTX III) from S. haddoni (Honma
et al., 2008) have been demonstrated to exhibit blocking activity against Kv1 potassium
channels as well as protease inhibitory activity.
10.3.3.2 Serine protease inhibitors

Marine microorganisms are widely recognized as new frontiers in natural product research.
In particular, the actinomycete bacteria and higher fungi from diverse marine environ-
ments are very prolific sources of structurally unique and biologically active metabolites.
Since the 1990s, more than a few hundred novel compounds have been annually isolated
from these organisms, which significantly contribute to both the chemical and biomedical
aspects of natural product research. The bioactivities of these marine-derived microbial
metabolites are highly diverse but with a focus on cytotoxic and antimicrobial activities.
Among the antimicrobial-related bioactivities, the sortase enzymes are regarded as being
a promising target for therapy because gram-positive bacteria proteins are displayed on
the cell surface with these enzymes (Bugni and Ireland, 2004; Cragg et al., 2009; Clancy
et al., 2010; Julianti et al., 2013).
Serine protease inhibitors are the largest and most widely distributed superfamily of
protease inhibitors (Hedstrom, 2002; Krowarsch et al., 2003; Di Cera, 2009), and based on
their possession of conserved functional motifs, they can be subdivided into many classes,
being the Kunitz-type inhibitors, the best characterized of them, probably due to their abun-
dance in several organisms (Lingaraju and Gowda, 2008; Yuan et al., 2008; Zhao et al., 2011;
Isaeva et al., 2012). The Kunitz-type motif consists of a polypeptide chain of ~60 amino acid
residues stabilized by three disulfide bridges (CI–CVI, CII–CIV, CIII–CV). Marine cyanobacte-
ria have emerged as a rich source of biologically and ecologically active secondary metabo-
lites, in particular peptides and depsipeptides (Gunasekera et al., 2009). The Kunitz-type
protease inhibitors are the best characterized family of serine protease inhibitors, probably
due to their abundance in several organisms. These inhibitors consist of a chain of ~60
amino acid residues stabilized by three disulfide bridges and were first observed in the
bovine pancreatic trypsin inhibitor (BPTI) like protease inhibitors, which strongly inhibit
trypsin and chymotrypsin. Canonical serine protease inhibitors have been the most exten-
sively studied so far, and 18 families are currently recognized (Laskowski et al., 2000).
Among them, the BPTI Kunitz family (PFAM: PF00014), which comprises extremely potent
inhibitors of serine proteases, is composed of more than 1000 different sequences isolated
from a variety of animals ranging from invertebrates to mammals (Kunitz and Northrop,
1936; Delfin et al., 1996). De Almeida Nogueira et al. (2013) investigated the interference of
the S. helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55–amino acid peptide, in
Trypanosoma cruzi serine peptidase activities, parasite viabilities, and parasite morpholo-
gies. Potent biological activity is often found in products isolated from marine organisms
because of their novel molecular structures (Donia and Hamann, 2003; Molinski et al.,
2009). Trabectedin (Yondelis), cytarabine (Ara-C), and eribulin (Halaven), which are known
as antitumor drugs, were developed from compounds found in marine organisms (Mayer
et al., 2010). Two new potent serine protease inhibitors, cyclotheonamides E2 and E3, have
been isolated from a marine sponge of the genus Theonella (Nakao et al., 1998). The effects
of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anem-
one S. helianthus (ShPI-I), on the viability and morphology of parasites in Leishmania culture
were studied. The N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and benzamidine
(Bza) inhibitors, which are potential Leishmania protease inhibitors, in all experimental
conditions reduced the parasite viability, with regard to time dependence. On the other
hand, N-tosyllysine chloromethyl ketone did not significantly affect the parasite viability
as it was a poor Leishmania enzyme inhibitor. Ultrastructural analysis demonstrated that

both Bza and TPCK induced changes in the flagellar pocket region with membrane altera-
tion, including bleb formation. However, TPCK effects were more pronounced than those
of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bod-
ies were visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine pro-
teases and the parasite viability (Silva-Lopez et al., 2007).
Also, there are reports of isolation of protease inhibitors, for example, the bioas-
say-guided isolation of serine protease inhibitors from a marine sponge, Coscinoderma
mathewsi, has yielded 1-methylherbipoline salts of halisulfate 1 and of suvanine (Kimura
et al., 1998). Two cyclodepsipeptides named kempopeptins A and B were isolated from a
collection of a Floridian marine cyanobacterium Lyngbya sp. that had previously afforded
the structurally related potent elastase inhibitors lyngbyastatin 7 and somamide B (Taori
et al., 2008).
A serine protease inhibitor, CvSI-1, was recently identified from the plasma of the east-
ern oyster, Crassostrea virginica (Xue et al., 2006). CvSI-1 is a 7609.6 Da protein consisting of
a 71–amino acid sequence with no homology to any known protein sequence in databases.
CvSI-1 inhibited subtilisin A, trypsin, and perkinsin, the major serine protease secreted by the
oyster protozoan parasite Perkinsus marinus, in a slow tight binding manner (Xue et al., 2006).
The Ras family of guanine nucleotide–binding proteins plays important roles in signal
transduction and the regulation of cell differentiation and proliferation. The Ras protein
requires posttranslational processing in order to associate with the plasma membrane and
to function in signal transduction or cellular transformation. The first processing step is cata-
lyzed by farnesyl protein transferase (FPT), which adds a farnesyl group to a cysteine residue
near the carboxy terminus of the Ras protein. Inhibition of FPT is a potential therapeutic target
for novel anticancer agents. Cembranolide diterpene, isolated from the soft coral Lobophytum
cristagalli, showed potent inhibitory activity against FPT with an IC50 value of 0.15 µM.
Serine, cysteine, and threonine proteases have many common active site features
including an active site nucleophile and a general base, which are often the target of irre-
versible inhibitors. So far, this group includes the majority of proteolytic enzymes and
many significant enzymes with involvement in human diseases. We will cover inhibitors
commonly considered to be irreversible. This includes inhibitors that form stable covalent
bonds with the enzyme. Covalent irreversible inhibitors of cysteine, serine, and threonine
proteases are capable of either alkylating or acylating their target enzymes. Alkylating
agents are very effective cysteine protease inhibitors, and they include chloromethyl
ketones, fluoromethyl ketones, diazomethyl ketones, acyloxymethyl ketones, epoxides,
and vinyl sulfones. The nucleophilic active site cysteine attacks an activated carbon and
forms an irreversible carbon–sulfur bond. Because the active site serine of serine proteases
is generally less nucleophilic than the corresponding catalytic cysteine, alkylating agents
primarily target cysteine proteases. An exception is the chloromethyl ketones, which are
capable of inhibiting serine proteases, although they have a slightly different mechanism
of inhibition. After a nucleophilic attack of the carbonyl of the inhibitor, the inhibitor alkyl-
ates the catalytic histidine. The enzyme can then be deacylated, but the alkylation of the
catalytic histidine results in a covalently bound inhibitor (Farady and Craik, 2010).
10.3.3.3 Threonine protease inhibitors

Threonine proteases are part of a multicomponent proteasome complex in microbial cells.
The archaebacterial proteasome has 14 active sites in the inner channel, one on each β-subunit.
The hydrolytic sites are spatially separated from the intracellular components. Recent reports
have indicated that the active site nucleophile is the hydroxyl group on the threonine at the
N-terminus of the β-subunit. The replacements of the terminal threonine by serine in archae-
bacterial proteasomes allow complete proteolytic activity. Therefore, the conservation of the
threonines in the active sites of all threonine proteases from bacteria to eukaryotes is unclear.
Looking at the diverse functions of the threonine proteases in bacteria and mammals, it is
evident that the phylogenetically ancient proteasome has undergone adaptations that favor
different functions in different physiological situations (Menon and Rao, 2012).
10.3.3.4 Aspartic protease inhibitors

Proteases are responsible either directly or indirectly for all bodily functions, including
cell growth, differentiation, and death (apoptosis), cell nutrition, intra- and extracellular
protein turnover (housekeeping and repair), cell migration and invasion, and fertilization
and implantation. These functions extend from the cellular level to the organ and organ-
ism levels to produce cascade systems such as homeostasis and inflammation and complex
processes at all levels of physiology and pathophysiology. Any system that encompasses
normal and abnormal bodily functions must have effective regulatory counterparts, that
is, protease inhibitors. Hence, the research interest in protease inhibitors has evoked tre-
mendous attention in many disciplines. Multicellular organisms possess endogenous pro-
tein protease inhibitors to control proteolytic activity. Most of these inhibitory proteins are
directed against serine proteases, although some are known to target cysteine, aspartyl,
or metalloproteases. Indeed, inhibitors of serine, cysteine, and metalloproteases are dis-
tributed ubiquitously throughout the biological world (Menon and Rao, 2012). Xylanases
(1,4-β-d-xylan xylanohydrolase) are glycosidases that catalyze the hydrolytic cleavage of
β-1,4-linked polymers of d-xylose. They have raised enormous interest in the past decade
in view of their application in clarification of juices and wines, conversion of renewable
biomass into liquid fuels, and development of environmentally sound prebleaching pro-
cesses in the paper and pulp industry (Kulkarni et al., 1999). Although extensive studies
have been carried out on the industrial applications of xylanases, there is a paucity of
reports on their molecular enzymology and clinical implications. Recently, glycosidases
have been studied with a clinical perspective of locating enzyme allergens (Tarvainen
et al., 1991). Some of these enzymes, including xylanases and cellulases, have been found
to cause occupational and nonoccupational allergies, such as respiratory and irritant con-
tact dermatitis. Therefore, from the biomedical point of view, inhibitors of this class of
enzymes will have tremendous importance in the near future. In addition, the inhibi-
tion of cellulolytic and hemicellulolytic enzymes has potential applications to prevent the
degradation of wood and cloth by the action of the hydrolytic enzymes present in the gut
of termites (Menon and Rao, 2012). A bifunctional low-molecular-weight, linear, peptidic
inhibitor API, from thermotolerant Bacillus licheniformis, is reported to exhibit a slow tight
binding inhibition against ChiA. The bifunctionality of the inhibitor can be defined as it
was previously reported to inhibit an aspartic protease, pepsin (Kumar and Rao, 2006).
Class-specific transition state analogs have been developed to interfere specifically
with the catalytic residues of each class of proteases. Aspartic protease inhibitors have long
been designed around substrate polypeptides, with a replacement of the scissile amide
bond with a noncleavable, transition-state isostere. The first specific inhibitor for aspartic
proteases, pepstatin A, was discovered from Actinomyces, as an inhibitor for pepsin. It also
showed strong inhibitory activity against several other aspartic proteases. Pepstatin A is a
peptide, but the scissile bond is replaced with a statin group [(3S,4S)-4-amino-3-hydroxyl-
6-methylheptanoic acid]. Instead of a trigonal carbonyl, statins have a chiral hydroxyl
group, giving it the ability to mimic the tetrahedral state of the substrate transition state
(Eder et al., 2007).
10.4 Conclusion
The family of protease inhibitors, although rather small, contains a number of validated and
potential drug targets making drug discovery efforts in this area very fruitful and exciting.
There have been substantive advances in our understanding of the use of protease inhibitors
as therapeutic agents. Several synthetic protease inhibitors have been approved by the FDA for
therapy of HIV and hypertension. These drugs represent prime examples of structure-based
drug design. Moreover, the inhibitory principles and compounds, which have been established
and discovered, now enable mechanism-based drug discovery across the whole family. The
sequencing of the human genome and the resulting knowledge on all human aspartic proteases
allow an exhaustive profiling of inhibitors for specificity toward all family members, thereby
reducing the risk of unwanted side effects. A number of natural and peptidomimetic inhibitors
performed well in different phases of clinical testing to treat other human disorders, includ-
ing cancer, inflammation, cardiovascular, neurodegenerative, and various infectious diseases.
Despite this impressive progress, there is much to learn about the cross talk between signal
transduction pathways and protease activation cascades. Additionally, the development of suc-
cessful protease inhibitors for clinical use is reliant on maximizing bioavailability, specificity,
and potency of inhibition of the target enzyme. Ideally, localizing protease inhibitors to a single
target area of the body may also help minimize the potential for complications and detrimental
side effects. There is the further issue of the development of drug resistance to protease inhibi-
tors in the face of a buildup of substrate pressure and selection of catalytically active mutant or
other salvage proteases that do not have complementarity for carefully designed inhibitors of
wild-type proteases. The future appears to still hold considerable promise for protease inhibi-
tors. We can anticipate new, overexpressed proteases from genomic/biochemical comparisons
made between normal/diseased cells, host/pathogen, healthy/unhealthy subjects leading to
more effective and efficient validation of proteases as drug targets. New advances in protein
chemistry will lead to faster production and greater quantities of pure recombinant proteases,
and advances in structural biology (crystallography, NMR spectroscopy) will produce faster
and more accurate inhibitor protease structures. Inhibitors (naturally occurring and synthetic)
have permitted detailed biochemical and crystallographic investigations to be made, but an
understanding of the selectivity of such inhibitors may be of just as much importance for the
design and synthesis of specific inhibitors for use therapeutically in controlling individual
aspartic proteases. The discovery of novel selective inhibitors can proceed only through the
combination of screening of chemical libraries, rational design, computational technology,
and exploration of natural compounds. The exploitation of vast microbial diversity will also
generate a large amount of biologic aspartic protease inhibitors. Furthermore, future research
into the synergistic capabilities of inhibitors will help elucidate the most effective combina-
tion therapies. These advances, together with more careful attention to inhibitor conformation,
mechanism of action, and drug-like composition, are expected to result in more potent, more
selective, and more bioavailable inhibitors with a higher probability of success in the clinic.
References
Antuch, W., Berndt, K. D., Chavez, M. A., Delfin, J., and Wuthrich, K. 1993. The NMR solution struc-
ture of a Kunitz type proteinase inhibitor from the sea anemone Stichodactyla helianthus. Eur J
Biochem 212(3):675–684.
Bellail, A. C., Hunter, S. B., Brat, D. J., Tan, C., and Van Meir, E. G. 2004. Microregional extracel-
lular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol
36(6):1046–1069.
Bergmann, W. and Burke, D. C. 1956. Contributions to the study of marine products. XL. The nucleo-
sides of sponges. 1 IV. Spongosine 2. J Org Chem 21(2):226–228.
Bergmann, W., Watkins, J. C., and Stempien Jr, M. F. 1957. Contributions to the study of marine prod-
ucts. XLV. Sponge nucleic acids, 1. J Org Chem 22(11):1308–1313.
Blunt, J. W., Copp, B. R., Hu, W. P., Munro, M. H., Northcote, P. T., and Prinsep, M. R. 2007. Marine
natural products. Nat Prod Rep 24(1):31–86.
Bode, W. and Huber, R. 1993. Natural protein proteinase inhibitors and their interaction with pro-
teinases. Eur J Biochem Rev 204:43–61.
Bugni, T. S. and Ireland, C. M. 2004. Marine-derived fungi: A chemically and biologically diverse
group of microorganisms. Nat Prod Rep 21(1):143–163.
Chambers, L. S., Black, J. L., Poronnik, P., and Johnson, P. R. 2001. Functional effects of protease-
activated receptor-2 stimulation on human airway smooth muscle. Am J Physiol 281:L1369–L1378.
Clancy, K. W., Melvin, J. A., and McCafferty, D. G. 2010. Sortase transpeptidases: Insights into mech-
anism, substrate specificity, and inhibition. Biopolymers 94(4):385–396.
Cragg, G. M., Grothaus, P. G., and Newman, D. J. 2009. Impact of natural products on developing
new anti-cancer agents. Chem Rev 109(7):3012–3043.
De Almeida Nogueira, N. P., Morgado-Diaz, J. A., Menna-Barreto, R. F., Paes, M. C., and da Silva-
Lopez, R. E. 2013. Effects of a marine serine protease inhibitor on viability and morphology of
Trypanosoma cruzi, the agent of chagas disease. Acta Trop 128(1):27–35.
De Hoog, G. S., Guarro, J., Gene, J., and Figueras, M. J. 2000. Atlas of Clinical Fungi. Centraalbureau
voor Schimmelcultures (CBS).
Debashish, G., Malay, S., Barindra, S., and Joydeep, M. 2005. Marine enzymes. Mar Biotechnol 1,
189–218.
Delfin, J., Martinez, I., Antuch, W., Morera, V., Gonzalez, Y., Rodriguez, R., Marquez, M. et al. 1996.
Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla
helianthus. Toxicon 34(11–12):1367–1376.
Demuth, H. U. 1990. Recent developments in inhibiting cysteine and serine proteases. J Enzyme Inhib
3(4):249–278.
Di Cera, E. 2009. Serine proteases. IUBMB Life 61(5):510–515.
Dixon, M. and Webb, E. C. 1979. Enzymes. Longam Group Limited, London, U.K.
Donia, M. and Hamann, M. T. 2003. Marine natural products and their potential applications as
anti-infective agents. Lancet Infect Dis 3(6):338–348.
Drag, M. and Salvesen G. S. 2010. Emerging principles in protease-based drug discovery. Nat Rev
Drug Discov 9(9):690–701.
Eder, J., Hommel, U., Cumin, F., Martoglio, B., and Gerhartz, B. 2007. Aspartic proteases in drug
discovery. Curr Pharm Des 13(3):271–285.
Farady, C. J. and Craik, C. S. 2010. Mechanisms of macromolecular protease inhibitors. Chembiochem
11(17):2341–2346.
Fear, G., Komarnytsky, S., and Raskin, I. 2007. Protease inhibitors and their peptidomimetic deriva-
tives as potential drugs. Pharmacol Ther 113(2):354–368.
Fitzpatrick, B. and O’Kennedy, R. 2004. The development and application of a surface plasmon
resonance-based inhibition immunoassay for the determination of warfarin in plasma ultra-
filtrate. J Immunol Methods 291(1–2):11–25.
Galea, K., Bavec, S., Turk, V., and Lenarcic, B. 2000. Cloning and expression of functional equistatin.
Biol Chem 381(1):85–88.
Glinsky, G. E. 1993. Cell adhesion and metastasis: Is the site specificity of cancer metastasis deter-
mined by leukocyte-endothelial cell recognition and adhesion? Crit Rev Oncol Hematol
14(3):229–278.
Gunasekera, S. P., Miller, M. W., Kwan, J. C., Luesch, H., and Paul, V. J. 2009. Molassamide, a depsi-
peptide serine protease inhibitor from the marine cyanobacterium Dichothrix utahensis. J Nat
Prod 73(3):459–462.
Hamato, N., Takano, R., Kamei-Hayashi, T., Imada, C., and Hara, S. 1992. Leupeptins produced by
the marine Alteromonas sp. B-10–31. Biosci Biotech Biochem 56:1316–1318.
Hedstrom, L. 2002. Serine protease mechanism and specificity. Chem Rev 102(12):4501–4524.
Honma, T., Kawahata, S., Ishida, M., Nagai, H., Nagashima, Y., and Shiomi, K. 2008. Novel peptide
toxins from the sea anemone Stichodactyla haddoni. Peptides 29(4):536–544.
Imada, C., Maeda, M., Hara, S., Taga, N., and Simidu, U. 1986. Purification and characterization
of subtilisin inhibitors ‘Marinostatin’ produced by marine Alteromonas sp. J Appl Microbiol
60(6):469–476.
Imada, C., Nobuo, T., and Masachika, M. 1985. Cultivation conditions for subtilisin inhibitor-
producing bacterium and general properties of the inhibitor marinostatin. Bull Japan Soc Sci Fish
505–810.
Ireland, P., Jolley, D., Giles, G., O’Dea, K., Powles, J., Rutishauser, I., and Williams, J. 1994. Development
of the Melbourne FFQ: A food frequency questionnaire for use in an Australian prospective
study involving an ethnically diverse cohort. Asia Pac J Clin Nutr 3(1):19–31.
Isaeva, M. P., Chausova, V. E., Zelepuga, E. A., Guzev, K. V., Tabakmakher, V. M., Monastyrnaya, M. M.,
and Kozlovskaya, E. P. 2012. A new multigene superfamily of Kunitz-type protease inhibitors
from sea anemone Heteractis crispa. Peptides 34(1):88–97.
Ishida, M., Minagawa, S., Miyauchi, K., Shimakura, K., Nagashima, Y., and Shiomi, K. 1997. Amino
acid sequences of Kunitz-type protease inhibitors from the sea anemone Actinia equina. Fish
Sci 63(5):794–798.
Jones, A. and Westlake, D. W. S. 1974. Regulation of chloramphenicol synthesis in Streptomyces sp.
3022 a. properties of arylamine synthetase, an enzyme involved in antibiotic biosynthesis. Can
J Microbiol 20(11):1599–1611.
Julianti, E., Lee, J. H., Liao, L., Park, W., Park, S., Oh, D. C., Oh, K. B., and Shin, J. 2013. New polyaro-
matic metabolites from a marine-derived fungus Penicillium sp. Org Lett 15(6):1286–1289.
Katz, E. and Weissbach, H. 1962. Biosynthesis of the actinomycin chromophore; enzymatic conver-
sion of 4-methyl-3-hydroxyanthranilic acid to actinocin. J Biol Chem 237(3):882–886.
Kim, J, Yu, W., Kovalski, K., and Ossowski, L. 1998. Requirement for specific proteases in cancer
cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 94(3):353–362.
Kimura, J., Ishizuka, E., Nakao, Y., Yoshida, W. Y., Scheuer, P. J., and Kelly-Borges, M. 1998. Isolation
of 1-methylherbipoline salts of halisulfate-1 and of suvanine as serine protease inhibitors from
a marine sponge, Coscinoderma mathewsi. J Nat Prod 61(2):248–250.
Kobori, H. and Taga, N. 1980. Extracellular alkaline phosphatase from marine bacteria: Purification
and properties of extracellular phosphatase from a marine Pseudomonas sp. Can J Microbiol
26(7):833–838.
Komatsu, K., Shigemori, H., Mikami, Y., and Kobayashi, J. 2000. Sculezonones A and B, two metabo-
lites possessing a phenalenone skeleton from a marine-derived fungus Penicillium species.
J Nat Prod 63(3):408–409.
Krowarsch, D., Cierpicki, T., Jelen, F., and Otlewski, J. 2003. Canonical protein inhibitors of serine
proteases. Cell Mol Life Sci 60(11):2427–2444.
Kulkarni, N., Shendye, A., and Rao, M. 1999. Molecular and biotechnological aspects of xylanases.
FEMS Microbiol Rev 23(4):411–456.
Kumar, A. and Rao, M. 2006. Biochemical characterization of a low molecular weight aspartic pro-
tease inhibitor from thermo-tolerant Bacillus licheniformis: Kinetic interactions with pepsin.
Biochim Biophys Acta 1760(12):1845–1856.
Kunitz, M. and Northrop, J. H. 1936. Isolation from beef pancreas of crystalline trypsinogen, tryp-
sin, a trypsin inhibitor, and an inhibitor-trypsin compound. J Gen Physiol 19(6):991–1007.
Laskowski Jr, M. I. C. H. A. E. L., Qasim, M. A., and Lu, S. M. 2000. Interaction of standard mechanism,
canonical protein inhibitors with serine proteinases. Protein–Protein Recognition (Kleanthous,
C., ed.), Oxford University Press, Oxford, U.K. pp. 228–279.
Lenarcic, B., Ritonja, A., Strukelj, B., Turk, B., and Turk, V. 1998. Equistatin, a new inhibitor of cys-
teine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain.
J Biol Chem 273(20):12682.
Lingaraju, M. H. and Gowda, L. R. 2008. A Kunitz trypsin inhibitor of Entada scandens seeds: Another
member with single disulfide bridge. Biochim Biophys Acta 1784(5):850–855.
Lipton, A. P. 2003. Isolation and application of marine natural products. 1–8.
Liras, P., Villanueva, J. R., and Martin, J. F. 1977. Sequential expression of macromolecule biosynthe-
sis and candicidin formation in Streptomyces griseus. J Gen Microbiol 102(2):269–277.
Macleod, R. A. 1968. On the role of inorganic ions in the physiology of marine bacteria. Adv Microbiol
Sea 1:95–126.
Maeda, M. and Taga, N. 1976. Extracellular nuclease produced by a marine bacterium. II.
Purification and properties of extracellular nuclease from a marine Vibrio sp. Can J Microbiol
22(10):1443–1452.
Martin, J. F. 1989. Molecular mechanisms for the control by phosphate of the biosynthesis of anti-
biotics and other secondary metabolites. Regulation of Secondary Metabolism in Actinomycetes:
213–237.
Mayer, A. M., Glaser, K. B., Cuevas, C., Jacobs, R. S., Kem, W., Little, R. D., McIntosh, J. M., Newman,
D. J., Potts, B. C., and Shuster, D. E. 2010. The odyssey of marine pharmaceuticals: A current
pipeline perspective. Trends Pharmacol Sci 31(6):255–265.
Menon, V. and Rao, M. 2012. Microbial aspartic protease inhibitors. 1–30.
Minagawa, S., Ishida, M., Shimakura, K., Nagashima, Y., and Shiomi, K. 1997. Isolation and amino
acid sequences of two Kunitz-type protease inhibitors from the sea anemone Anthopleura aff.
xanthogrammica. Comp Biochem Physiol B Biochem Mol Biol 118(2):381–386.
Minagawa, S., Ishida, M., Shimakura, K., Nagashima, Y., and Shiomi, K. 1998. Amino acid sequence
and biological activities of another Kunitz-type protease inhibitor from the sea anemone
Anthopleura aff. xanthogrammica. Fish Sci 64:157–161.
Mishima, T., Murata, J., Toyoshima, M., Fujii, H., Nakajima, M., Hayashi, T., and Saiki, I. 1998.
Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated poly-
saccharide derived from a blue-green alga, Spirulina platensis. Clin Exp Metastasis 16(6):541–550.
Mittl, P. R. and Grütter, M. G. 2006. Opportunities for structure-based design of protease-directed
drugs. Curr Opin Struct Biol 16(6):769–775.
Molinski, T. F., Dalisay, D. S., Lievens, S. L., and Saludes, J. P. 2009. Drug development from marine
natural products. Nat Rev Drug Discov 8(1):69–85.
Muller, W. E., Schroder, H. C., Wiens, M., Perovic-Ottstadt, S., Batel, R., and Muller, I. M. 2004.
Traditional and modern biomedical prospecting: Part II-the benefits: Approaches for a sustain-
able exploitation of biodiversity (secondary metabolites and biomaterials from sponges). Evid
Based Complement Alternat Med 1(2):133–144.
Nakao, Y., Oku, N., Matsunaga, S., and Fusetani, N. 1998. Cyclotheonamides E2 and E3, new
potent serine protease inhibitors from the marine sponge of the genus Theonella. J Nat Prod
61(5):667–670.
Rawlings, N. D., Tolle, D. P., and Barrett, A. J. 2004. Evolutionary families of peptidase inhibitors.
Biochem J 378(Pt 3):705–716.
Schweitz, H., Bruhn, T., Guillemare, E., Moinier, D., Lancelin, J. M., Beress, L., and Lazdunski, M.
1995. Kalicludines and kaliseptine two different classes of sea anemone toxins for voltage sen-
sitive K+ channels. J Biol Chem 270(42):25121–25126.
Silva-Lopez, R. E., Morgado-Diaz, J. A., Chavez, M. A., and Giovanni-De-Simone, S. 2007. Effects of
serine protease inhibitors on viability and morphology of Leishmania amazonensis promasti-
gotes. Parasitol Res 101(6):1627–1635.
Štrukelj, B., Lenarčič, B., Gruden, K., Pungerčar, J., Rogelj, B., Turk, V., and Jongsma, M. A. 2000.
Equistatin, a protease inhibitor from the sea anemone Actinia equina, is composed of three
structural and functional domains. Biochem Biophys Res Commun 269(3):732–736.
Taori, K., Paul, V. J., and Luesch, H. 2008. Kempopeptins A and B, serine protease inhibitors
with different selectivity profiles from a marine cyanobacterium, Lyngbya sp. J Nat Prod
71(9):1625–1629.
Tarvainen, K., Kanerva, L., Tupasela, O., Grenquist-Norden, B., Jolanki, R., Estlander, T., and Keskinen,
H. 1991. Allergy from cellulase and xylanase enzymes. Clin Exp Allergy 21(5):609–615.
Thakur, N. L., Hentschel, U., Krasko, A., Pabel, C. T., Anil, A. C., and Müller, W. E. 2003. Antibacterial
activity of the sponge Suberites domuncula and its primmorphs: Potential basis for epibacterial
chemical defense. Aquatic Microb Ecol 31(1):77–83.
Umezawa, S., Tatsuta, K., Fujimoto, K., Tsuchiya, T., and Umezawa, H. 1972. Structure of antipain, a
new sakaguchi positive product of Streptomyces. J Antibiot 25(4):267–270.
Wang, H. M., Pan, J. L., Chen, C. Y., Chiu, C. C., Yang, M. H., Chang, H. W., and Chang, J. S. 2010.
Identification of anti-lung cancer extract from Chlorella vulgaris CC by antioxidant property
using supercritical carbon dioxide extraction. Process Biochem 45(12):1865–1872.
Wunderer, G., Machleidt, W., and Fritz, H. 1981. The broad-specificity proteinase inhibitor 5 II from
the sea anemone Anemonia sulcata. Methods Enzymol 80:816–820.
Xue, Q. G., Waldrop, G. L., Schey, K. L., Itoh, N., Ogawa, M., Cooper, R. K., Losso, J. N., and La Peyre,
J. F. 2006. A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea
virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan
parasite Perkinsus marinus. Comp Biochem Physiol B Biochem Mol Biol 145(1):16–26.
Yuan, C. H., He, Q. Y., Peng, K., Diao, J. B., Jiang, L. P., Tang, X., and Liang, S. P. 2008. Discovery of a
distinct superfamily of Kunitz-type toxin (KTT) from tarantulas. PLoS One 3(10):e3414.
Zhao, R., Dai, H., Qiu, S., Li, T., He, Y., Ma, Y., Chen, Wu, Z., Li, Y. W., and Cao, Z. 2011. SdPI, the
first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom. PLoS One
6(11):e27548.
Zilinskas, R. A. and Hill, R. T. 1995. The global challenge of marine biotechnology: A status report
on the United States, Japan, Australia, and Norway. Maryland Sea Grant College.
Zykova, T. A. 1985. Amino-acid sequence of trypsin inhibitor IV from Radianthus macrodactylus.
Bioorg Khim 11:293–301.
chapter eleven
Ganoderma
A bioresource of antimicrobials
K. Rajesh and Dharumadurai Dhanasekaran
Contents
11.1 Introduction....................................................................................................................... 189
11.2 Classification of Ganoderma.............................................................................................. 190
11.3 History of Ganoderma........................................................................................................ 191
11.4 Medicinal Ganoderma........................................................................................................ 191
11.5 Bioactive substances in Ganoderma sp............................................................................ 192
11.5.1 Terpenoid compounds.......................................................................................... 192
11.5.2 Proteins and polysaccharides.............................................................................. 192
11.5.3 Antimicrobial compounds................................................................................... 193
11.6 Antibacterial activity........................................................................................................ 194
11.7 Antifungal activity............................................................................................................ 195
11.8 Antiviral activity............................................................................................................... 195
11.9 Antiprotozoal effect of Ganoderma sp............................................................................. 195
11.10 Current biomedical applications..................................................................................... 196
11.11 Future perspective............................................................................................................. 197
Acknowledgement....................................................................................................................... 197
References...................................................................................................................................... 198
11.1 Introduction
“Mushroom” is not a taxonomic category. The term “mushroom” should be used here
according to the definition of Chang and Miles (1992) as “a macro fungus with a distinc-
tive fruiting body, which can be hypogeous or epigeous, large enough to be seen with the
naked eye and to be picked by hand.” From a taxonomic point of view, mainly basidiomy-
cetes but also some species of ascomycetes belong to mushrooms. Mushrooms constitute
at least 14,000 and perhaps as many as 22,000 known species.
The natural products and herbal medicine industry have become increasingly popu-
lar over the past three decades (Hamburger and Hostettmann, 1991; Shu, 1998). The rec-
ognition of the value of traditional medical systems, particularly of Asian origin, and
identification of indigenous medicinal plants that have shown to have healing power
(Elvin Lewis, 2001) are factors that have had significant influence in the expansion of the
natural product industry. Furthermore, there is a constant search for new and effective
drugs, in order to overcome a number of pathogenic organism which are resistant to
many therapeutic products available in market.
189
Mushrooms have been a major focus of investigations for novel biologically active
compounds from natural resources, and in recent years, pharmaceutical companies have
spent a lot of time developing these natural products to produce more affordable and cost-
effective remedies (Farnsworth, 1994). In recent years, more mushrooms have been iso-
lated and identified, and the number of mushrooms being cultivated for food or medicinal
purposes has been increasing rapidly (Chang, 1995). Mushroom “nutriceuticals” are bio-
active compounds that are extractable from mushrooms, and they have nutritional and
medicinal features that may be used in the prevention and treatment of disease (Chang
and Buswell, 1996).
Ganoderma has been used in folk medicine in China and Japan for over 2000 years for
a wide range of ailments. Ganoderma, commonly known as Reishi mushroom, is one of the
most important medicinal mushrooms widely used in various countries. Ganoderma has
been regarded as a cure for all types of disease perhaps due to its demonstrated efficacy as
a popular remedy to treat a large number of diseases, namely, chronic hepatitis, arthritis,
hypertension, hyperlipidemia, bronchitis, asthma, gastic ulcer, diabetes, etc.; almost all
medicinal properties have been attributed to Ganoderma lucidum, and thus, it is known as
mushroom of immortality. Due to its ability to cure many different diseases, it received
names like “elixir of life” and food of god. Its intracellular and extracellular polysaccha-
ride shows inhibition of growth of several types of cancer cells. The different kinds of
polysaccharide derived from mushrooms and their effects on host immune system are
very important today.
Antimicrobial activity including antibacterial, antifungal, antiparasitic, and antiviral
agents is the third widespread therapeutic effect reported in mushrooms (Kettering et al.,
2005; Ngai and Ng, 2003; Obuchi et al., 1990; Okamoto and Li, 1993). Indeed, it is thought
that over 200 higher fungus species showed antimicrobial properties (Anke and Sterner,
1991; Gianetti et al., 1986; Wang et al., 1993; Yoon et al., 1995). The combined biological
properties of different mushroom genera such as G. lucidum, G. frondosa, Lentinus edodes,
Omphalotus illudens (Schwein.), P. betulinus, P. ostreatus, and Rozites caperatus were reported
by Wasser and Weis (1999b), and Ying et al. (1987) generated a broad spectrum of antimi-
crobial activities including antibacterial effects (Beltran-Garcia et al., 1997; Cherqui et al.,
1999; Donnelly et al., 1985; Dornberger et al., 1986; Min et al., 2000; Piraino, 2006; Wasser
and Weis, 1999; Ying et al., 1987). G. lucidum, through a laccase, showed a potent inhibitory
activity against HIV-1 reverse transcriptase. G. frondosa also demonstrates anti-HIV prop-
erties. Its action is simultaneously general and topical.
The limitations of traditional identification techniques indicate that alternative meth-
ods need to be developed for the identification of these fungi. With the development of
molecular biology, some new techniques have been applied to fungal classical taxonomy.
DNA fingerprinting techniques, however, would be allowed to identify the Ganoderma
species and cultivars, indicating that it is a useful tool for the valid protection of newly
bred cultivars.
11.2 Classification of Ganoderma

Ganoderma species belong to the kingdom of fungi, the division of Basidiomycota, the
class of Homobasidiomycetes, the order of Aphyllophorales, the family of Polyporaceae,
(Ganodermataceae), and the genus of Ganoderma (Chang, 1995; Wassser and Weis, 1999).
Fungi from the family of Polyporaceae are classified as they have many tiny holes on the
underside of the fruiting body, which are pores that contain the reproductive spores. They
have a woody or leathery feel, and the presence of these pores is an obvious characteristic
Chapter eleven: Ganoderma 191
that distinguishes polypores from other common types of mushrooms. Polypore does, like
other fungi, grow on wood as an expensive network of microscopic tubes known as myce-
lium. They degrade the wood over time and produce a fruiting body (or conk) on the sur-
face of the wood. Ganoderma species are among those fungi that can thrive under hot and
humid conditions and are usually found in subtropical and tropical regions (Moncalvo
and Ryvarden, 1998).
Ganoderma species are not classified as edible, as the fruiting bodies are always
thick, corky, and tough and do not have the fleshy texture characteristic of true edible
mushrooms such as the common white button mushroom Agaricus bisporus. Although
they are not c lassified as edible, several types of Ganoderma products are available on the
market including ground fruiting bodies or mycelium processed into capsule or tablet
form, extracts from fruiting body or mycelium dried and processed into capsule or tablet
form, Ganoderma beer, and Ganoderma hair tonics (Jong and Birmingham, 1992).
Within the genus Ganoderma, over 250 taxonomic names have been reported world-
wide (Moncalvo et al., 1995) including G. adspersum, G. applanatum, G. australe, G. boninense,
G. cupreum, G. incrassatum, G. lipsience, G. lobatum, G. lucidum, G. oerstedii, G. oregonense, G. pfeifferi,
G. platense, G. resinaceum, G. sessile, G. sinense, G. tornatum, G. tsugae, and G. webrianum.
11.3 History of Ganoderma

Ganoderma lucidum has been treasured in China and Japan for many thousands of years
(Willard, 1990). In Chinese, the mushroom is called “lingzhi”; in Japanese “Reishi,
Mannentake, or Sachitake”; and “Youngzhi” in Korean. Chinese tradition proclaims that
Ganoderma is also called “miraculous zhi” or “aunicious herb” and is usually considered to
“symbolize happy augury, and to bespeak good fortune, good health and longevity, even
immortality” (Wasson, 1968).
As early as 800 years ago in the Yuan Dynasty (1280–1368 A.D.), G. lucidum has been
represented in paintings, carvings, furniture, carpet design, jewellery, perfume bottles,
and many more creative artworks (Wasser and Weis, 1999). According to the two famous
Chinese herb medical books, Shen Nong Ben Cao Jing (25–220 A.D., Eastern Han Dynasty)
and Ben Cao Gang Mu (1590 A.D., Ming Dynasty), there were six known species of Ganoderma
(lingzhi) in China at that time, whereas now more than 250 species have been described
(Moncalvo et al., 1995).
11.4 Medicinal Ganoderma
G. lucidum (lingzhi) was the favorite species within the Ganodermataceae family as it was
believed to be the only mushroom containing therapeutic properties (Willard, 1990). In
the literature today, there is much confusion as to which is the true Ganoderma species.
The Japanese believed that the true Ganoderma was red and that a Ganoderma species with
different colors was a red Ganoderma that had become discolored due to changes in envi-
ronmental conditions such as temperature, humidity, and light. In China, they believed
that the true Ganoderma was black as there were reports of a black Ganoderma that had
unusual medicinal benefits not produced by the red mushroom (Mayzumi et al., 1997).
Chang (1995) suggested that Ganoderma (lingzhi) encompassed several Ganoderma spe-
cies, although most investigations and therapeutic practices refer to the species G. lucidum.
More recently, other species such as G. tsugae, G. boninense, G. capense, G. sinense, G. japonicum,
G. applanatum, G. tropicum, G. tenue, and G. luteum have become increasingly popular for the
investigation of medicinal properties. A number of reviews have described the bioactive
substances, medicinal effects, and health benefits of Ganoderma species (Chang, 1995; Chang
and Buswell, 1996; Chang and Miles, 1996; Jong and Birmingham, 1992; Mizuno et al., 1995).
It is also noted that the majority of the studies concerning the Ganodermataceae family
relate to the antitumour and antiviral effects, while the antioxidant properties associated
with this fungus have only recently become apparent (Mau et al., 2002; Yen and Wu, 1999;
Zhu et al., 1999). There appears to be limited information available that reports the antimi-
crobial properties of Ganoderma species.
11.5 Bioactive substances in Ganoderma sp.

Many bioactive compounds have been found in mushroom, some of which inhibit the
growth of cancer cell used under in vitro condition. These substances may be useful as
starting materials for the development of chemical therapeutic agents in cancer treatment
and other ailments (Mizuno, 1995). In addition, the modes of action of these compounds
are being investigated (Gao et al., 2002; Lei and Lin, 1992; Zhang et al., 2002). The fruit-
ing body, mycelia, and spores of G. lucidum contain approximately 400 different bioactive
compounds, which mainly include triterpenoids, polysaccharides, nucleotides, sterols,
steroids, fatty acids, proteins, peptides, and trace elements.
11.5.1 Terpenoid compounds

A new terpenoid, named ganosporeric acid A, was recently isolated from the ether-soluble
fraction of the spores. Min et al. (2000) reported the isolation of six new lanostane-type
triterpenes and also from the spores (ganodericacids g, d, e, z, Z, and y). Preliminary stud-
ies indicate that the spores contain considerably higher contents of ganoderic acids than
other parts of the fungus, and triterpene composition of the fruit body varies; spores also
contain triterpene lactones. The mode of action of triterpenes has been of interest to many
researchers (Gonzalez et al., 2002; Kimura et al., 2002; Lin et al., 2003). The triterpenes are
used to enhance the immune system, as similar to polysaccharides; triterpenes have been
shown to have direct cytotoxicity against tumor cells (Gonzalez et al., 2002). A list of the
important terpenoid bioactive components structure and their biological functions found
in Ganoderma species is given in Table 11.1 and Figure 11.1.
Ha et al. (2000) also isolated two lanosteroids from the basidiocarpe (fruiting body) of
this mushroom, one of which markedly increased the activity of NAD(P)H:quinone oxi-
doreductase. Since this enzyme takes part in xenobiotic metabolism, the determination of
its activity can be used to detect the antitumor chemopreventive potential of the product.
11.5.2 Proteins and polysaccharides

One of the most important proteins isolated from the mycelium of G. lucidum is lingzhi
(LZ-8) (Kino et al., 1989). LZ-8 is a polypeptide consisting of 110 amino acid residues with
an acetylated amino terminus and has a molecular mass of 12 kDa (Tanaka et al., 1989).
The native form of LZ-8, with a molecular mass of 24 kDa, is a homodimer of the LZ-8
polypeptide (Tanaka et al., 1989). This protein has been shown to have mitogenic activity
in vitro and immunomodulating activity in vivo (Haak-Frendscho et al., 1993; Kino et al.,
1989; Van der Hem et al., 1995).
More than 100 types of polysaccharides have been isolated from the fruiting body, spores,
and mycelia or separated from the broth of a submerged liquid culture of G. lucidum. These
polysaccharides are the major sources of G. lucidum’s pharmacologically active compounds.
Table 11.1 Major triterpenoid bioactive constituents in Ganoderma species and their function
S. no. Name of the species Effects Compound References
1. Ganoderma lucidum, Cytotoxicity Lucidenic acid N, Gao et al. (2002), Gonzalez
G. applanatum methyl lucidenate F, et al. (2002), Kimura et al.
lucialdehydes A–C, (2002), Lin et al. (2003),
and ganoderic alcohol Min et al. (2000), Su (1991)
2. Ganoderma pfeiferri Antiviral Ganoderic acid β, El-Mekkawy et al. (1998),
ganoderiol F, Min et al. (1998)
ganodermanotriol
3. Ganoderma lucidum Anticomplement Lucialdehydes Min et al. (2001)
activity
4. Ganoderma lucidum Hypolipidemic Ganoderan B Komoda et al. (1989),
(cholesterol Shiao (1992)
inhibitors)
5. Ganoderma lucidum Antiplatelet Ganodermic acid S Shiao (1992), Wang et al.
and G. japonicum aggregation (1993)
activity
6. Ganoderma lucidum Antioxidant Ganosporeric acid A, Zhu et al. (1999)
lucidenic acid A, and
ganoderic acids B
7. Ganoderma lucidum, Antibacterial Ganomycin A, Smania et al. (1999), Rajesh
Ganoderma pfeifferi, ganomycin B, and and Dhanasekaran (2014)
Ganoderma lanostanoid terpenoids
resinaceum,
Ganoderma sp.
DKR1
8. Ganoderma lucidum Antiinflammatory N-acetylglucosamine Giner-Larza et al. (2000)
and G. tsugae
9. Ganoderma lucidum Antiprotozoal Ganoderic acid, Adams et al. (2006)
ganodermanondiol,
23-hydroxyganoderic
acid S, and ganofuran B
10. Ganoderma sp. Antifungal Terpenoids Rajesh and Dhanasekaran
DKR1 (2014)
G. lucidum polysaccharides such as beta-d-glucans, heteropolysaccharides, and glycoprotein

have been isolated and characterized and are considered the major contributors of bioactiv-
ity of the mushroom.
11.5.3 Antimicrobial compounds

In recent years, there have been a significant number of human pathogenic bacteria becom-
ing resistant to antimicrobial drugs (Davis, 1994; Donadio et al., 2002). Antimicrobial
drug resistance is of major economic concern having an impact on physicians, patients,
health-care administrators, pharmaceutical producers, and the public (McGowan, 2001).
Therefore, bacterial and fungal pathogenic diseases are often difficult to treat (Baratta
et al., 1998).
For the past two decades, attention has turned to the extracts and biologically active
compounds used in traditional herbal medicine to expose their remedial effects and to seek
new lead compounds for development into therapeutic drugs (Cragg, 1997). In addition
OH
H 3C
OH CH3 O H O
H 3C O H3C H
O O
CH3 CH3
O H O H H
CH3 CH3
CH3O CH3 O
HO OH HO H O
H H
H3C CH3 H3C CH3
(a) (b)
O O O OH
H 3C
OH HC
CH3 H 3C O 3
O CH3
O
CH3 H CH3
O
CH3 CH3 O
O O HO O
H H
H3C CH3 H3C CH3
(c) (d)
H3C CH3
CH3
O O
O OH
CH3 H
OH
CH3
HO OH
H
H3C CH3
(e)
Figure 11.1 Structure of triterpenoids from Ganoderma lucidum: (a) lucidenic acid N, (b) ganoderic
acid H, (c) lucidenic acid E, (d) ganoderic acid AM, and (e) ganoderic acid C6.
to plants extracts as sources of antimicrobial agents, research is being performed on fungi

for their ability to prevent bacterial, viral, or fungal pathogens that are resistant to current
therapeutic agents (Wasser and Weis, 1999a). Fungi are well known for the production of
important antibiotic compounds such as penicillin; however, the occurrence of antibiotics
in the class of fungi known as basidiomycetes (the mushrooms) is less well documented,
and there are only few reviews that summarize the antibacterial activity from this type of
mushrooms (Gao et al., 2003; Wasser and Weis, 1999a,b; Zjawiony, 2004).
11.6 Antibacterial activity

Antibacterial activity has been observed against gram-positive bacteria from the fruiting
body extracts of G. lucidum (Kim et al., 1993), Ganoderma sp. DKR1 (Rajesh et al., 2014), and
G. orgonense. Furthermore, observed that seven Indonesian Ganoderma species inhibited
the growth of B. subtilis. Yoon et al. (1995) investigated the additive effect on the activ-
ity of an aqueous extract of G. lucidum with four known antibiotics and observed that
the antibacterial activity increased. The basidiomycetous mushrooms have been shown to
possess antibacterial activity against this group of bacteria. The triterpenes have a great
antibacterial effect (Pinducciu et al., 1995; Wilkens et al., 2002), and it is well documented
that triterpenes are one of the major constituents isolated from Ganoderma.
A few triterpenoids isolated from the fruit body of G. applanatum exhibited inhibitory
activity against gram-positive and gram-negative bacteria such as Bacillus cereus, Coryne
bacterium diphtheriae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphy
lococcus saprophyticus, and Streptococcus pyogenes (Smania et al., 1999). The a ntibacterial activity
of these compounds was determined by MIC and minimal bactericidal concentration (MBC).
Among the seven bacterial species tested, the gram positives were more sensitive (Rajesh
and Dhanasekaran, 2014) (MIC: 0.003–2.0 mg/mL; MBC: 0.06–4.0 mg/mL) than were the
gram negatives (MIC: 1.0–4.0 mg/mL; MBC: 2.0–4.0 mg/mL).
11.7 Antifungal activity

The antifungal drugs available today are not always successful in treating immune com-
promised patients due to the ineffectiveness or toxicity that many of them have on the host
(Seltrennikoff, 1995), and hence, there is a need for the identification of novel antifungal
agents. The basidiomycetes belong to the kingdom of fungi; they are thought to have weak
antifungal activities (Mizuno, 1995) and, therefore, have rarely been investigated for their
bioactivity as antifungal agents. It is only recently that they have become of interest due to
their secondary metabolites exhibiting a wide range of antimicrobial activities.
11.8 Antiviral activity

Recently, a number of reviews by Gao et al. (2003) were published on the antiviral value
of the genus Ganoderma, which summarized the major biologically active constituents
and their effect or mode of action toward a number of viruses such as herpes simplex
virus (HSV) and human immunodeficiency virus (HIV). Polysaccharide fractions from
G. lucidum exhibited activity against HSV-1 and HSV-2 (Eo et al., 1999). Lanostane-type
triterpenes from G. pfeifferi have also been shown to exhibit activity against HSV-1 as well
as inhibit the influenza A virus.
The fruiting bodies of Ganoderma lucidum are the source of antiviral triterpenoids.
Ganoderic acid β (a) isolated from the spores of G. lucidum showed significant anti-HIV-1
protease activity, with an IC50 value of 20 µM (Min et al., 1998). The same species also
produced ganoderiol F (b) and ganodermanontriol (c), which have anti-HIV-1 activity
(El-Mekkawy et al., 1998) (Figure 11.2).
Herbal medicines may have potential activity against Epstein-Barr virus (EBV) infec-
tion or EBV-mediated tumor promotion. A few polyoxygenated lanostanoid triterpenes
isolated from G. applanatum inhibited the 12-O-tetradecanoyl phorbol-13-acetate–induced
EBV early antigen expression in Raji cells (Chairul and Hayashi, 1994). Similar effects
have been observed with Zingiberaceae rhizomes, a commonly used traditional medi-
cine in Malaysia (Vimala et al., 1999). These results indicate that herbal medicines like as
Ganoderma may behave as antitumor agents.
11.9 Antiprotozoal effect of Ganoderma sp.

Protozoa are single-celled creatures with nuclei that show some characteristics usually associ-
ated with animals, most notably mobility and heterotrophy. According to the World Health
OH
H3C OH OH
O O
H 3C H H3C
CH3 CHH
3
O H H
CH3 CH3
CH3O CH3
HO H OH O
H H H
H3C CH3 H3C CH3
(a) (b)
OH
H3C
OH
H3C H OH
CHH 3
H
CH3
CH3
O H
H3C CH3
(c)
Figure 11.2 Structure of antiviral compound from Ganoderma species: (a) ganoderic acid β,
(b) ganoderiol F, and (c) ganodermanotriol.
Organization (WHO) estimates, approximately 300–500 million individuals are infected with
malaria, with death totals ranging from 1.5 to 3.5 million annually (Stanley, 1997). It is caused
by infection with protozoa of the genus Plasmodium consisting of four species of obligate intra-
cellular sporozoans: P. malariae, P. vivax, P. ovale, and P. falciparum. Of the four species, P. falci-
parum is the predominant species with about 400 million new cases and one million deaths
per year globally. P. falciparum is the most dangerous of these infections as P. falciparum malaria
has the highest rates of complications and mortality. Drug therapy, in particular compounds
arising from natural resources, is the major approach to manage malaria. The fight against
P. falciparum species has proven to be formidable with an increasing rise in drug-resistant
strains being isolated. Thus, novel compounds are sought for controlling malaria (Figure 11.3).
Interestingly, the extract from G. lucidum showed inhibitory activity against Plasmodium
falciparum (a pyrimethamine-resistant malarial parasite) (Lovy et al., 1999). Other mush-
rooms including Polyporus umbellatus, Russula xerampelina, Trametes versicolor, Lentinula
aurantiacum, Laetiporus sulphureus, Boletus variipes, Boletus queletii, Grifola frondosa, and
Lentinula edodes also demonstrated activity against Plasmodium falciparum (Lovy et al., 1999).
11.10 Current biomedical applications

A number of biomedical applications of medicinal mushrooms (Borchers et al., 1999; Mizuno,
1995; Wasser and Weis, 1999) and Ganoderma species were reported earlier (Su, 1991). Recently,
a review was published on the immunomodulating effects of G. lucidum, which outlined
the mode of actions of a number of biologically extracted compounds (Zhou and Gao, 2002).
Ganoderma is best known for its immune stimulatory effects in aiding cancer treatment and for
OH
H3C H OH
H 3C
O O CH3
H3C H 3C H OH
CH3H CH3H
O H H
CH3 CH3
H
CH3 OH CH3
O H H OH O H
H3C CH3 H3C CH3
(a) (b)
O
H3C CH3
OH
CH3
CH3
CH3
CH3 H
O CH3
CH3
O HO O
H
H3C CH3 HO
(c) (d)
Figure 11.3 Structure of antiprotozoval compound from Ganoderma lucidum: (a) ganoderic acid,
(b) ganodermanondiol, (c) 23-hydroxyganoderic acid S, and (d) ganofuran B.
its anti-HIV activity. Over the past decade, there has been an increasing amount of research
to investigate Ganoderma species for new biomedical applications. Some of the applications
for which the mushroom extracts and constituents have been found to play important roles
include the following: hepatoprotective activity, hypoglycaemic activity, hypolipidimic activ-
ity, antihistamine release, anti-inflammatory properties, antiplatelet aggregation activity, anti-
complement activity, antiviral activity, enzyme inhibition, and in the healing of open skin
wounds.
11.11 Future perspective

In recent years, more varieties of mushrooms have been isolated and identified, and the
number of mushrooms being cultivated for food or medicinal purposes has been increas-
ing rapidly (Chang, 1995). Mushroom “nutriceuticals” are bioactive compounds that are
extractable from mushrooms, and they have nutritional and medicinal features that may
be used in the prevention and treatment of disease (Chang and Buswell, 1996). Several
nutriceutical products have been isolated from medicinal mushrooms, and three of these,
which are carcinostatic polysaccharides drugs, have been developed from mushroom in
many countries. With particular focus on Ganoderma species, it is apparent that most of the
available data on active extracts and compounds relate to the pharmacological effects on
tumor cells, which appear to be based on the enhancement of the host’s immune system.
Acknowledgement
The valuable support of Department of Microbiology, Bharathidasan University,
Tiruchirappalli is greatly acknowledged.
References
Adams, M., Ettl, S., Kunert, O., Wube, A.A., Haslinger, E., Bucar, F., and Bauer, R. 2006.
Antimycobacterial activity of geranylated furocoumarins from Tetradium daniellii. Planta Med
72: 1132–1135.
Anke, H. and Sterner, O. 1991. Comparison of the antimicrobial and cytotoxic activities of twenty
unsaturated sesquiterpene dialdehydes from plants and mushrooms. Planta Medica 57:
344–346.
Baratta, M.T., Dorman, H.J.D., Deans, S.G., Figueiredo, A.C., Barroso, J.G., and Ruberto, G. 1998.
Antimicrobial and antioxidant properties of some commercial essential oils. Flav Frag J 13:
235–244.
Beltran-Garcia, M.J., Estarron-Espinosa, M., and Ogura, T. 1997. Volatile compounds secreted by the
Oyster mushroom and their antibacterial activities. J Agric Food Chem 45: 4049–4052.
Borchers, A.T., Stern, J.S., Hackman, R.M., Keen, C.L., and Gershwin, M.E. 1999. Mushrooms, tumors,
and immunity. Proc Soc Exp Biol Med 221: 281–293.
Chairul, C.S.M. and Hayashi, Y. 1994. Lanostanoid triterpenes from Ganoderma applanatum.
Phytochemistry 35: 1305–1308.
Chang, A.W. and Miles, P.G. 1996. Biomedical research and the application of mushroom nutriceuti-
cals from Ganoderma lucidum. Proceedings of the 2nd International Conference on Mushroom Biology
and Mushroom Products, pp. 161–175. University Park, PA.
Chang, S.T. 1995. Ganoderma—The leader in production and technology of mushroom neutraceuti-
cals. In: Kim BK, Kim IH, Kim YS, eds., Proceedings of the 6th International Symposium. Recent
Advances in Ganoderma lucidum Research, pp. 43–52.
Chang, S.T. and Buswell, J.A. 1996. Mushroom nutriceuticals. World J Microbiol Biotechnol 12: 473–476.
Chang, S.T. and Miles, P.G. 1992. Mushroom biology—A new discipline. Mycologist 6: 64–65.
Cherqui, F., Rapior, S., and Cuq, P. 1999. Les activités biologiques de Lepista nebularis (Batsch:Fr.)
Harm. Ann Soc Hortic Hist Nat Hérault 139(3): 75–86.
Cragg, W.J. 1997. Phytochemicals-guardians of our health. J Am Diet Assoc 97: S199–S204.
Davis, J. 1994. Inactivation of antibiotic and the dissemination of resistance genes. Science 264: 375–382.
Donadio, S., Carrano, L., Brandi, L., Serina, S., Soffientini, A., Raimondi, E., Montanini, N.,
Sosio, M., and Gualerzi, C.O. 2002. Targets and assays for discovering novel antibacterial
agents. J Biotechnol 99: 175–185.
Donnelly, D.M.X., Abe, F., Coveney, D., and Fukuda, N. 1985. Antibacterial sesquiterpene aryl esters
from Armillaria mellea. J Nat Prod 48(1): 10–16.
Dornberger, K., Ihn, W., Schade, W., and Tresselt, D. 1986. Antibiotics from Basidiomycetes evidence
for the occurrence of the 4-hydroxybenzenediazonium ion in the extracts of Agaricus xantho-
dermus Genevier (Agaricales). Tetrahedron Lett 27(5): 559–560.
El-Mekkawy, S., Meselhy, M.R., Nakamura, N., Tezuka, Y., Hattori, M., Kakiuchi, N., and Otake, T.
1998. Anti-HIV-1 and anti-HIV-1-protease substances from Ganoderma lucidum. Phytochemistry
49(6), 1651–1657.
Eo, S.K., Kim, Y.S., Lee, C.K., and Han, S.S. 1999. Antitheraputic activates of various protein bound
polysaccharides isolated from Ganoderma lucidum. J Ethnopharmacol 68: 175–181.
Farnsworth, N.R. 1994. Ethnopharmacology and drug development. In Ethnobotany and the Search for
New Drugs, Vol. 185, (ed. G.T. Prance), pp. 42–59. John Wiley & Sons, Chichester, U.K.
Gao, Y., Zhou, S., Wen, J., Huang, M., and Xu, A. 2002. Mechanism of the antiulcerogenic effect of
Ganoderma lucidum polysaccharides on indomethacin induced lesions in the rat. Life Sci 72:
731–745.
Gao, Y., Zhou, S., Huang, M., and Xu, A., 2003. Antibacterial and antiviral value of the genus
Ganoderma P. Karst. species (aphyllophoromycetideae): A review. Int J Med Mushroom 5: 235–246.
Gianetti, B.M., Steffan, B., Steglich, W., Kupka, J., and Anke, T. 1986. Antibiotics from basidiomy-
cetes. Part 24. Antibiotics with a rearranged hirsutane skeleton from Pleurotus hypnophilus
(Agaricales). Tetrahedron 42(13): 3587–3593.
Giner-Larza, E.M., Máñez, S., Giner, R.M., Recio, M.C., Prieto, J.M., Cerdá-Nicolás, M., and Rios, J.L.
2002. Anti-inflammatory triterpenes from Pistacia terebinthus galls. Planta Med 68(4): 311–315.
Gonzalez, A.G., Leon, F., Rivera, A., Padran, J.I., Quintana, J., Estevez, F., and Bermejo, J. 2002. New
lanostanoids from the fungus Ganoderma concinna. J Nat Prod 65: 417–421.
Ha, T.B., Gerhäuser, C., Zhang, W.D., Ho-Chong-Line, N., and Fouraste, I. 2000. New lanostanoids
from Ganoderma lucidum that induce NAD(P) H:quinone oxidoreductase in cultured hepalcic7
murine hepatoma cells. Planta Med 66: 681–684.
Haak-frendscho, M., Kino, K., Sone, T., and Jardieu, P. 1993. Ling Zhi-8: A novel T cell mitogen induces
cytokine production and upregulation of ICAM-1 expression. Cell Immunol 150: 101–113.
Hamburger, M. and Hostettmann, K. 1991. Bioactivity in plants: The link between phytochemistry
and medicine. Phytochemistry 30: 3864–3874.
Jong, S.C. and Birmingham, J.M. 1992. Medicinal benefits of the mushroom Ganoderma. Adv Appl
Microbiol 37: 101–134.
Kettering, M., Valdivia, C., Sterner, O., Anke, H., and Thines, E. 2005. Heptemerones A-G, seven
novel diterpenoids from Coprinus heptemerus: Producing organism, fermentation, isolation and
biological activities. J Antibio 58(6): 390–396.
Kim, B.K., Cho, H.Y., Kim, J.S., Kim, H.W., and Choi, E.C. 1993. Studies on constituents of higher
fungi of Korea. Antitumor components of the cultured mycelia of Ganoderma lucidum. Kor J
Pharmacol 24: 203–212.
Kimura, Y., Taniguchi, M., and Baba, K. 2002. Antitumor and antimetastatic effects on liver of trit-
erpenoid fractions of Ganoderma lucidum: Mechanism of action and isolation of an active sub-
stance. Anticancer Res 22: 3309–3318.
Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. 1989.
Isolation and characterization of new immunomodulatory protein, (LZ-8), from Ganoderma
lucidum. J Biol Chem 264: 472–478.
Komoda, Y., M. Shimizu, Sonoda, Y., and Sato, Y. 1989. Ganoderic acid and derivatives as cholesterol
synthesis inhibitors. Chem Pharm Bull (Tokyo) 37: 531–533.
Lei, L.S. and Lin, Z.B. 1992. The effect of Ganoderma polysaccharides on T cell subpopulations and
production of interleukin 2 in mixed lymphocyte response. Acta Pharmaceut Sin 27: 331–335.
Lin, S.B., Li, C.H., Lee, S.S., and Kan, L.S. 2003. Triterpene-enriched extracts from Ganoderma lucidum
inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen-activated
protein kinase and G2-phase cell cycle arrest. Life Sci 72: 2381–2390.
Lovy, A., Knowles, B., Labbe, R., and Nolan, L. 1999. Activity of edible mushrooms against the
growth of human T4 leukemic cancer cells, HeLa cervical cancer cells, and Plasmodium falci-
parum. J Herb Spice Med Plant 6: 49–57.
Mau, J.L., Lin, H.C., and Chen, C.C. 2002. Antioxidant properties of several medicinal mushrooms.
J Agric Food Chem 50: 6072–6077.
Mayzumi, F., Okamoto, H., and Mizuno, T. 1997. Cultivation of Ganoderma lucidum. Food Rev Int 13:
365–382.
McGowan, Jr, J.E. 2001. Economic impact of antimicrobial resistance. Emerg Infect Dis 7(2): 286.
Min, B.S., Gao, J.J., Nakamura, N., and Hattori, M. 2000. Triterpenes from the spores of Ganoderma
lucidum and their cytotoxicity against meth-A and LLC tumor cells. Chem Pharm Bull (Tokyo)
48: 1026–1033.
Min, B.S., Nakamura, N., Miyashiro, H., Bae, K.W., and Hattori, M. 1998. Triterpenes from the spores
of Ganoderma lucidum and their inhibitory activity against HIV-1 protease. Chem Pharm Bull
(Tokyo) 46(10): 1607–1612.
Min, B.S., Gao, J.J., Hattori, M., Lee, H.K., and Kim, Y.H. 2001. Anticomplement activity of terpenoids
from the spores of Ganoderma lucidum. Planta Med 67: 811–814.
Mizuno, T. 1995. Bioactive biomolecules of mushrooms: Food function and medicinal effect of
mushroom fungi. Food Rev Int 11: 7–21.
Mizuno, T., Wang, G., Zhang, J., Kawagishi, H., Nishitoba, T., and Li, J. 1995. Reishi Ganoderma lucidum
and Ganoderma tsugae: Bioactive substances and medicinal effects. Food Rev Int 11: 151–166.
Moncalvo, J.F. and Ryvarden, F. 1998. Nomenclature of ganodermataceae. Syn Fungorum 11: 1–109.
Moncalvo, J.M., Wang, H., and Hseu, R.S. 1995. Gene phylogeny of the Ganoderma lucidum com-
plex based on ribosomal DNA sequences. Comparison with traditional taxonomic characters.
Mycol Res 99: 1489–1499.
Ngai, P.H. and Ng, T.B. 2003. Lectin, a novel and potent antifungal protein from shitake mushroom
with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase
and proliferation of leukemia cells. Life Sci 73: 63–74.
Obuchi, T., Kondoh, H., Watanabe, N., Tamai, M., Omura, S., Yang, J.S., and Liang, X.T. 1990.
Armillaric acid, a new antibiotic produced by Armillaria mellea. Planta Medica 56: 198–201.
Okamoto, H. and Li, J. 1993. Antitumor active polysaccharides from the Chinese mushroom
Songshan lingzhi, the fruiting body of Ganoderma tsugae. Biosci Biotechnol Biochem 57: 894–900.
Pinducciu, G., Serra, C., Cagetti, M.G., Cotti, M., Deidda, D., Pinza, M., and Pompei, R. 1995. Selective
antibacterial activity of triterpene derivatives. Med Microbiol Lett 4: 83–90.
Piraino, F.F. 2006. Emerging antiviral drugs from medicinal mushrooms. Int J Med Mush 8(2): 101–114.
Rajesh, K. and Dhanasekaran, D. 2014. Phytochemical screening and biological activity of medicinal
mushroom Ganoderma species. Malaya J Biosci 1(2): 67–75.
Rajesh, K., Dhanasekaran, D., and Panneerselvam, A. 2014. Isolation and taxonomic characterization
of medicinal mushroom Ganoderma sp. Acad J Microbiol Res 2(2): 061–071.
Seltrennikoff, C. 1995. Antifungal Drugs: (1,3)-Beta-Glucan-Synthase Inhibitors, pp. 6–8. Springer-
Verlag, Heidelberg, Germany.
Shiao, M.S. 1992. Triterpenoid natural products in the fungus Ganoderma lucidum. J Chin Chem Soc
39: 669–674.
Shu, Y.Z. 1998. Recent natural products based drug development: A pharmaceutical industry per-
spective. J Nat Prod 61: 1053–1071.
Smania, A., Delle Monache, F., Smania, E.F., and Cuneo, R.S. 1999. Antibacterial activity of steroidal
compounds isolated from Ganoderma applanatum (Pers.) Pat. (Aphyllophoromycetideae) fruit
body. Int J Med Mushr 1: 325–330.
Stanley, J. 1997. Malaria. Emerg Med Clin North Am 15: 113–155.
Su, C.H. 1991. Taxonomy and physiology active compounds of Ganoderma—A review. J Taipei Med
Coll 20: 1–16.
Tanaka, S., Ko, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., and Tsunoo, H. 1989.
Complete amino acid sequence of an immunomodulatory protein, (LZ-8). An immuno-
modulator from a fungus, Ganoderma lucidum having similarity to immunoglobin variable
region. J Biol Chem 264: 16372–16377.
Van der Hem, L.G., Van der Vliet, J.A., Bocken, C.F., Kino, K., Hoitsma, A.J., and Tax, W.J. 1995. Ling
Zhi-8: Studies of a new immunomodulating agent. Transplantation 60: 438–443.
Vimala, S., Norhanom, A.W., and Yadav, M. 1999. Anti-tumour promoter activity in Malaysian
ginger rhizobia used in traditional medicine. Br J Cancer 80: 110–116.
Wang, G., Zhang, J., Mizuno, T., Zhuang, C., Ito, H., Mayuzumi, H., Okamoto, H., and Li, J. 1993.
Antitumor active polysaccharides from the Chinese mushroom Songshan lingzhi, the fruiting
body of Ganoderma tsugae. Biosci Biotechnol Biochem 57: 894–900.
Wasser, S.P. and Weis, A.L. 1999a. Therapeutic effects of substances occurring in higher basidio
mycete’s mushrooms: A modern perspective. Crit Rev Immunol 19: 65–96.
Wasser, S.P. and Weis, A.L. 1999b. General description of the most important medicinal higher
basidiomycetes mushroom. Int J Med Mush 1: 351–370.
Wasson, R.G. 1968. Soma—Divine Mushroom of Immortality, 381 p. Harcourt, Brace and World, Inc.,
New York.
Wilkens, M., Alarcon, C., Urzua, A., and Mendoza, A. 2002. Characterization of the bactericidal
activity of natural diterpene kaurenoic acid. Plata Medica 68: 452–454.
Willard, T. 1990. Reishi Mushroom. Herb of Spiritual Potency and Medicinal Wonder, 167 p. Sylvan Press,
Seattle, WA.
Yen, G.C. and Wu, J.Y. 1999. Antioxidant and radical scavenging properties of extracts from
Ganoderma tsugae. Food Chem 65: 375–379.
Ying, J., Mao, X., Ma, Q., Zong, Z., and Wen, H. 1987. Icons of Medicinal Fungi from China (ed. X.
Yuehan) 575 p. Science Press, Beijing, China.
Yoon, J.O., Min, T.J., and Yoon, H. 1995. An antibacterial lectin from Lampteromyces japonicus “LJAP”
(Lampteromyces japonicus antibiotic protein). Han’guk Kyunhakhoechi 23(1): 46–52.
Zhang, G.L., Wang, Y.H., Ni, W., Teng, H.L., and Lin, Z.B. 2002. hepatoprotective role of Ganoderma
lucidum polysaccharides against BCG- induced immune liver injury in mice. World J
Gastroenterol 8: 728–733.
Zhou, S. and Gao, Y. 2002. The immunomodulating effects of Ganoderma lucidum (Curt.:Fr.) P. Karst.
(Ling Zhi, Reishi mushroom) (Aphyllophoromycetideae). Int J Med Mush 4: 1–11.
Zhu, M., Chang, Q., Wong, L.K., Chong, F.S., and Li, R.C. 1999. Triterpene antioxidents from
Ganoderma lucidum. Phytother Res 13: 529–531.
Zjawiony, J.K. 2004. Biologically active compounds from aphyllophorales (polypore) fungi. J Nat
Prod 67: 300–310.
chapter twelve
Marine cyanobacteria
A prolific source of antimicrobial
natural products
Natesan Sivakumar and Gopal Selvakumar
Contents
12.1 Introduction......................................................................................................................... 203
12.2 Cyanobacteria...................................................................................................................... 204
12.3 Marine cyanobacteria as a source of natural products................................................. 205
12.4 Bioactive antimicrobial compounds from marine cyanobacteria............................... 207
12.4.1 Antibacterial activity.............................................................................................. 207
12.4.2 Antifungal activity of cyanobacteria................................................................... 210
12.4.3 Antiprotozoan metabolites.................................................................................... 213
12.4.4 Antiviral activity..................................................................................................... 214
12.4.5 Antiquorum compounds....................................................................................... 217
12.5 Screening of bioactive compounds from cyanobacteria............................................... 218
12.6 Biosynthetic genes for secondary metabolites................................................................ 220
12.7 Barbamide biosynthetic pathway.....................................................................................222
12.8 Conclusion........................................................................................................................... 224
References...................................................................................................................................... 224
12.1 Introduction
Antibiotics are essential for the treatment of various microbial infectious diseases.
However, emergence of antibiotic-resistant microorganisms is one of the major problems
in recent years. For example, penicillin resistance in Staphylococcus spp. emerged rapidly.
Due to increasing antibiotic-resistant microorganisms, a new antibiotic is required, which
is active against antibiotic-resistant pathogens. In response to bacterial resistance, the phar-
maceutical industry has produced a remarkable range of antibiotics (Coates and Hu, 2007).
In addition, currently available drugs are effective against only one-third of the diseases
as a result of increased antibiotic resistance in pathogens. Therefore, identification of new
metabolites is urgently required for the development of new drugs. In recent decades, the
main focus on natural products from marine microbial sources has been increased. The
majority of new chemical compounds introduced as drugs during the period of 1981–2002
are of natural origin (Newman et al., 2003).
Recently, high-throughput screening like genome-based bioinformatics tools is used
to design new bioactive compounds. In spite of these technological advances, the num-
ber of new entities reaching the market has declined from 53 to only 26 within a time
span (1996–2005) of 9 years (Singh et al., 2011). As a result, insufficient numbers of new
203
therapeutic drugs are available in the market. To fulfill the demand for new therapeutic
drugs, scientists have been searching novel bioactive compounds from microbial origin.
Among the producers of commercially important metabolites, cyanobacteria have proven
to be a prolific source for the vast majority of compounds discovered until now (Burja
et al., 2001; Kumar et al., 2013). In this chapter, we have made an attempt to consolidate the
recent researches in the field of cyanobacterial bioactive metabolites as antibiotic, antifun-
gal, antimalarial, antiviral, and anti–quorum sensing activities.
12.2 Cyanobacteria
Cyanobacteria are highly diverse groups of gram-negative oxygenic photoautotrophic
prokaryotic microorganisms also known as blue-green algae. The name “cyanobacte-
ria” comes from the color of the bacteria (Adams, 2002). These organisms can inhabit a
wide range of habitats including freshwater, marine, and soil environments, as well as
extreme habitats (Taton et al., 2006). The morphology of cyanobacteria varies from unicel-
lular to filamentous or colonial forms (Figure 12.1). The colonies are often surrounded by a
1.5 µm
5 µm
10 µm
(a) (b) (c)
14 µm
1.5 µm 10.5 µm
(d) (e) (f)
5 µm
4 µm
5 µm
(g) (h) (i)
Figure 12.1 Photomicrographs of cyanobacterial species: (a) Lyngbya confervoides AU0045, (b)

Phormidium ambiguum AU0015, (c) Gloeocapsa sp. AU0033, (d) Phormidium tenue AU0028, (e) Oscillatoria
subbrevis AU0026, (f) Chroococcus sp. AU0052, (g) Microcystis sp. AU0036, (h) Aphanocapsa littoralis
AU0055, and (i) Oscillatoria princeps AU0012.
Chapter twelve: Marine cyanobacteria 205
Siderophores Antibacterial Antimalarial
Protease Cyanobacterial Photo

inhibitors bioactive protective
compounds
Anticancer Phytohormones Antifungal
Figure 12.2 Bioactivity of cyanobacterial compounds.
mucilaginous or gelatinous sheath, depending on environmental conditions. Some of the

filamentous cyanobacteria have three types of cell morphology, namely, vegetative cells,
akinetes, and thick-walled heterocysts. They are either free living or associated with other
living organisms to form symbiosis or communalistic relationship. The cyanobacteria can
form bloom under certain environmental conditions.
In recent years, significant importance has been given to cyanobacterial biotechnology
(Thajuddin and Subramanian, 2005). Marine cyanobacteria are one of the richest sources
of known and novel bioactive compounds including toxins with wide pharmaceutical
applications (Browitzka, 1995; Singh et al., 2011). During recent decades, attention is drawn
toward cyanobacterial bioactive metabolites (Gademann and Portmann, 2008), prob-
ably secondary metabolites (Figure 12.2). The secondary metabolites from cyanobacteria
include a range of compounds showing toxicity on animals and antibacterial, antifun-
gal, antiinflammatory, antimalarial, antiprotozoal, antituberculous, antiviral, and antitu-
mor activities (Mayer et al., 2009). More than 800 secondary metabolic compounds have
been isolated from cyanobacteria and named on the basis of chemical structure, bioassay
method, and their toxicological targets (Pearson et al., 2010).
Cyanobacteria can grow in extreme environmental conditions containing highly
diverse bioactive compounds with a broad range of activity. In general, cyanobacteria
do not produce specific class of compounds; its secondary metabolite spectrum includes
40.2% lipopeptides, 5.6% amino acids, 4.2% fatty acids, 4.2% macrolides, and 9.4% amides
and others (Figure 12.3; Burja et al., 2001). The bioactivity spectrum (antibacterial, antifun-
gal, antiviral, and cytotoxic) of the metabolites can be explained on the basis of their ubiq-
uitous occurrence and long evolutionary history. Broad activity spectrum of the secondary
metabolites may clearly indicate the pharmaceutical potential of cyanobacteria (Devillers
et al., 2007).
12.3 Marine cyanobacteria as a source of natural products

Marine cyanobacteria have been considered a well-known source of structurally diverse
and biologically active natural products (Nunnery et al., 2010) such as phenols, fatty acids,
terpenes, lipopeptides, sterols, and alkaloids. Many cyanobacteria produce compounds
that are generally considered as secondary metabolites. Wide diversity of the natural prod-
uct is a result of the cyanobacterial biosynthetic genes, having the ability to synthesize
various classes of compounds such as linear peptides, linear lipopeptides, depsipeptides
(Han et al., 2005), cyclic peptides, glicomacrolides (Teruya et al., 2009), cyclic depsipeptides
No activity
Anticancer 15
13
Others
8
Antibiotic
Cytotoxic 12
41
Antiviral
4
Antifungal
8
Figure 12.3 Bioactivity of marine cyanobacterial compounds. (From Burja, A.M. et al., Tetrahedron,
57, 9347, 2001.)
(Soria-Mercado et al., 2009), and fatty acid amides (Chang et al., 2011). They have poten-
tially useful biological activities such as cytotoxic, antiviral, antibiotic, antimycotic, multi-
drug resistance reversing, immunosuppressive, anti-inflammatory, and enzyme inhibition
properties (Priyadarshani and Rath, 2012; Mukherjee et al., 2013). Cyanobacterial species
accumulate high amounts of these bioactive compounds in their biomass, which leach into
their surrounding environment (Mukherjee et al., 2013). Most of the cyanobacterial metab-
olites have cytotoxic activity, for example, lyngbyatoxins B and C produced by Lyngbya
majuscula (Aimi et al., 1990), microcystins (Dawson, 1998), hermitamides A and B from
L. majuscula (Tan et al., 2000), apratoxin D produced by Lyngbya sp. (Pérez Gutiérrez et al.,
2008a,b), symplocamide A isolated from Symploca sp. (Linington et al., 2008), malyngamide
2 (Malloy et al., 2011), and antillatoxin (Okura et al., 2013). Organic solvent extracts exhibit
antimicrobial activity, for example, extracellular diterpenoids from Nostoc commune (Jaki
et al., 1999, 2000) have antibacterial activity. Ether- and water-soluble fractions obtained
from L. majuscula have been reported to have antibacterial action against Pseudomonas
fluorescens, Micrococcus pyogenes, and Mycobacterium smegmatis (Starr et al., 1962). Extract
from Phormidium fragile has antagonistic effect against Bacillus subtilis (Palaniselvam, 1998).
Phormidium corium has exhibited inhibition zone (46 mm) against Staphylococcus aureus
(Madhumathi et al., 2011). Extract from Tolypothrix sp. reported to have inhibition zone
(17 and 21 mm) against Bacillus cereus and Staphylococcus epidermidis (Zeeshan et al., 2010),
and Plectonema boryanum and Anabaena variabilis inhibited (17 and 12 mm) S. epidermidis
(Suhail et al., 2011).
More than 50% of the marine cyanobacteria are potentially exploitable for extracting
bioactive substances that are effective in killing cancer cells by inducing apoptotic death
(Tidgewell et al., 2010). Extensive studies revealed that cyanobacterial species, especially
those belonging to the genus Lyngbya, are a prolific source of unique bioactive natural
Table 12.1 Different classes of cyanobacterial secondary metabolites

Organism Compound Class of compound
Lyngbya semiplena Wewakpeptins Cyclic depsipeptides
Lyngbya bouillonii Alotamide A
L. majuscula Pitipeptolides C and D, lagunamides A and B,
apratoxin D, homodolastatin, lagunamide C,
malyngolide dimmer, palmyramide A,
pitipeptolides A and B
Lyngbya sordida Apratoxin D
Lyngbya confervoides Obyanamide, largamides A–C, tiglicamides
A–C
Lyngbya sp. Palau’amide, ulongapeptin, kempopeptins
A and B
L. bouillonii Bouillomides A
L. semiplena Lyngbyastatins
Leptolyngbya sp. Coibamide A
Symploca sp. Largazole, tasipeptins A and B
Oscillatoria margaritifera Veraguamides
Dichothrix utahensis Molassamide
Oscillatoria sp. Largamides A–H Cyclic peptides
L. confervoides Pompanopeptins A and B
Tychonema sp. Brunsvicamides A–C
Symploca sp. Belamide A Tetrapeptide
Phormidium spp. Caylobolide B Macrolactone
L. majuscula Caylobolide A
Lyngbya sp. Lyngbyaloside B, biselyngbyaside Glycosylated macrolide
L. majuscula Isomalyngamide A
L. majuscula Antillatoxin B, carmabins A and B, hoiamides, Lipopeptide
(+)-kalkitoxin
Lyngbya sp. Malyngamide
Symploca sp. Tasiamide
L. majuscula Dragonamide E Linear lipopeptide
molecules (Gerwick et al., 2001). For example, L. majuscula yielded over 30% of all marine
cyanobacterial metabolites, reflecting its notable biosynthetic capacity with regard to natu-
ral products production, and are also deemed as “super” producer of secondary metabo-
lites (Gerwick et al., 2001). Some of the bioactive secondary metabolites of cyanobacteria
are listed in Table 12.1 and Figure 12.4.
12.4 Bioactive antimicrobial compounds

from marine cyanobacteria
12.4.1 Antibacterial activity
Increasing the incidence of drug-resistant bacterial pathogens, diseases may become
untreatable (posed therapeutic challenges and are of great concern worldwide). Hence,
there is a need to search for new antimicrobials to combat antibiotic-resistant strains
O
CH3
NH
CH3 CH3
N
NH CH3
O O
O NH O CH3
O O HN O H3C
O O CH3
N N N N HN O
NH NH
O O
O OH
1 2
CH3 O
CH3 CH3 CH3
H3C CH3
N O H
H CH3
H 3C N H
O
H
H N CH3
O
H
CH3 O
H H
3
4 13 16 5΄
S CH3 CH3 CH3
1 4΄
H2C N 1΄ 3΄ CH3
3 N 5 7 9 11
CH3 CH3 O
14 15
4
Me Me O
N N O
O N
O O S
Me
O
Me
HO
HO O O OMe O
MeO
OH 6
5
Figure 12.4 Some marine cyanobacterial secondary metabolites: (1) belamide A, (2) brunsvi-
camide C, (3) antillatoxin A, (4) kalkitoxin, (5) biselyngbyaside 1, (6) alotamide A. (Continued)
O
N O
N N
H
O N
O
HN O
O OH O O
OH
8R΄
O O HO 9R΄
4S΄ 6R΄
7΄S N OH
H H
O
H CI
8
O
N PMB
N
O HN
N O O
14 11
O 13
3 5 7
H
O N Cl3C N 9
OH 12
S O 15
37 1 N S
9 10 16 17
HN O
O
O
O N O O
O
N
O
O
11
Figure 12.4 (Continued) Some marine cyanobacterial secondary metabolites: (7) lagunamide,
(8) malyngamide 2, (9) apratoxin D, (10) herbamide B, and (11) palmyramide A.
of pathogenic bacteria. Marine habitat has been described recently as a “particularly

promising” (Fischbach and Walsh, 2009) source of novel antibacterial marine natural
products. Marine cyanobacteria are considered being one of the potential organisms use-
ful to mankind in various ways, which contains diverse bioactive compounds responsible
for antibacterial activity (Kreitlow et al., 1999; Biondi et al., 2008).
Various researchers studied the effect of cyanobacterial metabolites as antibacterial
substances. Methanol extracts and culture supernatants of cyanobacteria exhibited sig-
nificant antibacterial effect (Ghasemi et al., 2003; Bhateja et al., 2006). Ethyl acetate extract
of Spirulina platensis can inhibit some gram-positive and gram-negative bacteria (Ozdemir
et al., 2004). Organic extracts of Anabaena sp. have antibacterial properties against Escherichia
coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, and S. aureus (Kaushik
et al., 2009). Madhumathi et al. (2011) reported that organic extracts of Oscillatoria laetevi-
rens, Phormidium corium, Lyngbya martensiana, Chroococcus minor, and Microcystis aeruginosa
have active activities against B. subtilis, S. aureus, Streptococcus mutans, E. coli, Micrococcus
mutans, and K. pneumoniae. The ethanol extracts of Phormidium sp. and Microcoleus sp. at vari-
ous concentrations (0.2, 0.06, 0.03, and 0.015 g/mL) showed the antibacterial activity against
Streptococcus enteritidis and E. coli (Thummajitsakul et al., 2012). Acetone extract of Spirulina
subsalsa NTRI02 and ethanol extract of Oscillatoria pseudogeminata NTRI 03 showed high
inhibitory activity against P. aeruginosa (MTCC 2453) and S. aureus (MTCC 96) (Reehana
et al., 2012). Crude hydrophilic and lipophilic extracts of Anabaena, Nostoc, Scytonema, and
Microcystis species showed activity against Pseudomonas sp. (Yadav et al., 2012). Oscillatoria
boryanum has active activities against S. mutans (Chauhan et al., 2011). Culture liquids of
Gloeocapsa sp. and Synechocystis sp. have the widest spectrum of activity with minimal inhib-
itory concentration (MIC) ranging from 1.56 to 12.5 mg/mL against Streptococcus pyogenes
(Najdenski et al., 2013). Leptolyngbya sp. had antimicrobial activity individually and in syn-
ergy with antibiotics against gram-positive and gram-negative bacteria (Abazari et al., 2013).
Only few antibacterial compounds from cyanobacteria have been structurally char-
acterized. Noscomin (Figure 12.5(1)) from Nostoc commune showed antibacterial activ-
ity against B. cereus, S. epidermidis, and E. coli (Mundt et al., 2003). Ambiguine isonitriles
(Figure 12.5(2)) H and I (alkaloids) isolated from a marine cyanobacterium Fischerella sp.
(Raveh and Carmeli, 2007) have antibacterial activity against B. subtilis and Staphylococcus
albus. Carbamidocyclophanes (Figure 12.5(4)) are paracyclophanes isolated from Nostoc sp.
CAVN 10 showed moderate antibacterial activity against S. aureus (Luke Simmons et al.,
2008). Pérez Gutierrez et al. (2008a) screened two new antibacterial norbietane diterpe-
noids (Figure 12.5(5)) from cyanobacterium Micrococcus lacustris, showing antibacterial
activity against S. aureus, S. epidermidis, S. typhi, Vibrio cholerae, B. subtilis, B. cereus, E. coli,
and K. pneumoniae. Phycobiliproteins isolated from Synechocystis sp., Arthrospira fusiformis,
Porphyridium aerugineum, and Porphyridium cruentum have antimicrobial activity (Najdenski
et al., 2013). The fatty acids of Synechocystis sp. inhibited the growth of B. cereus and
E. coli. Cyanobacterial exopolysaccharides (EPSs) are having antibacterial activity. EPSs
of Synechocystis sp., Gloeocapsa sp., and Rhodella reticulata have high antibacterial activity
(Najdenski et al., 2013). Table 12.2 shows the cyanobacterial compounds and their targeted
bacteria.
12.4.2 Antifungal activity of cyanobacteria

Fungal infections caused by Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis
carinii, Aspergillus sp., and Candida sp. represent an increasing threat to human health. The
prevalence of these fungal infections has increased significantly during the past decades
R
CO2H O
Me OH NC NH
R Me S H
Me H R H Me H
R S CI
R S Me Me H
OH
HO
H N N
Me H H
Me
1 2 3
Me
R5 R1
HO OH Me
OH R2
Me
R3 HO OH
OH
R4 R5
Me Me O
Me Me
4 5
O
CH3
NH
CH3 CH3
N
NH CH3
O O
O NH O CH3
O HN O H3C
Me O CH3
O O
N—
—C Me HN
NH NH
O
O
HO OH
6 7
HO
COOR H3C COOH
H3C
H
8 9
c
COOR
H3C
c
10
Figure 12.5 Antibacterial metabolites from cyanobacteria: (1) noscomin, (2) ambiguine 1 isonitrile,
(3) hapalindole T, (4) carbamidocyclophanes, (5) norbietane diterpenoid, (6) phenolic compound,
(7) brunsvicamide C, (8) α-dimorphecolic acid, (9) 13-hydroxy-9Z, 11E-octadecadienoic acid, and
(10) coriolic acid.
(Vicente et al., 2003). Commercially available antifungal drugs have some side effects.
Marine metabolites can be exploited in a number of different ways for the development
of new antifungal drugs. The compounds extracted from the source either can be directly
used as drugs as leads for the design of novel synthetic products, or as leads for the design
of a novel mode of action available as new screening targets. Marine cyanobacteria have
Table 12.2 Antibacterial activity of cyanobacterial compounds

Organism Compound Targeted organism
Nostoc commune Noscomin B. cereus, S. epidermidis, E. coli
Nostoc muscorum Phenolic compound B. subtilis, B. cereus, E. coli,
S. typhi, S. aureus
Nostoc sp. CAVN 10 Caramida cyclophanes S. aureus
Fischerella sp. Ambiguine 1 isonitrile E. coli, Staphylococcus albus,
B. subtilis
Hapalindole T S. aureus, P. aeruginosa, S. typhi,
E. coli
Lyngbya sp. Pahayokolide A Bacillus sp.
Oscillatoria redekei HUB051, Fatty acid B. subtilis, Micrococcus flavus
Synechocystis sp. and S. aureus
M. lacustris Norbietane diterpenoids S. aureus, S. epidermidis, S. typhi,
Vibrio cholerae, B. subtilis,
B. cereus, E. coli, and
K. pneumoniae
Synechocystis sp., Arthrospira Phycobiliproteins Antibacterial activity
fusiformis, Porphyridium
aerugineum, and Porphyridium
cruentum
Synechocystis sp., Gloeocapsa sp., EPSs B. cereus, E. coli
and Rhodella reticulata
a number of novel metabolites having antifungal activities. However, antifungal activ-

ity is also reported with crude extract and pure compounds from cyanobacteria such as
Anabaena, Nostoc, Aphanocapsa, Synechocystis, Synechococcus, Oscillatoria, Nodularia, and
Calothrix (Volk and Furkert, 2006; Kim, 2006; Drobac-Čik et al., 2007; Pawar and Puranik,
2008; Ghazala et al., 2010).
Organic solvent extracts of cyanobacteria have antifungal activity. The extract from
Nostoc paludosum shows activity against Candida albicans (Ramachandra Rao, 1994).
Diethyl ether and acetone extract of Spirulina platensis had the highest antifungal activ-
ity (Ozdemir et al., 2004). Methanol extracts of Phormidium valderianum and P. fragile
showed anticandidal activity (Sundararaman and Nagaraja, 2006). Likewise, acetone
extract of Phormidium corium, methanol extract of Lyngbya maintensiana and diethyl
ether extract of M. aeruginosa showed the largest inhibition zone tested against fun-
gal pathogens (Madhumathi et al., 2011). Pyridine and n-butanol extracts of Oscillatoria
subbrevis, O. amphibia, and O. chlorina gave maximum activity against Aspergillus wentii
and Candida albicans (Prabakaran, 2011). Methanol extracts of Oscillatoria salina actively
inhibit Fusarium solani and Phormidium tenue against Rhizoctonia solani (Sakthivel and
Kathiresan, 2012). The cyanobacteria Lyngbya aestuarii and Aphanothece bullosa are found
to be a potent source of antifungal activity (Kumar et al., 2013). P. fragile exhibited poten-
tial antifungal activity against Aspergillus flavus, Candida albicans, and Trichoderma viride
(Senthil Kumar et al., 2013).
Rath and Priyadarshani (2013) reported that crude extracts of Phormidium tenue and
Phormidium sp. had high antifungal activity.
The antimicrobial activity of the cyanobacterial extract could be due to the pres-
ence of different secondary metabolites that include polypeptides and lipopeptides
Table 12.3 Antifungal agents from cyanobacteria

Source Compound
L. majuscula Tanikolide, lyngbyabellin B, hectochlorin,
almiramides A–C, dragonamide E
Cyanobacterial mat assemblage Majusculoic acid
Scytonema sp. Scytoscalarol
Tolypothrix sp. Hassallidin A
Nodularia harveyana Norharmane
Nostoc insulare 4′-Dihydroxy biphenyl
Fischerella muscicola Fischerellin A
Synechocystis Antifungal
Aphanothece bullosa Antifungal
(Burja, et al., 2001); amides and alkaloids (Ghasemi et al., 2004); heptadecane and tet-
radecane (Ozdemir et al., 2004); fatty acids, tetramine, spermine, and piperazine
(Prashantkumar et al., 2006; Shanab, 2007); flavonoids, triterpenoids, phenolic com-
pounds (Figure 12.5(6)), and free hydroxyl group (Yu et al., 2009); and metabolites such
as tannin, alkaloids, protein, and flavonoids (Zeeshan et al., 2010). Hassallidin A, a gly-
cosylated lipopeptide from cyanobacterium Hassallia sp., has antifungal activity (Neuhof
et al., 2005). Table 12.3 and Figure 12.6 show few cyanobacterial secondary metabolites
used as antifungal compounds. The marine cyanobacterial genera Lyngbya is known to
produce more than 200 compounds including antifungal such as lobocyclamides A–C
and lyngbyabellin B (Milligan et al., 2000; MacMillan et al., 2002). Although the cyano-
bacteria Nodularia harveyana for norharmane (9H-pyrido[3,4-b]indole), Nostoc insulare for
4,4′-dihydroxy biphenyl (Hagmann and Jiittner, 1996), Fischerella muscicola for fischerellin
A, and Synechocystis for partially purified AK3 as antifungal compounds (Hagmann and
Jiittner, 1996; Yoon and Lee, 2009).
12.4.3 Antiprotozoan metabolites

Due to the limited availability of effective pharmaceutical products and serious side effects
of the available therapy for protozoan infections, the search for useful antiprotozoan metab-
olites from natural resources is necessary (Table 12.4). Cyanobacteria are prolific producers
of structurally distinct and biologically active antiprotozoan metabolites (Figure 12.7).
The Panamanian International Cooperative Biodiversity Group has reported the isola-
tion of five classes of antiprotozoal compounds from cyanobacteria (Tan et al., 2006). The
crude extracts of Nostoc commune and Rivularia biasolettiana are active against Plasmodium
falciparum, Trypanosoma brucei rhodesiense, and Leishmania donovani (Broniatowska
et al., 2011). The crude extract of Lyngbya aestuarii (25.6 mg/mL) and Aphanothece bullosa
(24.0 mg/mL) has antileishmanial activity (Kumar et al., 2013).
Marine cyanobacteria are extremely rich in diverse lipopeptide natural products, many
of which have potent biological activities (Gerwick et al., 2001). For example, bioassay-
guided fractionation of the lipophilic extract of the marine cyanobacterium Phormidium
ectocarpi yielded hierridin B, and 2,4-dimethoxy-6-heptadecyl-phenol showed antiplasmo-
dial activity toward P. falciparum (Papendorf et al., 1998). The discovery of several unique
linear peptides with antiparasitic activity includes the highly modified lipopeptide gal-
linamide A with antimalarial activity (IC50 = 8.4 μM) (Linington et al., 2009), dragonamide
E with antileishmanial activity (IC50 = 5.1 μM) (Balunas et al., 2010), and the almiramides
A–C also with promising antileishmanial activity (Sanchez et al., 2010; Uzair et al., 2012).
Besides these compounds, the protease inhibitor nostocarboline (Barbaras et al., 2008), an
alkaloid isolated from Nostoc sp., was also found to be active against T. brucei, Trypanosoma
cruzi, Leishmania donovani, and P. falciparum (IC50 = 0.5–0.194 mM). Two new lipopeptides
such as viridamides A and B are having antiprotozoal activity, isolated from Oscillatoria
nigroviridis (Luke Simmons et al., 2008). Aerucyclamide C isolated from M. aeruginosa PCC
7806 has also been found to be active against T. brucei and the already known aerucy-
clamide B against P. falciparum (Portmann et al., 2008). In addition, new acyl praline
derivatives, tumonoic acid I, from the marine cyanobacterium Blennothrix cantharidosmum
displayed (IC50 2 mM) moderate antimalarial activity (Clark et al., 2008).
12.4.4 Antiviral activity

The wide outbreaks of deadly viral diseases like dengue, human immunodeficiency
virus (HIV, acquired immune deficiency syndrome), and Ebola hemorrhagic fever may
have dramatic consequences. Hence, potent and safe antiviral agents are urgently needed
OH
HO HO
H O O OH
N H
H N N
N N
O H H
O O OH
O
O O
NH
O NH2
H 2N HN
N O
O HN O
O
O
O
HO
HO
HO
OH 1
O
HN NH2
HN HN
O S NH
N
S N
O OH
O CI CI
O
O
HO
2 3
Figure 12.6 Antifungal secondary metabolites from cyanobacteria: (1) hassallidin A, (2) lyngbya-
bellin B, (3) scytoscalarol. (Continued)
O O
N N N N NH2
O O O
4
R2 O R3 O O
R1 N N N N
N R4
O O O
N
OH
N H H
H
6 Br
7
Figure 12.6 (Continued) Antifungal secondary metabolites from cyanobacteria: (4) dragonamide,
(5) almiramide, (6) norharmane, and (7) majusculoic acid.
Table 12.4 Antiprotozoan compounds from cyanobacteria

Source Compound Activity against
Symploca sp. Gallinamide A, symplocamide A P. falciparum, Trypanosoma cruzi,
Leishmania donovani
Blennothrix cantharidosmum Tumonoic acid I P. falciparum
M. aeruginosa PCC 7806 Aerucyclamide C Antileishmanial, T. brucei
Oscillatoria nigroviridis Viridamides A and B Antiprotozoal
Trypanosoma cruzi, Leishmania
mexicana, P. falciparum
Nostoc sp. Nostocarboline T. brucei, Trypanosoma cruzi,
Leishmania donovani, and
P. falciparum
Phormidium ectocarpi Hierridin B and P. falciparum
2,4-dimethoxy-6-heptadecyl-
phenol
Lyngbya sp. Dragonamide E Antileishmanial
L. majuscula Almiramides A–C Antileishmanial
Fischerella ambigua Ambigol C Trypanosoma rhodesiense,
P. falciparum
in these situations. The scientist searching novel natural products derived from marine
cyanophytes for antiviral activity has yielded a considerable number of active crude aque-
ous and organic solvent extracts. Marine cyanobacteria also appear to be a rich source
of new antiviral compounds. Gustafson et al. (1989) used a tetrazolium-based microcul-
ture to screen extracts of marine cyanobacterial cultures, Lyngbya sp. (Tripathi et al., 2010),
Lyngbya lagerheimii, and Phormidium tenue for inhibition of HIV-1 (Gustafson et al., 1989).
O
S
N
O H
N N
O O O
NH HN O
N N N
N N NH2
O O O
S
1 2
CI N+ CH3
N
H
3
HO
O O
H
O O N
N N N O
H
O 4 O O
O
5
R2 O R3 O O
R1 N N N
N N R4
O O O
Figure 12.7 Cyanobacterial secondary metabolites showing antiprotozoan activity: (1) venturamide
A, (2) dragonamide B, (3) nostocarboline, (4) hierridin B, (5) gallinamide A, and (6) almiramide.
Anti-influenza virus activity was found in extracts of Lyngbya prepared from early
exponential growth phase cells (Armstrong et al., 1991). In the University of Hawaii,
researchers screen around 10% of (n = 600) cyanobacterial extracts tested live in virus test
systems for inhibition of HSV-2 and HIV type 1 (HIV-1). However, a smaller percentage of
(2.5%) extracts were active against respiratory syncytial virus (Patterson et al., 1993). Lau
et al. (1993) screened 2% (among 900 strains) of cultured cyanobacteria having antireverse
transcriptase activity against avian myeloblastosis virus and HIV-1.
So far, few antiviral compounds were isolated from marine cyanobacteria (Table 12.5).
The antiviral cancer polysaccharides such as spirulan and Ca spirulan were from Spirulina
sp. that showed potent and broad-spectrum activity against HIV-1, HIV-2, Influenza, and
a series of other enveloped viruses (Feldman et al., 1999). These sulfated polysaccharides
prevent virus–cell attachment and fusion with host cells (CD4+) and inhibit the reverse
transcriptase activity of HIV-1 (like azidothymidine). The natural sulfoglycolipids from
cyanobacteria Scytonema sp. are also reported to inhibit HIV reverse transcriptase and
DNA polymerases (Loya et al., 1998).
A polypeptide cyanovirin-N (CV-N) (11 kDa; 101 amino acid) is a carbohydrate-
binding protein produced by Nostoc ellipsosporum showing potent in vitro and in vivo
activities against HIV and other lentiviruses (Boyd et al., 1997). CV-N is a cyanobacte-
rial protein with potent neutralizing activity against HIV-1 high affinity with gp120, and
Table 12.5 Antiviral compounds from marine cyanobacteria

Organism Compound Activity against
Spirulina sp. Spirulan HIV-1 and HIV-2 (antireverse
transcriptase activity),
influenza, HSV
Nostoc flagelliforme Nostoflan HSV-1 (HF), HSV-2, HCMV,
influenza, adenovirus type 2
Nostoc ellipsosporum CV-N HIV-1, HIV-2, HSV-6, SIV, FIV
Scytonema varium Scytovirin N HIV-1
Scytonema sp. Sulfoglycolipid HIV-1
Trichodesmium erythraeum Debromoaplysiatoxin, Chikungunya virus
anhydrodebromoaplysiatoxin, and
3-methoxydebromoaplysiatoxin
Microcystis ichthyoblabe Ichthyopeptins A and B Influenza A
it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with

HIV-1 entry. CV-N has blocking action at the level of gp120 interaction with coreceptor
and the chemokine receptor CXCR4 as an entry receptor (Dey et al., 2000). Thus, CV-N has
broad-spectrum antiviral activity, for example, (1) CV-N impairs both CD4-dependent and
CD4-independent binding of sgp120 to the target cells, (2) CV-N blocks the sCD4-induced
binding of sgp120 with cell-associated coreceptor CXCR4, and (3) CV-N dissociates bound
sgp120 from target cells (Mori and Boyd, 2001).
Another anti-HIV protein scytovirin (cyanobacteria, Scytonema varium) has two
carbohydrate-binding sites. This unique protein (95 residues) contains five structurally
important intrachain disulfide bonds, which makes unusual clusters with aromatic resi-
dues of sites, suggesting a binding mechanism similar to other known hevein-like carbo-
hydrate-binding proteins but differing in carbohydrate specificity. Scytovirin is used to
inhibit HIV entry and prophylactic anti-HIV applications (McFeeters et al., 2007). It binds
to the envelope glycoprotein of HIV (gp120, gp160, and gp41) and inactivates the virus in
low nanomolar concentrations (Bokesch et al., 2003; Xiong et al., 2006, 2010). Nostoflan is an
acidic polysaccharide from Nostoc flagelliforme that exhibits potent virucidal activity against
herpes simplex virus 1 (Kenji et al., 2005).
In addition, two cyclic depsipeptides, ichthyopeptins A and B, were also isolated from
Microcystis ichthyoblabe. They showed antiviral activity against influenza A virus with an
IC50 value of 12.5 mg/mL (Zainuddin et al., 2007). Deepak Kumar et al. (2014) screened five
different compounds from Trichodesmium erythraeum. The debrominated analogs such as
debromoaplysiatoxin, anhydrodebromoaplysiatoxin, and 3-methoxydebromoaplysiatoxin
displayed dose-dependent inhibition of Chikungunya virus (Gupta et al., 2014).
12.4.5 Antiquorum compounds

The most widely used method of antifouling activity in marine environments involves the
use of highly toxic compounds, which affects the marine life. Hence, finding an alternative
form of biofilm control is essential. Many potential marine-derived antifoulants have been
identified from organisms that are naturally exposed to larvae of fouling organisms and
produce antifouling chemicals (Tan et al., 2010).
Cyanobacteria produce a variety of potential natural bioactive metabolites that may
have to prevent biofouling by colonizing organisms. Generally, metabolites with antibiotic,
algicidal, cytotoxic, and enzyme-inhibiting activities may represent antiquorum a ctivities

on both micro- and macrofoulents. Molecules such as amides, alkaloids, terpenoids,
fatty acids, lipopeptides, lactones, steroids, and pyrroles display antifouling activities.
Phycotoxins and related products from cyanobacteria may serve as m aterials for anti-
fouling applications (Dahms et al., 2006). Recently, more than 21 different antifouling
substances from 27 strains of cyanobacteria have been reported (Dahms et al., 2006). For
example, cyanobacteria L. majuscula produced many useful secondary metabolites that
have antifeed properties, and they may inhibit invertebrate larval settlement. The metab-
olite dolistain 16 (EC50 at 0.003 µg/mL) showed high antisettlement activities against
barnacle cyprids (Tan et al., 2010), honaucins A–C, potent inhibitors of bacterial quo-
rum sensing (Choi et al., 2012), ellagic acid (Huber et al., 2003), malyngolide (Dobretsov
et al., 2010), and microcolins A and B and kojic acid that inhibits the LuxR-based reporter
induced by N-3-oxo-hexanoyl-l-homoserine lactone (Dobretsov et al., 2011). Honaucin
A and its two natural analogs exhibit potent inhibition of both bioluminescence and
a quorum sensing–dependent phenotype, in Vibrio harveyi BB120 (Choi et al., 2012).
Malyngamides A and B, which are structurally different from malyngamide C, inhibited
quorum sensing–dependent violacein production by Chromobacterium violaceum CV017
(Dobretsov et al., 2011) and inhibited Las-R-based bacterial luminescence (Kwan et al.,
2010). Dobretsov et al. (2011) reported that kojic acid effectively inhibits the bacterial den-
sity (3.2 fold) and diatom density (4.7 fold) on glass slide at 330 μM concentration. Kojic
acid has tyrosinase inhibitory action (Burdock et al., 2001), by which it inhibits fouling
organisms. The mechanism of this quorum inhibition remains unknown, but it was sug-
gested that antibiotics can change bacterial membrane permeability, thus affecting flux of
quorum signals (Skindersoe et al., 2008).
The discovery of marine cyanobacterial compounds with macrofoulent antisettle-
ment activities were revealed as a potential source of natural antifoulants controlling
biofouling. A number of natural products, including hantupeptin C (Figure 12.8a), isoma-
jusculamide (Figure 12.8e), and majusculamide A, have moderate barnacle antisettlement
activities (Tan and Goh, 2009). The marine cyanobacterium Schizothrix calcicola produced
the compound ypaoamide (Figure 12.8b), which prevents feeding of two species of rab-
bitfishes (Nagle and Paul, 1998). Pitipeptolide A from L. majuscula has chemical defense
against various grazers (Cruz-Rivera and Paul, 2007) and larval recruitments of Acropora
surculosa and Pocillopora damicornis (Kuffner and Paul, 2004). Lyngbyatoxin A and debro-
moaplysiatoxin inhibit various marine predators (Capper et al., 2006). Brown et al. (2004)
demonstrated antifouling property of marine cyanobacterial (Kyrtuthrix maculans) com-
pound maculalactone A that has toxicity against various species of barnacle larvae such
as Tetraclita japonica, Balanus amphitrite, and Ibla cumingii.
12.5 Screening of bioactive compounds from cyanobacteria

Considering the great diversity of natural products from marine cyanobacteria, the appro-
priate methods can be rapidly used to screen different bioactive compounds from the
source of great interest. To design this screening methodology, different parameters have
to be considered, which include the nature of the bioactive compounds (soluble property,
heat resistance, or molecular weight) and their activity (Falch et al., 1995). The screening of
bioactive metabolites from the cyanobacterial source begins with suitable extraction tech-
nique, based on either expected or targeted bioactive metabolites (Figure 12.9).
Generally, solvent extraction method is used to obtain certain compounds from the
source materials (Plaza et al., 2010). A large number of bioactive compounds have been
N
O
O
O O N
N O O
O H H OCH3 O
N O
N N
O H
O
HO
(a) (b)
O O
H
O N N
N N
OH
O O O N
OAc
O
(c) (d) O
OCH3
O N
OCH3 O
N O O
(e) CI H OCH3 O
(f )
Figure 12.8 Chemical structure of cyanobacterial secondary metabolites as antifouling compounds:

(a) hantupeptin C, (b) ypaoamide, (c) malyngolide, (d) microcolin B, (e) isomalyangamide A, and
(f) maculalactone A.
traditionally extracted with organic solvents (hexane, ethanol, water, methanol, acetone,
diethyl ether, petroleum ether), either single solvent method or fractionation methods
(Starmans and Nijhuis, 1996; Plaza et al., 2010; Tokuoka et al., 2010). These organic solvents
can be employed for the extraction of both polar and nonpolar organic compounds such as
alkaloids, phenols, aromatic hydrocarbons, fatty acids, and oils (El Hattab et al., 2007; Plaza
et al., 2010). The parameters such as influence of solvents, temperatures, and pressures
might have a significant influence on the outcome of the extraction process. Invariably,
solvent extraction is advantageous compared to other methods due to low processing cost
and ease of operation. However, this method uses toxic solvents and requires an evapora-
tion/concentration step for recovery (Joana Gil-Chávez et al., 2013).
After the extraction obtained using diverse conditions, these extracts must be tested for
biological activities by performing the appropriate functional activity assay(s) (Figure 12.9),
for example, antibacterial and antifungal screening can be done with agar spot assay, well
diffusion assay, and disk diffusion assay. When an organic solvent extract shows bioactiv-
ity, the bioactive compounds may be screened and characterized subsequently.
Once the target biological activities have been confirmed, the next step involves
chemical characterization of the bioactive components present in the initial extract.
Marine
cyanobacteria
Solvent Screening of
extraction bioactivity
Functional Bioactivity in vitro

characterization assays
Chemical Advanced analytical

characterization methods
Figure 12.9 Basic scheme showing the proposed work flow for the screening of bioactive com-
pounds from marine cyanobacteria.
Primarily, bioactive compounds should be traced by qualitative detection methods such as

phenols (Harborne, 1998; Biglari et al., 2008), amino acids, sterols and saponins (Lacaille-
Dubois and Wagner, 1996; Shiau et al., 2009), alkaloids (Shamsa et al., 2008), flavonoids
(Mansouri et al., 2005; Wang et al., 2008), tannins (ferric chloride test), carbohydrates, pro-
teins, and fatty acids (standard protocols). The chemistry of bioactive metabolites like tox-
ins is known, and that has made possible the development of high-throughput analysis
methods such as gas chromatography (GC), high-performance liquid chromatography
(HPLC), and liquid chromatography–mass spectrometry (LC/MS). Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry has been proven as a fast screen-
ing method for the detection of cyanobacterial peptide toxins and bioactive compounds
(Erhard et al., 1997; Fastner et al., 2001; Welker et al., 2004). For example, to identify the indi-
vidual compound, separation is needed and identification methods such as TLC, HPLC,
FT-IR, and GC with mass detection (Meriluoto et al., 2004; Meriluoto and Codd, 2005) and
LC/MS (Sivonen, 2000) are required.
Purified compounds’ functional characterization should be assessed through the
application of fast in vitro assays directed to the confirmation of the sought biological
properties, for instance, antioxidant capacity assays and antimicrobial activity assays.
Based on the pharmacological importance, bioactive secondary metabolites are synthe-
sized and used for various purposes.
12.6 Biosynthetic genes for secondary metabolites

In the past two decades, biosynthesis gene clusters were assigned to an increasing num-
ber of these cyanobacterial natural products (Welker and von Döhren, 2006; Jones et al.,
2009). Microbial peptides are produced by two types of biosynthetic pathways: by ribo-
somal synthesis and subsequent posttranslational modification and processing or by giant
complex multidomain enzymes and the nonribosomal peptide synthetases (NRPSs) with-
out the help of ribosomes and mRNA. Nonribosomal peptides are often cyclic and/or
branched structures that contain nonproteinogenic amino acids including d-amino acids,
with N-methyl and N-formyl groups, or glycosylated, acylated, halogenated, or hydroxyl-

ated. Nonribosomal peptides are often dimers or trimers. The biosynthetic gene of NRPS
consists of modules, each being responsible for the incorporation of a single amino acid.
In general, each NRPS complex contains three modules such as (a) initiation module,
[F/NMT]-A-PCP-; elongation module, -(C/Cy)-[NMT]-A-PCP-[E]-; and termination mod-
ule, -(TE/R). The order of these modules typically follows a colinearity rule; the succession
of modules corresponds to the order of amino acids in the final product. Each module
contains a number of domains; each domain plays an important role to add and modify
the amino acids by their own functions.
The domains of NRPS modules are as follows: F, formylation (optional); A, ade-
nylation (required in a module); PCP, and peptide-carrier protein with attached
4′-phosphopantetheine (required in a module); C, condensation forming the amide bond
(required in a module); Cy, cyclization into thiazolines or oxazolines (optional); Ox, oxi-
dation of thiazolines or oxazolines to thiazoles or oxazoles (optional); Red, reduction of
thiazolines or oxazolines to thiazolidines or oxazolidines (optional); E, epimerization into
d-amino acids (optional); NMT, N-methylation (optional); TE, termination by a thioesterase
(only found once in a NRPS); and R, reduction to terminal aldehyde or alcohol (optional).
A minimal module is composed of an amino acid–activating adenylation (A) domain,
a peptidyl carrier (PCP) domain carrying the phosphopantetheine cofactor and a conden-
sation (C) domain (Figure 12.10) (Koglin and Walsh, 2009).
Currently, there are well over 300 proteinogenic and nonproteinogenic secondary
metabolites, being reported from marine cyanobacteria (Uzair et al., 2012). These natural
products represent great structural diversity, belonging to the polyketide synthase (PKS)
and NRPS, as well as hybrid PKS–NRPS structural classes (Gerwick et al., 2001). To date,
only few putative biosynthetic PKS–NRPS gene clusters of marine cyanobacterial mol-
ecules have been reported including barbamide, jamaicamide, and curacin A (Chang et al.,
2002, 2004; Edwards et al., 2004).
KR Terminal modules (module n)

A Loading module Modules n–x
C MT
PCP C A PC E TE
P AT ACP KS AT ACP TE
S S
O O S
R1 R2 O
N R
NH2 H
O O
NH2 R
+ amino KR HO
R1
acid PPT R
+ ATP Minimum configuration DH
R
Optional domain
Minimum configuration
ER
Optional domain
(a) (b)
Figure 12.10 Schematic representation of enzymatic domains in (a) nonribosomal peptide synthe-
tases and (b) polyketide synthase gene clusters. Abbreviations: A, adenylation domain; ACP, acyl
carrier protein; AT, acyltransferase; C, condensation domain; PCP, peptidyl carrier protein; DH,
dehydratase; MT, methyltransferase; E, epimerase; KS, ketosynthase; KR, ketoreductase; ER, enoyl
reductase; and TE, thioesterase.
In contrast, ribosomal biosynthesis of peptides has limitations to a minimum of 20

proteinogenic amino acids. This group of peptides nevertheless displays a high diversity
and a considerable biosynthetic and bioactive potential. The ribosomal prepeptides are
typically composed of a leader peptide and a core peptide. Associated posttranslational
modification enzymes (PTMs) catalyze different types of macrocyclizations of the core
peptide and side-chain modifications of amino acids. Peptide maturation further requires
cleavage of the leader peptide by processing proteases (PPs) frequently combined with
transport across the plasma membrane (Oman and van der Donk, 2010).
Cyanobacterial macrolides like lyngbouillosides A, B, and C and the related com-
pounds have attracted a lot of attention over the past decade due to their intriguing archi-
tecture, their natural scarcity, and their potential biological activities (El Marrouni et al.,
2013). They are produced by modular-type PKSs resembling NRPS with respect to their
modular nature. The well-studied PKS assembly is the animal fatty acid synthase (Jenke-
Kodama et al., 2005) and consists of ketosynthase, acyltransferase, ketoreductase, dehy-
dratase, enoyl reductase, and acyl carrier protein domains (Staunton and Weissman, 2001).
Parts of the domains (KR, DH, ER) are optionally used leading to a different reduction
state of the keto groups of polyketides.
12.7 Barbamide biosynthetic pathway

The marine cyanobacteria L. majuscula produces several bioactive secondary metabo-
lites that include barbamide, curacin A, carmabin A, antillatoxin B, and malyngamide
(Ramaswamy et al., 2006). Biosynthesis of these secondary metabolites may have unique
structural features.
The genetic architecture and catalytic domain organization of the barbamide gene
cluster (bar) is generally colinear and contains 12 open reading frames (ORFs) (Chang
et al., 2002) (Figure 12.8). It is synthesized by a mixed PKS/NRPS system with unusual
features that include a stand-alone PCP and unusual adenylation (A) domains, as well as
a PKS module that is encoded on two separate ORFs. In addition, the biosynthetic sys-
tem encodes several tailoring enzymes involved in (1) the chlorination, α-oxidation, and
decarboxylation of leucine to form a trichloroisovaleric acid moiety and (2) the oxidative
decarboxylation of the cysteine residue at the end of the assembly line to form the terminal
thiazole ring (Figure 12.11).
1. The BarA-BarE and BarJ proteins are involved in the conversion of leucine to the tri-
chloroisovaleric acid.
2. BarA contains a PCP domain.
3. BarD dwells a leucine/trichloroleucine-specific A domain, which activates leucine
and is required for covalent bond formation between leucine and BarA.
4. BarC shares high sequence homology with known thioesterases (TE). BarC can medi-
ate the release of leucine from BarA.
5. BarB1 and BarB2 are believed to be the putative halogenases (Figure 12.8). The con-
version of l-leucine to the chlorinated α-keto derivative occurs by the action of BarB1
and BarB2, while the substrate is bound as a thioester intermediate to BarA.
6. BarE contains an A domain that shows high activity for both the nonchlorinated and
trichlorinated α-keto derivatives of l-leucine, as well as modest activation of trichlo-
roleucine. The chlorinated α-keto intermediate could then be released and incorpo-
rated as the loading module in BarE to initiate barbamide biosynthesis.
Halogenation and Decarboxylation—ketide Decarboxylation thiazole
α-oxidation extension O-methylation NRPS formation resistance genes
Bar E Bar F Bar G
Bar C
Bar A
Bar B1
Bar B2
Bar H
Bar I
Bar D
Bar J
Bar K
PCP Hal Hal TE A C A PCP KS AT OM ACP C A MT PCP Cy A PCP TE DG LAO
S S
S S S S
O O O SH OH
Chapter twelve: Marine cyanobacteria
NH2 O OCH3 NH O
N–CH3
CCI3 CCI3 N
S
Or OCH3 N–CH3 Bar G
Bar B1 CCI3
OCH3 N–CH3
Bar B2
CCI3
OCH3
PCP PCP CCI3
BarJ Barc OH
S S O BarH
O O O
NH2 O CCI3
CCI3 CCI3
N
S
N–CH3
Barbamide
OCH3
CCI3
Figure 12.11 Barbamide biosynthetic gene cluster, an example for nonribosomal peptide synthetase–polyketide synthase hybrid biosynthesis.
223
7. In addition, BarJ, which shares homology with l-amino-acid oxidases, is proposed to

function as the α-oxidase that converts trichloroleucine to the α-keto derivative.
8. BarG is predicted to catalyze the addition of N-methyl phenylalanine and cysteine
followed by cleavage from the enzyme complex by the internal TE domain residing
in BarG. The released product must then undergo oxidative decarboxylation, pos-
sibly mediated by BarH, to form the final product, barbamide.
The genetic architecture of the bar cluster is supported by several stable isotope feed-
ing experiments that demonstrate that barbamide is derived from acetate; two
S-adenosylmethionine-derived methyl groups; and three amino acids l-leucine,
l-phenylalanine, and l-cysteine (Sitachitta and Gerwick, 1998; Sitachitta et al., 2000;
Williamson et al., 2004).
12.8 Conclusion
Cyanobacteria are one of the sources of known novel bioactive compounds including tox-
ins, polysaccharides, sterols, and vitamins with wide pharmaceutical applications. Many
compounds from cyanobacteria are useful for the welfare of mankind. At present, few
compounds and their analogs identified from cyanobacteria are in clinical trials, and
some of them have passed different phases of clinical trials to prove their candidacy as
potential drugs. Even if a single cyanobacterium, for example, Lyngbya sp., has produced
more than 100 metabolites, we can think what kind of novelty and potentiality the cya-
nobacterium is having. Research is needed to unfold other hidden bioactive principles
of marine cyanobacteria. One exciting feature of the biosynthetic gene clusters identified
from marine cyanobacteria is the absence of self-resistance, regulatory, or transport genes.
Recent chemical studies have shown that marine cyanobacteria are capable of producing a
wide range of other non-PKS–NRPS molecules. Thus, there is a need for extensive research
in this new emerging field for antimicrobial drug discovery.
References
Adams, D. G. 2002. Cyanobacteria in symbiosis with hornworts and liverworts. In: Rai, A. N.,
Bergman, B., and Rasmussen, U., eds., Cyanobacteria in Symbiosis. Dordrecht, the Netherlands:
Kluwer Academic Publishers, pp. 117–135.
Aimi, N., Odaka, H., Sakai, S., Fujiki, H., Suganuma, M., Moore, R. E., and Patterson, G. M. L. 1990.
Lyngbyatoxins B and C, two new irritants from Lyngbya majuscula. J Nat Prod 53(6): 1593–1596.
Armstrong, J. E., Janda, K. E., Alvarado, B., and Wright, A. E. 1991. Cytotoxin production by marine
Lyngbya strain (cyanobacterium) in large scale laboratory bioreactors. J Appl Phycol 3(3): 277–282.
Balunas, M. J., Linington, R. G., Tidgewell, K., Fenner, A. M., Ureña, L. D., Togna, G. D., Kyle, D. E.,
and Gerwick, W. H. 2010. Dragonamide E, a modified linear lipopeptide from Lyngbya majus-
cula with antileishmanial activity. J Nat Prod 73(1): 60–66.
Barbaras, D., Kaiser, M., Brun, R., and Gademann, K. 2008. Potent and selective antiplasmodial
activity of the cyanobacterial alkaloid nostocarboline and its dimers. Bioorg Med Chem Lett 18:
4413–4415.
Bhateja, P., Mathur, T., Pandya, M., Fatma, T., and Rattan, A. 2006. Activity of blue–green microalgae
extracts against in vitro generated Staphylococcus aureus with reduced susceptibility to van-
comycin. Fitoterpia 77: 233–235.
Biglari, F., AlKarkhi, A. F., and Easa, A. M. 2008. Antioxidant activity and phenolic content of vari-
ous date palm (Phoenix dactylifera) fruits from Iran. Food Chem 107(4): 1636–1641.
Biondi, N., Tredici, M. R., Taton, A., Wilmotte, A., Hodgson, D. A., Losi, D., and Marinelli, F. 2008.
Cyanobacteria from benthic mats of Antarctic lakes as a new source of bioactivities. J Appl
Microbiol 105: 105–115.
Bokesch, H. R., O’Keefe, B. R., McKee, T. C., Pannell, L. K., Patterson, G. M., Gardella, R. S., and Boyd,
M. R. 2003. A potent novel anti-HIV protein from the cultured cyanobacterium. Scytonema
varium. Biochemistry 42: 2578–2584.
Boyd, M. R., Gustafson, K. R., McMahon, J. B., Shoemaker, R. H., O’Keefe, B. R., Mori, T., and
Henderson, L. E. 1997. Discovery of cyanovirin-N, a novel human immune deficiency virus
inactivating protein that binds viral surface envelope glycoprotein gp120: Potential application
to microbicide development. Antimicrob Agents Chemother 41: 1521–1530.
Broniatowska, B., Allmendinger, A., Kaiser, M., Montamat-Sicotte, D., Hingley-Wilson, S., Lalvani,
A., and Tasdemir, D. 2011. and cytotoxic potential of cyanobacterial (blue–green algal) extracts
from Ireland. Nat Prod Commun 6(5): 689–694.
Browitzka, M. A. 1995. Microalgae as sources of pharmaceuticals and other biologically active com-
pounds. J Appl Phycol 7: 3–15.
Brown, G. D., Wong, H. F., Hutchinson, N., Lee, S. C., Chan, B. K., and Williams, G. A. 2004.
Chemistry and biology of maculalactone A from the marine cyanobacterium Kyrtuthrix macu-
lans. Phytochem Rev 3: 381–400.
Burdock, G. A., Soni, M. G., and Carabin, I. G. 2001. Evaluation of health aspects of kojic acid in food.
Regul Toxicol Pharmacol 33: 80–101.
Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., and Wright, P. C. 2001. Marine
cyanobacteria—A prolific source of natural products. Tetrahedron 57: 9347–9377.
Capper, A., Cruz-Rivera, E., Paul, V. J., and Tibbetts, I. R. 2006. Chemical deterrence of a marine
cyanobacterium against sympatric and non-sympatric consumers. Hydrobiologia 553: 319–326.
Chang, R. L., Ghamsari, L., Manichaikul, A., Hom, E. F., Balaji, S., Fu, W., and Papin, J. A. 2011.
Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal
metabolism. Mol Syst Biol 7: 518.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Sherman, D. H. 2002. The
barbamide biosynthetic gene cluster: A novel marine cyanobacterial system of mixed
polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an
unusual trichloroleucyl starter unit. Gene 296: 235–247.
Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., and Gerwick, W. H. 2004.
Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product
from the tropical marine cyanobacterium Lyngbya majuscula. J Nat Prod 67: 1356–1367.
Chauhan, R., Silambarasan, S., and Abraham, J. 2011. Biodiversity of marine cyanobacteria and its
antibacterial activity. IJPI’S J Biotechnol Biother 1(4): 15–19.
Choi, H., Mascuch, S. J., Villa, F. A., Byrum, T., Teasdale, M. E., Smith, J. E., and Gerwick, W. H. 2012.
Honaucins A–C, potent inhibitors of inflammation and bacterial quorum sensing: Synthetic
derivatives and structure-activity relationships. Chem Biol 19: 589–598.
Clark, B. R., Engene, N., Teasdale, M. E., Rowley, D. C., Matainaho, T., Valeriote, F. A., and Gerwick, W.
H. 2008. Natural products chemistry and taxonomy of the marine cyanobacterium. Blennothrix
cantharidosmum. J Nat Prod 71: 1530–1537.
Coates, A. R. M. and Hu, Y. 2007. Novel approaches to developing new antibiotics for bacterial infec-
tions. Br J Pharma 152: 1147–1154.
Cruz-Rivera, E. and Paul, V. J. 2007. Chemical deterrence of a cyanobacterial metabolite against gen-
eralized and specialized grazers. J Chem Ecol 33: 213–217.
Dahms, H. U., Ying, X., and Pfeiffer, C. 2006. Antifouling potential of cyanobacteria: A mini-review.
Biofouling 22(5–6): 317–327.
Dawson, R. M. 1998. The toxicology of microcystins. Toxicon 36(7): 953–962.
Devillers, J., Doré, J. C., Guyot, M., Poroikov, V., Gloriozova, T., Lagunin, A., and Filimonov, D. 2007.
Prediction of biological activity profiles of cyanobacterial secondary metabolites. SAR QSAR
Environ Res 18: 629–643.
Dey, B., Lerner, D. L., Lusso, P., Boyd, M. R., Elder, J. H., and Berger, E. A. 2000. Multiple antiviral
activities of cyanovirin-N: Blocking HIV type1 gp120 interaction with CD4 co receptor and
inhibition of diverse enveloped viruses. J Virol 74: 4562–4569.
Dobretsov, S., Teplitski, M., Alagely, A., Gunasekera, S. P., and Paul, V. J. 2010. Malyngolide from the
cyanobacterium Lyngbya majuscula interferes with quorum sensing circuitry. Environ Microbiol
Rep 2: 739–744.
Dobretsov, S., Teplitski, M., Bayer, M., Gunasekera, S., Proksch, P., and Paul, V. J. 2011. Inhibition of
marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27(8): 893–905.
Drobac-Čik, A. V., Dulić, T. I., Stojanović, D. B., and Svirčev, Z. B. 2007. The importance of extremo-
phile cyanobacteria in the production of biological active compounds. Proc Nat Sci Matica
Srpska Novi Sad 112: 57–66.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M. A., and Gerwick,
W. H. 2004. Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide
neurotoxins from the marine cyanobacterium Lyngbya majuscula. Chem Biol 11: 817–833.
El Hattab, M., Culioli, G., Piovetti, L., Chitour, S. E., and Valls, R. 2007. Comparison of various extrac-
tion methods for identification and determination of volatile metabolites from the brown alga
Dictyopteris membranacea. J Chromatogr A 1143(1–2): 1–7.
El Marrouni, A., Kolleth, A., Lebeuf, R., Gebauer, J., Prevost, S., Heras, M., and Cossy, J. 2013.
Lyngbouilloside and related macrolides from marine cyanobacteria. NPC Nat Prod Commun
8: 1–8.
Erhard, M., von Döhren, H., and Jungblut, P. 1997. Rapid typing and elucidation of new secondary
metabolites of intact cyanobacteria using MALDI-TOF mass spectrometry. Nat Biotechnol 15:
906–909.
Falch, B. S., König, G. M., Wright, A. D., Sticher, O., Angerhofer, C. K., Pezzuto, J. M., and Bachmann,
H. 1995. Biological activities of cyanobacteria: Evaluation of extracts and pure compounds.
Planta Med 61: 321–328.
Fastner, J., Erhard, M., and von Döhren, H. 2001. Determination of oligopeptide diversity within a
natural population of Microcystis spp. (cyanobacteria) by typing single colonies by matrix–
assisted laser desorption ionization–time of flight mass spectrometry. Appl Environ Microbiol
67: 5069–5076.
Feldman, S. C., Reynaldi, S., Stortz, C. A., Cerezo, A. S., and Damonte, E. B. 1999. Antiviral properties
of fucoidan fractions from Leathesia difformis. Phytomedicine 6: 335–340.
Fischbach, M. A. and Walsh, C. T. 2009. Antibiotics for emerging pathogens. Science 325(5944): 1089–1093.
Gerwick, W. H., Tan, L., and Sitachitta, N. 2001. The Alkaloids, Vol. 57. San Diego, CA: Academic Press,
pp. 75–184.
Ghasemi, Y., Yazdi, M. T., Shafiee, A., Amini, M., Shokravi, S., and Zarrini, G. 2004. Parsiguine, A
novel antimicrobial substance from Fischerella ambigua. Pharmacol Biol 2: 318–322.
Ghasemi, Y., Yazdi, M. T., Shokravi, S., Soltani, N., and Zarrini, G. 2003. Antifungal and antibacte-
rial activity of paddy-fields cyanobacteria from the north of Iran. J Sci Islamic Rep Iran 14(3):
203–209.
Ghazala, B., Naila, B., and Shameel, M. 2010. Fatty acids and biological activities of crude extracts of
freshwater algae from Sindh. Pak J Bot 42: 1201–1212.
Gupta, D. K., Kaur, P., Leong, S. T., Tan, L. T., Prinsep, M. R., and Chu, J. J. H. 2014. Anti-chikungunya
viral activities of aplysiatoxin-related compounds from the marine cyanobacterium
Trichodesmium erythraeum. Mar Drugs 12(1): 115–127.
Gustafson, K. R., Cardellina, J. H., Fuller, R. W., Weislow, O. S., Kiser, R. F., Snader, K. M., and Boyd,
M. R. 1989. AIDS antiviral Sulfolipids from cyanobacteria (blue-green-algae). J Natl Cancer Inst
81(16): 1254–1258.
Hagmann, L. and Jiittner, F. 1996. Fischerellin A, a novel photosystem-II-inhibiting allelochemical
of the cyanobacterium Fischerella muscicola with antifungal and herbicidal activity. Tetrahedron
Lett 37: 6539–6542.
Han, B., Goeger, D., Maier, C. S., and Gerwick, W. H. 2005. The wewakpeptins, cyclic depsipeptides
from a Papua New Guinea collection of the marine cyanobacterium Lyngbya semiplena. J Org
Chem 70: 3133–3139.
Harborne, J. B. 1998. Phytochemical Methods: A Guide to Modern Techniques of Latent Analysis. London,
U.K.: Chapman and Hall Publishers, p. 278.
Huber, B., Eberl, L., Feucht, W., and Polster, J. 2003. Influence of polyphenols on bacterial biofilm
formation and quorum-sensing. Z Naturforsch C 58: 879–884.
Jaki, B., Orjala, J., Heilmann, J., Linden, A., Vogler, B., and Sticher, O. 2000. Novel extra cellular
diterpenoids with biological activity from the cyanobacterium Nostoc commune. J Nat Prod 63:
339–343.
Jaki, B., Orjala, J., and Sticher, O. 1999. A novel extracellular diterpenoid with antibacterial activity
from the cyanobacterium Nostoc commune. J Nat Prod 62: 502–503.
Jenke-Kodama, H., Sandmann, A., Muller, R., and Dittmann, E. 2005. Evolutionary implications of
bacterial polyketide synthases. Mol Biol Evol 22(10): 2027–2039.
Joana Gil-Chávez, G., Villa, J. A., Fernando Ayala-Zavala, J., Basilio Heredia, J., Sepulveda, D., Yahia,
E. M., and González-Aguilar, G. A. 2013. Technologies for extraction and production of bioac-
tive compounds to be used as nutraceuticals and food ingredients: An overview. Compr Rev
Food Sci Food Safety 12(1): 5–23.
Jones, A. C., Gu, L., Sorrels, C. M., Sherman, D. H., and Gerwick, W. H. 2009. New tricks from ancient
algae: Natural product biosynthesis in marine cyanobacteria. Curr Opin Chem Biol 13: 216–223.
Kaushik, P., Chauhan, A., and Goyal, P. 2009. Screening of Lyngbya majuscula for potential antibacte-
rial activity and HPTLC analysis of active methanolic extract. J Pure Appl Microbiol 3: 169–174.
Kim, J. D. 2006. Screening of cyanobacteria (blue–green algae) from rice paddy soil for antifungal
activity against plant pathogenic fungi. Microbiology 34(3): 138–142.
Koglin, A. and Walsh, C. T. 2009. Structural insights into nonribosomal peptide enzymatic assembly
lines. Nat Prod Rep 26: 987–1000.
Kreitlow, S., Mundt, S., and Lindequist, U. 1999. Cyanobacteria—A potential source of new biologi-
cally active substance. J Biotechnol 70: 61–73.
Kuffner, I. B. and Paul, V. J. 2004. Effects of the benthic cyanobacterium Lyngbya majuscula on larval
recruitment of the reef corals Acropora surculosa and Pocillopora damicornis. Coral Reefs 23: 455–458.
Kumar, M., Tripathi, M. K., Srivastava, A., Gour, J. K., Singh, R. K., Tilak, R., and Asthana, R. K. 2013.
Cyanobacteria, Lyngbya aestuarii and Aphanothece bullosa as antifungal and antileishmanial
drug resources. Asian Pac J Trop Biomed 3(6): 458–463.
Lacaille-Dubois, M. A. and Wagner, H. 1996. A review of the biological and pharmacological activi-
ties of saponins. Phytomedicine 2(4): 363–386.
Lau, A. F., Siedlecki, J., Anleitner, J., Patterson, G. M., Caplan, F. R., and Moore, R. E. 1993. Inhibition
of reverse transcriptase activity by extracts of cultured blue–green algae (cyanophyta). Planta
Med 59(2): 148–151.
Linington, R. G., Clark, B. R., Trimble, E. E., Almanza, A., Ureña, L. D., Kyle, D. E., and Gerwick, W. H.
2009. Antimalarial peptides from marine cyanobacteria: Isolation and structural elucidation of
gallinamide A. J Nat Prod 72: 14–17.
Linington, R. G., Edwards, D. J., Shuman, C. F., McPhail, K. L., Matainaho, T., and Gerwick, W. H.
2008. Symplocamide A, a potent cytotoxin and chymotrypsin inhibitor from the marine
Cyanobacterium Symploca sp. J Nat Prod 71(1): 22–27.
Loya, S., Reshef, V., Mizrachi, E., Silberstein, C., Rachamim, Y., Carmeli, S., and Hizi, A. 1998. The
inhibition of the reverse transcriptase of HIV-1 by the natural sulfoglycolipids from cyanobac-
teria: Contribution of different moieties to their high potency. J Nat Prod 61: 891–895.
Luke Simmons, T., Engene, N., Ureña, L. D., Romero, L. I., Ortega-Barría, E., Gerwick, L., and
Gerwick, W. H. 2008. Viridamides A and B, lipodepsipeptides with antiprotozoal activity from
the marine cyanobacterium Oscillatoria nigroviridis. J Nat Prod 71: 1544–1550.
MacMillan, J. B., Ernst-Russell, M. A., de Ropp, J. S., and Molinski, T. F. 2002. Lobocyclamides A–C.
Lipopeptides from a cryptic cyanobacterial mat containing Lyngbya confervoides. J Org Chem
67: 8210–8215.
Madhumathi, V., Deepa, P., Jeyachandran, S., Manoharan, C., and Vijayakumar, S. 2011. Antimicrobial
activity of cyanobacteria isolated from freshwater lake. Int J Microbiol Res 2: 213–216.
Malloy, K. L., Villa, F. A., Engene, N., Matainaho, T., Gerwick, L., and Gerwick, W. H. 2011.
Malyngamide 2, an oxidized lipopeptide with nitric oxide inhibiting activity from a Papua
New Guinea marine cyanobacterium. J Nat Prod 74(1): 95–98.
Mansouri, A., Embarek, G., Kokkalou, E., and Kefalas, P. 2005. Phenolic profile and antioxidant
activity of the Algerian ripe date palm fruit (Phoenix dactylifera). Food Chem 89: 411–420.
Mayer, A. M., Rodríguez, A. D., Berlinck, R. G., and Hamann, M. T. 2009. Marine pharmacology in
2005–6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-
inflammatory, antimalarial, antiprotozoal, antituberculosis and antiviral activities; affecting
the cardiovascular, immune and nervous systems and other miscellaneous mechanisms of
action. Biochim Biophys Acta 1790(5): 283–308.
McFeeters, R. L., Xiong, C., O’Keefe, B. R., Bokesch, H. R., McMahon, J. B., Ratner, D. M., and Byrd,
R. A. 2007. The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors. J Mol
Biol 369(2): 451–461.
Meriluoto, J. and Codd, G. A. 2005. Toxic: Cyanobacterial Monitoring and Cyanotoxin Analysis. Åbo,
Finalnd: Åbo Akademi University Press (Turku), ISBN 951–765–259–3, p. 149.
Meriluoto, J., Karlsson, K., and Spoof, L. 2004. High-throughput screening of ten microcystins and
nodularins, cyanobacterial peptide hepatotoxins, by reversed-phase liquid chromatography–
electrospray ionisation mass spectrometry. Chromatographia 59: 291–298.
Milligan, K. E., Marquez, B. L., Williamson, R. T., and Gerwick, W. H. 2000. Lyngbyabellin B. A toxic
and antifungal secondary metabolite from the marine cyanobacterium Lyngbya majuscula. J
Nat Prod 63: 1440–1443.
Mori, T. and Boyd, M. R. 2001. Cyanovirin-N, a potent human immunodeficiency virus-inactivating
protein, blocks both CD4-dependent and CD4-independent binding of soluble gp120 (sgp120)
to target cells, inhibits sCD4-induced binding of sgp120 to cell-associated CXCR4 and dissoci-
ates bound sgp120 from target cells. Antimicrob Agents Chemother 45(3): 664–672.
Mukherjee, P., Banerjee, I., Khatoon, N., and Pal, R. 2013. Cyanobacteria as elicitor of pigment in
ornamental fish Hemigrammus caudovittatus (Buenos Aires Tetra). J Algal Biomass Utln 4(3):
59–65.
Mundt, S., Kreitlow, S., and Jansen, R. 2003. Fatty acids with antibacterial activity from the cyanobac-
terium Oscillatoria redekei HUB051. J Appl Phycol 15: 263–267.
Nagle, D. G. and Paul, V. J. 1998. Chemical defense of a marine cyanobacterial bloom. J Exp Mar Biol
Ecol 225: 29–38.
Najdenski, H. M., Gigova, L. G., Iliev, I. I., Pilarski, P. S., Lukavský, J., Tsvetkova, I. V., and Kussovski,
V. K. 2013. Antibacterial and antifungal activities of selected microalgae and cyanobacteria.
Int J Food Sci Technol 48(7): 1533–1540.
Neuhof, T., Schmieder, P., Preussel, K., Dieckmann, R., Pham, H., Bartl, F., and von Döhren, H. 2005.
Hassallidin A, a glycosylated lipopeptide with antifungal activity from the cyanobacterium
Hassallia sp. J Nat Prod 68(5): 695–700.
Newman, D. J., Cragg, G. M., and Snader, K. M. 2003. Natural products as sources of new drugs over
the period 1981–2002. J Nat Prod 66: 1022–1037.
Nunnery, J. K., Mevers, E., and Gerwick, W. H. 2010. Biologically active secondary metabolites from
marine cyanobacteria. Curr Opin Biotechnol 21: 787–793.
Okura, K., Matsuoka, S., and Inoue, M. 2013. The bulky side chain of antillatoxin is important for
potent toxicity: Rational design of photoresponsive cytotoxins based on SAR studies. Chem
Commun (Camb) 49(73): 8024–8026.
Oman, T. J. and van der Donk, W. A. 2010. Follow the leader: The use of leader peptides to guide
natural product biosynthesis. Nat Chem Biol 6: 9–18.
Ozdemir, G., Ulku Karabay, N., Dalay, M. C., and Pazarbasi, B. 2004. Antibacterial activity of volatile
component and various extracts of Spirulina platensis. Phytother Res 18(9): 754–757.
Palaniselvam, V. 1998. Epiphytic cyanobacteria of mangrove: Ecological, physiological and bio-
chemical studies and their utility as biofertilizer and shrimp-feed. PhD thesis, Annamalai
University, Chidambaram, Tamil Nadu, India, p. 141.
Papendorf, O., König, G. M., and Wright, A. D. 1998. Hierridin B and 2,4-dimethoxy-
6-heptadecyl-phenol, secondary metabolites from the cyanobacterium Phormidium ectocarpi
with antiplasmodial activity. Phytochemistry 49: 2383–2386.
Patterson, G. M. L., Baker, K. K., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Larsen, L. K., Levine, I. A.
et al. 1993. Antiviral activity of cultured blue-green algae (Cyanophyta). J Phycol 29: 125–130.
Pawar, S. T. and Puranik, P. R. 2008. Screening of terrestrial and freshwater halotolerant cyanobac-
teria for antifungal activities. World J Microbiol Biotechnol 24: 1019–1025.
Pearson, L., Mihali, T., Moffitt, M., Kellmann, R., and Neilan, B. 2010. On the chemistry, toxicology
and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrosper-
mopsin. Mar Drugs 8: 1650–1680.
Pérez Gutiérrez, M., Suyama, T. L., Engene, N., Wingerd, J. S., Matainaho, T., and Gerwick, W. H.
2008a. Apratoxin D, a potent cytotoxic cyclodepsipeptide from papua new guinea collections
of the marine cyanobacteria Lyngbya majuscula and Lyngbya sordida. J Nat Prod 71(6): 1099–1103.
Pérez Gutiérrez, R. M., Flores, A. M., Solís, R. V., and Jimenez, J. C. 2008b. Two new antibacterial
norabietane diterpenoids from cyanobacterium. Micrococcus lacustris. J Nat Med 62: 328–331.
Plaza, M., Santoyo, S., Jaime, L., Reina, G. G. B., Herrero, M., Señoráns, F. J., and Ibáñez, E. 2010.
Screening for bioactive compounds from algae. J Pharm Biomed Anal 51(2): 450–455.
Portmann, C., Blom, J. F., Kaiser, M., Brun, R., Jüttner, F., and Gademann, K. 2008. Isolation of aerucy-
clamides C and D and structure revision of microcyclamide 7806A, heterocyclic ribosomal
peptides from Microcystis aeruginosa PCC 7806 and their antiparasite evaluation. J Nat Prod 71:
1891–1896.
Prabakaran, M. 2011. In vitro antimicrobial potentials of marine Oscillatoria species. Asian J Plant Sci
Res 1: 58–64.
Prashantkumar, P., Angadi, S. B., and Vidyasagar, G. M. 2006. Antimicrobial activity of blue-green
and green algae. Indian J Pharm Sci 68: 647–648.
Priyadarshani, I. and Rath, B. 2012. Commercial and industrial applications of micro algae—A
review. J Algal Biomass Utln 3(4): 89–100.
Ramachandra Rao, C. S. V. 1994. Antimicrobial activity of cyanobacteria. Indian J Mar Sci 23: 55–56.
Ramaswamy, A. V., Flatt, P. M., Edwards, D. J., Simmons, T. L., Han, B., and Gerwick, W. H. 2006. The
secondary metabolites and biosynthetic gene clusters of marine cyanobacteria. Applications
in biotechnology. In: Proksch, P. and Muller, W. E. G., eds., Frontiers in Marine Biotechnology.
Horizon Biosci 175–224.
Rath, B. and Priyadarshani, I. 2013. Antibacterial and antifungal activity of marine cyanobacteria
from Odisha Coast. Int J Curr Trends Res 2(1): 248–251.
Raveh, A. and Carmeli, S. 2007. Antimicrobial ambiguines from the cyanobacterium Fischerella sp.
collected in Israel. J Nat Prod 70: 196–201.
Reehana, N., Parveez Ahamed, A., and Thajuddin, N. 2012. In vitro studies on bactericidal effect of
selected cyanobacteria against bacterial pathogens. Int J Med Res 1(7): 345–347.
Sakthivel, K. and Kathiresan, K. 2012. Antimicrobial activities of marine cyanobacteria isolated
from mangrove environment of south east coast of India. J Natl Prod 5: 147–156.
Sanchez, L. M., Lopez, D., Vesely, B. A., Della Togna, G., Gerwick, W. H., Kyle, D. E., and Linington,
R. G. 2010. Almiramides A–C: Discovery and development of a new class of leishmaniasis lead
compounds. J Med Chem 53: 4187–4197.
Senthil Kumar, N. S., Sivasubramanian, V., and Mukund, S. 2013. Antimicrobial and Antifungal
activity of extracts of Phormidium fragile Gomont. J Algal Biomass Utln 4(3): 66–71.
Shamsa, F., Monsef, H., Ghamooshi, R., and Verdian-rizi, M. 2008. Spectrophotometric determina-
tion of total alkaloids in some Iranian medicinal plants. Thai J Pharm Sci 32: 17–20.
Shanab, S. M. M. 2007. Bioactive Allelo-chemical compounds from Oscillatoria species (Egyptian
Isolates). Int J Agric Biol 9: 617–621.
Shiau, I. L., Shih, T. L., Wang, Y. N., Chen, H. T., Lan, H. F., Lin, H. C., and Murase, Y. 2009.
Quantification for saponin from a soapberry in cleaning products by a chromatographic and
two colorimetric assays. J Faculty Agri Kyushu Univ 54: 215–221.
Singh, R. K., Tiwari, S. P., Rai, A. K., and Mohapatra, T. M. 2011. Cyanobacteria: An emerging source
for drug discovery. J Antibiot 64: 401–412.
Sitachitta, N. and Gerwick, W. H. 1998. Grenadadiene and grenadamide, cyclopropyl-containing fatty
acid metabolites from the marine cyanobacterium Lyngbya majuscula. J Nat Prod 61: 681–684.
Sitachitta, N., Williamson, R. T., and Gerwick, W. H. 2000. Yanucamides A and B, two new depsi-
peptides from an assemblage of the marine cyanobacteria Lyngbya majuscula and Schizothrix
species. J Nat Prod 63: 197–200.
Sivonen, K. 2000. Chapter 26, Freshwater cyanobacterial neurotoxins: Ecobiology, chemistry and
detection. In: Botana, L. M., ed., Seafood and Freshwater Toxins. New York: Marcel Dekker, Inc.,
pp. 567–582.
Skindersoe, M. E., Alhede, M., Phipps, R., Yang, L., Jensen, P. O., Rasmussen, T. B., and Givskov, M.
2008. Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents
Chemother 52: 3648–3663.
Soria-Mercado, I. E., Pereira, A., Cao, Z., Murray, T. F., and Gerwick, W. H. 2009. Alotamide A, a novel
neuropharmacological agent from the marine cyanobacterium Lyngbya bouillonii. Org Lett 11:
4704–4707.
Starmans, D. A. and Nijhuis, H. H. 1996. Extraction of secondary metabolites from plant material: A
review. Trends Food Sci Technol 7: 191–197.
Starr, T. J., Deig, E. F., Church, K. K., and Allen, M. B. 1962. Antibacterial and antiviral activities of
algal extracts studied by acridine orange staining. Tex Rep Biol Med 20: 271–278.
Staunton, J. and Weissman, K. J. 2001. Polyketide biosynthesis: A millennium review. Nat Prod Rep
18: 380–416.
Suhail, S., Biswas, D., Farooqui, A., Arif, J. M., and Zeeshan, M. 2011. Antibacterial and free radical
scavenging potential of some cyanobacterial strains and their growth characteristics. J Chem
Pharm Res 3: 472–478.
Sundararaman, M. and Nagaraja, B. K. 2006. Investigation of marine cyanobacteria for antifungal
activity against Candida albicans. Seaweed Res Util 28(1): 183–187.
Tan, G., Gyllenhaal, C., and Soejarto, D. D. 2006. Biodiversity as a source of anticancer drugs. Curr
Drug Targets 7: 265–277.
Tan, L. T. and Goh, B. P. L. 2009. Chemical ecology of marine cyanobacterial secondary metabolites:
A mini-review. J Coast Dev 13(1): 1–9.
Tan, L. T., Goh, B. P. L., Tripathi, A., Lim, M. G., Dickinson, G. H., Lee, S. S. C., and Teo, S. L. M.
2010. Natural antifoulants from the marine cyanobacterium Lyngbya majuscula. Biofouling 26(6):
685–695.
Tan, L. T., Okino, T., and Gerwick, W. H. 2000. Hermitamides A and B, toxic malyngamide-type
natural products from the marine cyanobacterium Lyngbya majuscula. Nat Prod 63(7): 952–955.
Taton, A., Grubisic, S., Balthasart, P., Hodgson, D. A., Laybourn-Parry, J., and Wilmotte, A. 2006.
Biogeographical distribution and ecological ranges of benthic cyanobacteria in East Antarctic
lakes. FEMS Microbiol Ecol 57(2): 272–289.
Teruya, T., Sasaki, H., Fukazawa, H., and Suenaga, K. 2009. Bisebromoamide, a potent cytotoxic
peptide from the marine cyanobacterium Lyngbya sp.: Isolation, stereostructure and biological
activity. Org Lett 11: 5062–5065.
Thajuddin, N. and Subramanian, G. 2005. Cyanobacterial biodiversity and potential applications in
biotechnology. Curr Sci 89: 47–57.
Thummajitsakul, S., Silprasit, K., and Sittipraneed, S. 2012. Antibacterial activity of crude extracts of
cyanobacteria Phormidium and Microcoleus species. Afr J Microbiol Res 6(10): 2574–2579.
Tidgewell, K., Engene, N., Byrum, T., Media, J., Valeriote, F. A., and Gerwick, W. H. 2010. Evolved
diversification of a modular natural product pathway: Apratoxins F and G, two cytotoxic cyclic
depsipeptides from a Palmyra collection of Lyngbya bouillonii. Chem Bio Chem 11: 1458–1466.
Tokuoka, M., Sawamura, N., Kobayashi, K., and Mizuno, A. 2010. Simple metabolite extraction
method for metabolic profiling of the solid-state fermentation of Aspergillus oryzae. J Biosci
Bioeng 110(6): 665–669.
Tripathi, A., Puddick, J., Prinsep, M. R., Lee, P. P. F., and Tan, L. T. 2010. Hantupeptins B and C, cyto-
toxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula. Phytochemistry
71: 307–311.
Uzair, B., Tabassum, S., Rasheed, M., and Rehman, S. F. 2012. Exploring marine cyanobacteria for
lead compounds of pharmaceutical importance. Sci World J 2012: 1–10.
Vicente, M. F., Basilio, A., Cabello, A., and Pelaez, F. 2003. Microbial natural products as a source of
antifungals. Clin Microbiol Infect 9(1): 15–32.
Wang, Y. C., Chuang, Y. C., and Hsu, H. W. 2008. The flavonoid, carotenoid and pectin content in
peels of citrus cultivated in Taiwan, Food Chem 106(1): 277–284.
Welker, M., Christiansen, G., and Von Döhren, H. 2004. Diversity of coexisting Planktothrix (cyano-
bacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopep-
tides. Arch Microbiol 182(4): 288–298.
Welker, M. and Von Döhren, H. 2006. Cyanobacterial peptides—Nature’s own combinatorial bio-
synthesis. FEMS Microbiol Rev 30(4): 530–563.
Williamson, R. T., Singh, I. P., and Gerwick, W. H. 2004. Taveuniamides: New chlorinated toxins
from a mixed assemblage of marine cyanobacteria. Tetrahedron 60: 7025–7033.
Xiong, C., O’Keefe, B. R., Byrd, R. A., and McMahon, J. B. 2006. Potent anti-HIV activity of scytovirin
domain 1 peptide. Peptides 27: 1668–1675.
Xiong, S., Fan, J., and Kitazato, K. 2010. The antiviral protein cyanovirin-N: The current state of its
production and applications. Appl Microbiol Biotechnol 86(3): 805–812.
Yadav, S., Sinha, R. P., and Tyagi, M. B. 2012. Antimicrobial activity of some cyanobacteria. Int J
Pharm Pharm Sci 4(3): 631–635.
Yoon, Y. S. and Lee, C. G. 2009. Partial purification and characterization of a novel antifungal com-
pound against Aspergillus spp. from Synechocystis sp. PCC 6803. Biotechnol Bioprocess Eng 14:
383–390.
Yu, H., Jia, S., and Dai, Y. 2009. Growth characteristics of the cyanobacterium Nostoc flagelliforme in
photoautotrophic, mixotrophic and heterotrophic cultivation. J Appl Phycol 21: 127–133.
Zainuddin, E. N., Mentel, R., Wray, V., Jansen, R., Nimtz, M., Lalk, M., and Mundt, S. 2007. Cyclic
depsipeptides, ichthyopeptins A and B, from Microcystis ichthyoblabe. J Nat Prod 70: 1084–1088.
Zeeshan, M., Suhail, S., Biswas, D., Farooqui, A., and Arif, J. M. 2010. Screening of selected cyano-
bacterial strains for phytochemical compounds and biological activities in vitro. Biochem Cell
Arch 10: 163–168.
chapter thirteen
Antimicrobial and natural compounds

from edible mushrooms
Annamalai Panneerselvam, V. Ambikapathy, and M. Nithya
Contents
13.1 Introduction.........................................................................................................................234
13.2 Mushroom............................................................................................................................234
13.2.1 Edible mushrooms.................................................................................................. 236
13.2.1.1 Other edible wild species....................................................................... 237
13.2.1.2 Conditionally edible species................................................................... 238
13.2.2 Medicinal mushrooms........................................................................................... 238
13.2.2.1 Ganoderma lucidum................................................................................... 238
13.2.2.2 Medical applications................................................................................ 239
13.2.3 Poisonous mushrooms........................................................................................... 241
13.2.3.1 Old and new world hallucinogenic mushrooms................................. 241
13.2.3.2 Amanita muscaria....................................................................................... 241
13.3 Nutritional content of edible mushrooms....................................................................... 242
13.3.1 Nutrients in button mushrooms........................................................................... 242
13.3.2 Great weight loss food............................................................................................ 243
13.3.3 Excellent source of potassium............................................................................... 243
13.3.4 Disease-fighting properties................................................................................... 243
13.3.5 Metabolism support nutrient................................................................................ 244
13.3.6 Great source of heart-healthy copper................................................................... 244
13.4 Bioactive compounds of edible mushrooms................................................................... 244
13.4.1 Alkaloids.................................................................................................................. 245
13.4.1.1 Psilocybin mushrooms............................................................................ 245
13.4.1.2 Psilocybin biochemistry.......................................................................... 245
13.4.1.3 Psilocybin and medicine......................................................................... 245
13.4.1.4 Psilocybin effects...................................................................................... 245
13.4.1.5 Adverse effects to psilocybin................................................................. 246
13.4.2 Flavonoids................................................................................................................ 247
13.4.3 Phenols..................................................................................................................... 247
13.4.4 Sterols....................................................................................................................... 248
13.4.5 Terpenes................................................................................................................... 248
13.4.6 Triterpenes............................................................................................................... 249
233
13.4.7 Saponins................................................................................................................... 249

13.4.8 Tannins..................................................................................................................... 249
13.4.9 Anthraquinones...................................................................................................... 249
13.4.9.1 Red from mushrooms.............................................................................. 249
13.4.9.2 Blue from mushrooms............................................................................. 249
13.5 Antimicrobial compounds and their applications......................................................... 250
13.6 Conclusion........................................................................................................................... 251
Acknowledgment......................................................................................................................... 251
References...................................................................................................................................... 251
13.1 Introduction
A mushroom (or toadstool) is the fleshy, spore-bearing fruiting body of a fungus, typically
produced aboveground on soil or on its food source. The standard for the name “mush-
room” is the cultivated white button mushroom, Agaricus bisporus; hence, the word “mush-
room” is most often applied to those fungi (Basidiomycota, Agaricomycetes) that have a
stem (stipe), a cap (pileus), and gills (lamellae, sing, lamella) or pores on the underside
of the cap. These pores or gills produce microscopic spores that help the fungus spread
across the ground or its occupant surface (Figure 13.1).
Mushroom describes a variety of gilled fungi, with or without stems, and the term is
used even more generally, to describe both the fleshy fruiting bodies of some Ascomycota
and the woody or leathery fruiting bodies of some Basidiomycota, depending on the con-
text of the word.
Forms deviating from the standard morphology usually have more specific names,
such as “puffball,” “stinkhorn,” and “morel,” and gilled mushrooms themselves are often
called “agarics” in reference to their similarity to Agaricus or their place Agaricales. By
extension, the term “mushroom” can also designate the entire fungus when in culture, the
thallus (called a mycelium) of species forming the fruiting bodies called mushrooms, or
the species itself (Dickinson and Lucas, 1982).
13.2 Mushroom
Mushrooms are mostly Basidiomycetes and gilled. Their spores, called basidiospores, are
produced on the gills and fall in a fine rain of powder from under the caps as a result.
At the microscopic level, the basidiospores are shot-off basidia and then fall between the
gills in the dead-air space. As a result, for most mushrooms, if the cap is cut off and placed
gill-side-down overnight, a powdery impression reflecting the shape of the gills (or pores,
or spines, etc.) is formed (when the fruit body is sporulating). The color of the powdery
print, called a spore print, is used to help classify mushrooms and can help to identify
them. Spore print colors include white (most common), brown, black, purple-brown, pink,
yellow, and creamy, but almost never blue, green, or red.
While modern identification of mushrooms is quickly becoming molecular, the stan-
dard methods for identification are still used by most and have developed into a fine art
harking back to medieval times and the Victorian era, combined with microscopic exam-
ination. The presence of juices upon breaking, bruising reactions, odors, tastes, shades
of color, habitat, habit, and season are all considered by both amateur and professional
mycologists. Tasting and smelling mushrooms carry their own hazards because of poisons
and allergens. Chemical tests are also used for some genera.
Chapter thirteen: Antimicrobial and natural compounds from edible mushrooms 235
Oyster mushroom (yellow) Oyster mushroom (white)
Milky mushroom (Calocybe indica)

(a)
Milky mushroom—ventral view (Calocybe indica)

(b)
Figure 13.1 Mushroom: close view of (a) stalk and (b) Gills plate.
In general, identification to genus can often be accomplished in the field using a local
mushroom guide. Identifying species, however, requires more effort; one must remember
that a mushroom develops from a button stage into a mature structure, and only the latter
can provide certain characteristics needed for the identification of the species. However,
overmature specimens lose features and cease producing spores. Many novices have mis-
taken humid water marks on paper for white spore prints or discolored paper from oozing
liquids on lamella edges for colored spored prints.
13.2.1 Edible mushrooms

Edible mushrooms are the fleshy and edible fruit bodies of several species of macrofungi
(fungi that bear fruiting structures that are large enough to be seen with the naked eye).
They can appear either belowground (hypogeous) or aboveground (epigeous) where they
may be picked by hand. Edibility may be defined by criteria that include the absence of
poisonous effects on humans and desirable taste and aroma.
Edible mushrooms are consumed by humans as comestibles for their nutritional value,
and they are occasionally consumed for their supposed medicinal value. Mushrooms
consumed by those practicing folk medicine are known as medicinal mushrooms. While
hallucinogenic mushrooms (e.g., psilocybin mushrooms) are occasionally consumed for
recreational or religious purposes; they can produce severe nausea and disorientation and
are therefore not commonly considered edible mushrooms.
Edible mushrooms include many fungal species that are either harvested wild or cul-
tivated. Easily cultivatable and common wild mushrooms are often available in markets,
and those that are more difficult to obtain (such as the prized truffle and matsutake) may
be collected on a smaller scale by private gatherers. Some preparations may render certain
poisonous mushrooms fit for consumption.
Before assuming that any wild mushroom is edible, it should be identified. Proper iden-
tification of a species is the only safe way to ensure edibility. Some mushrooms that are
edible for most people can cause allergic reactions in some individuals, and old or improp-
erly stored specimens can cause food poisoning. Deadly poisonous mushrooms that are
frequently confused with edible mushrooms and responsible for many fatal poisonings
include several species of the Amanita genus, in particular, Amanita phalloides, the death cap.
Mushrooms growing in polluted locations can accumulate pollutants such as heavy metals.
Some species are difficult to cultivate; others (particularly mycorrhizal species) have
not yet been successfully cultivated. Some of these species are harvested from the wild
and can be found in markets. When in season, they can be purchased fresh, and many spe-
cies are sold dried as well. The following species are commonly harvested from the wild:
• Boletus edulis or edible Boletus, native to Europe, known in Italian as fungo porcino
(plural “porcini,” pig mushroom), in German as Steinpilz (stone mushroom), in
Russian as “white mushroom,” in Albanian as wolf mushroom, and in French the
cèpe. It also known as the king bolete and is renowned for its delicious flavor. It is
sought after worldwide and can be found in a variety of culinary dishes.
• Cantharellus cibarius (the chanterelle), the yellow chanterelle is one of the best and most
easily recognizable mushrooms and can be found in Asia, Europe, North America,
and Australia. There are poisonous mushrooms that resemble it, though these can be
confidently distinguished if one is familiar with the chanterelle’s identifying features.
• Cantharellus tubaeformis, the tube chanterelle or yellow leg.
• Clitocybe nuda, Blewit (or Blewitt).
• Cortinarius caperatus the gypsy mushroom (recently moved from the genus Rozites).
• Craterellus cornucopioides, trompette de la mort or horn of plenty.
• Grifola frondosa, known in Japan as maitake (also “hen of the woods” or “sheep’s
head”); a large, hearty mushroom commonly found on or near stumps and bases of
oak trees and believed to have Macrolepiota procera properties.
• Gyromitra esculenta, this “false morel” is prized by the Finns. This mushroom is
deadly poisonous if eaten raw, but highly regarded when parboiled.
• Hericium erinaceus, a tooth fungus; also called “lion’s mane mushroom.”
• Hydnum repandum, sweet tooth fungus, hedgehog mushroom, urchin of the woods.
• Lactarius deliciosus, saffron milk cap, consumed around the world and prized in Russia.
• Morchella species (morel family), morels belong to the ascomycete grouping of fungi.
They are usually found in open scrub, woodland, or open ground in late spring.
When collecting this fungus, care must be taken to distinguish it from the poisonous
false morels, including Gyromitra esculenta.
• Morchella conica var. deliciosa
• Morchella esculenta var. rotunda
• Tricholoma matsutake the matsutake, a mushroom highly prized in Japanese cuisine.
• Tuber species (the truffle), truffles have long eluded the modern techniques of domesti-
cation known as trufficulture. Although the field of trufficulture has greatly expanded
since its inception in 1808, several species still remain uncultivated. Domesticated
truffles include
• Tuber borchii
• Tuber brumale
• Tuber indicum—Chinese black truffle
• Tuber macrosporum—white truffle
• Tuber mesentericum—Bagnoli truffle
• Tuber uncinatum—Black summer truffle
13.2.1.1 Other edible wild species

Many wild species are consumed around the world. The species that can be identified
in the field (without use of special chemistry or a microscope) and therefore safely eaten
vary widely from country to country, even from region to region. This list is a sampling of
lesser-known species that are reportedly edible.
Lactarius salmonicolor
• Amanita caesarea (Caesar’s mushroom)

• Armillaria mellea
• Boletus badius
• Chroogomphus rutilus (pine spikes or spike caps)
• Calvatia gigantea (giant puffball)
• Calocybe gambosa (St George’s mushroom)
• Clavariaceae species (coral fungus family)
• Clavulinaceae species (coral fungus family)
• Coprinus comatus, the shaggy mane; must be cooked as soon as possible after harvest-
ing or the caps will first turn dark and unappetizing and then deliquesce and turn to
ink, hence not being found in markets for this reason
• Corn smut
• Cortinarius variecolor
• Fistulina hepatica (beefsteak polypore or the ox tongue)
• Hygrophorus chrysodon
Auricularia auricula-judae
• Lactarius salmonicolor
• Lactarius subdulcis (mild milkcap)
• Lactarius volemus
• Laetiporus sulphureus (sulfur shelf), also known by names such as the “chicken mush-
room” and “chicken fungus” and is a distinct bracket fungus popular among mush-
room hunters
• Leccinum aurantiacum (red-capped scaber stalk)
• Leccinum scabrum (birch bolete)
• Lepiota procera
• Macrolepiota procera parasol mushroom, globally being widespread in temperate regions
• Polyporus squamosus (dryad’s saddle and pheasant’s back mushroom)
• Polyporus mylittae
• Ramariaceae species (coral fungus family)
• Rhizopogon luteolus
• Russula, some members of which are edible
• Sparassis crispa, also known as “cauliflower mushroom”
• Suillus bovinus
• Suillus granulatus
• Suillus luteus
• Suillus tomentosus
• Tricholoma terreum
13.2.1.2 Conditionally edible species

Amanita muscaria, a conditionally edible species: There are a number of fungi that are consid-
ered choice by some and toxic by others. In some cases, proper preparation can remove
some or all of the toxins (Rubel and Arora, 2008):
• Amanita muscaria is edible if parboiled to leach out toxins. Fresh mushrooms cause
vomiting, twitching, drowsiness, and hallucinations due to the presence of musci-
mol. Although present in A. muscaria, ibotenic acid is not in high enough concentra-
tion to produce any physical or psychological effects unless massive amounts are
ingested.
• Coprinopsis atramentaria is edible without special preparation. However, consumption
with alcohol is toxic due to the presence of coprine. Some other Coprinus spp. share
this property.
• Gyromitra esculenta is eaten by some after it has been parboiled; however, mycologists
do not recommend it. Raw Gyromitra are toxic due to the presence of gyromitrin, and
it is not known if all of the toxin can be removed by parboiling.
• Lactarius spp.: Apart from L. deliciosus that is universally considered edible, other
Lactarius spp. that are considered toxic elsewhere in the world are eaten in some east-
ern European countries and Russia after pickling or parboiling (Arora, 1986).
• Verpa bohemica: Considered choice by some, it even can be found for sale as a “morel,”
but cases of toxicity have been reported. Verpas contain toxins similar to gyromitrin
and similar precautions applied.
13.2.2 Medicinal mushrooms

13.2.2.1 Ganoderma lucidum
Medicinal mushrooms are mushrooms or extracts used or studied as possible treat-
ments for diseases. Some mushroom materials, including polysaccharides, glycoproteins,
and proteoglycans, modulate immune system responses and inhibit tumor growth in
preliminary research, whereas other isolates showed potential cardiovascular, antiviral,
antibacterial, antiparasitic, anti-inflammatory, and antidiabetic properties. Currently, sev-

eral extracts have widespread use in Japan, Korea, and China, as adjuncts to radiation
treatments and chemotherapy, even though clinical evidence of efficacy in humans has not
been confirmed (Smith et al., 2002).
Historically, mushrooms have long been thought to hold medicinal value, espe-
cially in traditional Chinese medicine. They have been studied in modern medical
research since the 1960s, where most studies use extracts, rather than whole mush-
rooms. Only a few specific extracts have been tested for efficacy in laboratory research.
Polysaccharide-K and lentinan are among the extracts best understood from in vitro
research of animal models such as mice or early-stage human pilot studies (Borchers
et al., 2008).
Preliminary experiments showed that glucan-containing mushroom extracts may
affect the function of the innate and adaptive immune systems, functioning as biore-
sponse modulators. In some countries, the extracts of polysaccharide-K, schizophyllan,
polysaccharide peptide, or lentinan are government-registered adjuvant cancer therapies.
13.2.2.2 Medical applications

13.2.2.2.1 Antimicrobial isolates and derivatives Immunomodulatory protein isolated
from Ganoderma lucidum. Antibiotics retapamulin, tiamulin, and valnemulin are deriva-
tives of the mushroom isolate pleuromutilin. Plectasin, austrocortilutein, austrocortirubin,
coprinol, oudemansin A, strobilurin, illudin, pterulone, and sparassol are antibiotics iso-
lated from mushrooms. Researchers have isolated a number of antifungal, antiviral, and
antiprotozoan isolates from mushrooms (Engler et al., 1998) (Table 13.1).
Table 13.1 Enzyme inhibitors from mushroom

Mushroom Isolate/extract/metabolite Enzyme inhibited
Pleurotus ostreatus Lovastatin HMG-CoA reductase
Hypholoma sublateritium Clavaric acid Farnesyl transferase
Polyozellus multiplex Polyozellin, thelephoric acid, Prolyl endopeptidase
kynapcins
Lentinus edodes Eritadenine S-adenosyl-l-homocysteine
hydrolase
Coprinopsis atramentaria 1-Aminocyclopropanol Acetaldehyde dehydrogenase
Inonotus obliquus Extract Dipeptidyl peptidase-4
Grifola frondosa Extract Alpha-glucosidase
Trametes versicolor Extract Alpha-amylase
Pholiota squarrosa, Daedalea Extract (Pholiota squarrosa), Xanthine oxidase
quercinol (Daedalea quercina)
Phellinus linteus Phellinstatin Enoyl-ACP reductase
Phellinus linteus Hispidin and hypholomine B Neuraminidase
Various Extract 5-Alpha reductase
Various Extract Aromatase
Various Peptides Angiotensin-converting enzyme
Daedalea quercina Quercinol Cyclooxygenase 2
Daedalea quercina Quercinol Horseradish peroxidase
13.2.2.2.2 Anticancer research Bristol-Myers Squibb manufactures paclitaxel using

Penicillium raistrickii. In 2014, researchers reported creating a transgenic Flammulina velu-
tipes that expresses the gene used to synthesize the paclitaxel precursor baccatin III (Han
et al., 2014).
Mushroom isolates researched for anticancer activity include clavaric acid, a farne-
syl transferase inhibitor; asparaginase, a Flammulina velutipes isolate; irofulven and acyl-
fulvene, derivatives of illudin S, conjugated linoleic acid, an Agaricus isolate; grifolin, an
Albatrellus confluens isolate; and clitocine, a Leucopaxillus giganteus isolate.
Some countries have approved mushroom extracts lentinan, polysaccharide-K, and poly-
saccharide peptide as immunologic adjuvants (Ina et al., 2013). There is some evidence of this
use having effectiveness in prolonging and improving the quality of life for patients with
certain cancers, although the Memorial Sloan Kettering Cancer Center observes that “well
designed, large scale studies are needed to establish the role of lentinan as a useful adjunct to
cancer treatment.” According to Cancer Research UK, “there is currently no evidence that any
type of mushroom or mushroom extract can prevent or cure cancer” (Gao et al., 2009).
The mushrooms credited with success against cancer belong to the genera Phellinus,
Pleurotus, Agaricus, Ganoderma, Clitocybe, Antrodia, Trametes, Cordyceps, Xerocomus, Calvatia,
Schizophyllum, Flammulina, Suillus, Inonotus, Inocybe, Funlia, Lactarius, Albatrellus, Russula,
and Fomes. The anticancer compounds play crucial role as reactive oxygen species inducer,
mitotic kinase inhibitor, antimitotic inhibitor, angiogenesis inhibitor, and topoisomerase
inhibitor, leading to apoptosis and eventually checking cancer proliferation. This review
updates the recent findings on the pharmacologically active compounds, their antitumor
potential, and the underlying mechanism of biological action in order to raise awareness
for further investigations to develop cancer therapeutics from mushrooms.
13.2.2.2.3 Antidiabetic research Many mushroom isolates act as DPP-4 inhibitors,

alpha-glucosidase inhibitors, and alpha-amylase inhibitors in vitro. Ternatin is a mush-
room isolate that suppresses hyperglycemia (Lo and Wasser, 2011).
13.2.2.2.4 Psychotropic research Psychotropic medicines created from ergot alka-

loids include cafergot, dihydroergotamine, methysergide, methylergometrine, hydergine,
nicergoline, lisuride, bromocriptine, cabergoline, and pergolide. Polyozellus multiplex syn-
thesizes prolyl endopeptidase inhibitors polyozellin, thelephoric acid, and kynapcins,
while Hericium erinaceus isolates erinacine and hericenone promote nerve growth factor
synthesis and myelination in vitro.
Neurotrophic mushroom isolates include l-theanine, tricholomalides, scabronines,
and termitomycesphins. Many mushrooms synthesize the partial, nonselective, serotonin
receptor agonist/analog psilocin.
13.2.2.2.5 Nutritional research Mushrooms are the only food source of statins like
lovastatin (Atli et al., 2013). Only fungi and animals can synthesize vitamin D. Mushrooms
have been verified creating D2 (ergocalciferol), D4 (22-dihydroergocalciferol), and vitamin D1
(lumisterol + D2) (Keegan et al., 2013). Mushrooms are a rare source of ergothioneine
(Weigand-Heller et al., 2012) that contain ACE inhibitor peptides and are a source of pre-
biotic dietary fiber. Mushrooms also contain a variety of chemicals like lovastatin, cordy-
cepin, inotilone, quercinol, antcin B, antrodioxolanone, and benzocamphorin F having
preliminary research evidence for anti-inflammatory activity. Mushroom mycelia can be
used to enhance the concentrations of γ-aminobutyric acid, ergothioneine, and other anti-
oxidants in bread (Ulziijargal et al., 2013; Postemsky and Curvetto, 2014).
13.2.3 Poisonous mushrooms

Many mushroom species produce secondary metabolites that can be toxic, mind altering,
antibiotic, antiviral, or bioluminescent. Although there are only a small number of deadly
species, several others can cause particularly severe and unpleasant symptoms. Toxicity
likely plays a role in protecting the function of the basidiocarp: the mycelium has expended
considerable energy and protoplasmic material to develop a structure to efficiently distribute
its spores. One defense against consumption and premature destruction is the evolution of
chemicals that render the mushroom inedible, either causing the consumer to vomit the meal
(emetics) or to learn to avoid consumption altogether. In addition, due to the ability of mush-
rooms to absorb heavy metals, including those that are radioactive, European mushrooms
may, to date, include toxicity from the 1986 Chernobyl disaster and continue to be studied.
13.2.3.1 Old and new world hallucinogenic mushrooms

Most fungi are not deadly to humans, and many are perfectly edible (and are quite deli-
cious); however, some species are poisonous and contain potent neurotoxins. Placing a silver
coin in a pan of cooking mushrooms to see if it turns black is not a reliable method of test-
ing poisonous mushrooms. Unless you understand fungal terminology and know how to
use a good taxonomic key (such as Mushrooms Demystified by David Arora, 1986), the staff at
Wayne’s Word® do not encourage self-indulgence on wild mushrooms. The beautiful, red,
fly agaric mushroom (A. muscaria) is unmistakable with its bright red cap covered with white
scales. It contains the toxic alkaloid, muscimol, which is derived from ibotenic acid, which
is an amino acid. In Europe, the mushrooms were reportedly left in open dishes to kill flies;
however, according to some authorities, the flies are merely stunned or stupefied by the
toxin and may even regain control and fly away. Although it is poisonous to humans, there
are other species of Amanita that are much more dangerous and are potentially lethal if
ingested. Some of these dangerously poisonous species are death cap (A. phalloides), death
angel (A. ocreata), and panther amanita (A. pantherina). Fortunately, these latter deadly poison-
ous species are not bright red and are seldom confused with A. muscaria; however, they may
be confused with other edible mushrooms by inexperienced gourmets. According to David
Arora, species may be one of the most common causes of mushroom poisoning in the Pacific
Northwest. David Isaak stated in a recent e-mail message that in the Pacific Northwest, a
number of people gather the panther for culinary purposes, cooking it in several changes of
water to remove most of the psychoactive materials (Allen and Merlin, 1992).
13.2.3.2 Amanita muscaria

When ingested by humans, A. muscaria may produce visions and delirium, and it is per-
haps one of the oldest known hallucinogens. Recent studies suggest that this mushroom
was the mysterious God-narcotic “Divine Soma” of ancient India. Thousands of years ago,
Aryan conquerors who swept across India worshiped soma, drinking it in religious cer-
emonies. Many hymns in the Indian Rig Veda are devoted to Soma and describe the mush-
room and its effects. According to the Rig Veda, Soma is without leaves, seeds, or branches,
but with a head and stalk or pillar (the structure of a mushroom); its dazzling red skin
is like the hide of the bull (the red cap); and its dress like that of a sheep, with woolly
fragments remaining when the envelop bursts (the outer membranous envelop called the
universal veil breaks as the stalk grows upward, leaving white remnants on the red cap).
This is a remarkably accurate description of the fly agaric mushroom (A. muscaria). There
are reports of Siberian tribesmen who ingested the mushroom to get intoxicated. Since the
active chemical (muscimol) passes through the body relatively unaltered, others would
drink the urine from these men to get high. This way a few mushrooms could inebriate
many people relatively safely and efficiently. Lapland shamans eat fly agaric mushrooms
for enlightenment, and some authors have postulated that this may have given rise to the
flying reindeer and the red- and white-costumed Santa Claus legends.
Apparently, not everyone agrees that the Divine Soma is A. muscaria. According to
Terrence McKenna, the active alkaloid in fly agaric mushrooms (muscimol) does not pro-
duce the psychoactive effects described in the Rig Veda and other literature. Terrence
McKenna has continued to question the effects of A. muscaria and suggested other p ossible
candidates. Based on firsthand experience with these hallucinogens, he has suggested
that a psilocybin “magic mushroom,” such as Stropharia cubensis, is the true Divine Soma.
In fact, he also states that the use of mind-altering psilocybin mushrooms by ancient
humans in Africa may have been a catalyst in the development of language and religion
in primitive cultures.
A. muscaria was apparently one of the sacred hallucinogenic mushrooms of the Incas,
Mayans, and Aztecs. (Other New World psychedelic genera included Psilocybe, Panaeolus,
Conocybe, and Stropharia.) For the Indians of Mexico and Central and South America,
partaking of these mushrooms was a deeply religious experience, enabling them to com-
municate with their gods. Cortez reported a mushroom (resembling A. muscaria) being
eaten during the coronation of Montezuma, and in Guatemala, stone carvings dating
back to 1000 BC depict curious figures with umbrellalike tops resembling the caps and
stalks of an Amanita mushroom. Mushrooms are also depicted in ancient Peruvian ves-
sels and in the Mexican codices. One drawing shows an animal-like messenger from
god offering the sacred Amanita to a ruler seated on a throne. And, a fresco in a Roman
Catholic Church in Plaincourault (Indre), France, depicts Adam and Eve on either side of
a tree of knowledge that is unequivocally a branched Amanita mushroom. Some scholars
believe that the original story of Alice’s Adventures in Wonderland, where Alice speaks
to a green caterpillar who is seated on a red- and white-capped mushroom, is actually
the interpretation of a mushroom experience by the author Rev. C.L. Dodgson of Christ
Church College in Oxford (better known by his pen name Lewis Carroll). Another hal-
lucinogenic “high” that is commonly depicted in paintings and children’s stories is the
infamous, “politically incorrect” picture of a witch flying on a broom—the effects of a
portion made from the deadly alkaloids of several solanaceous herbs, including jimson-
weed (Datura stramonium).
13.3 Nutritional content of edible mushrooms

Mushrooms are a low-calorie food usually eaten cooked or raw and as garnish to a meal.
Dietary mushrooms are a good source of B vitamins, such as riboflavin, niacin, and pan-
tothenic acid, and essential minerals, such as selenium, copper, and potassium. Fat, carbo-
hydrate, and calorie contents are low, with the absence of vitamin C and sodium. There are
approximately 20 calories in an ounce of mushrooms (Table 13.2).
When exposed to ultraviolet light, natural ergosterols in mushrooms produce
vitamin D2, a process now exploited for the functional food retail market.
13.3.1 Nutrients in button mushrooms

White button mushrooms, the popular ones available in all the grocery stores, have a sur-
prising amount of nutrients including niacin, riboflavin, folate, phosphorus, iron, panto-
thenic acid, zinc, potassium, copper, magnesium, vitamin B6, selenium, and thiamin.
Table 13.2 Nutritional value of edible mushroom (100 g)

Energy 113 kJ (27 kcal)
Carbohydrates 4.1 g
Fat 0.1 g
Protein 2.5 g
Thiamine (vitamin B1) 0.1 mg (9%)
Riboflavin (vitamin B2) 0.5 mg (42%)
Niacin (vitamin B3) 3.8 mg (25%)
Pantothenic acid (B5) 1.5 mg (30%)
Vitamin C 0 mg (0%)
Calcium 18 mg (2%)
Phosphorus 120 mg (17%)
Potassium 448 mg (10%)
Sodium 6 mg (0%)
Zinc 1.1 mg (12%)
Source: USDA Nutrient Database.
Percentages are roughly approximated using U.S. recommenda-
tions for adults.
In addition, white button mushroom extract has been found to reduce the size of some
cancer tumors and slow down the production of some cancer cells. It is most prominently
linked to reducing the risk of breast and prostate cancers (Koyyalamudi et al., 2009).
13.3.2 Great weight loss food

For those who are always looking for nutritious weight loss foods to pack into our diets,
mushrooms are a less well-known option. Mushrooms are low in calories, carbohydrates,
fats, and sodium. However, like watermelon, they are very high in water content (80%–
90% water) and fiber that makes them a great diet food.
13.3.3 Excellent source of potassium

Most people think that bananas are the food of high potassium, but it may surprise you to
learn that mushrooms out rank bananas on the potassium chart. Potassium helps the body
process sodium and lower blood pressure, so people with hypertension or high risk of stroke
can enjoy tremendous health benefits from a regular dose of mushrooms in their diet.
13.3.4 Disease-fighting properties

All mushrooms are an excellent source of the antioxidant, and the selenium works with
vitamin E to protect cells from damaging free radicals. Some studies also indicate that anti-
oxidants are some of the best nutrients for preventing and fighting cancers. Like almonds,
mushrooms are becoming more popular for their cancer-fighting and disease-protecting
properties.
Shitake mushrooms in particular are also high in the beta-glucan lentinan. Lentinan
has been linked with strengthening the immune system and helping combat illnesses that
attack the immune system like AIDS. In addition, mushroom extract has been linked to
some treatments for both migraines and mental disorders.
13.3.5 Metabolism support nutrient

The human metabolism relies on a healthy dose of protein, fiber, and vitamin B to keep it func-
tional and robust. Mushrooms rank high in all three of these metabolism-supporting nutrients.
13.3.6 Great source of heart-healthy copper

Copper is one of the less talked about minerals that is essential to the body, but that the
body cannot make on its own. Copper has properties that help protect our cardiovascular
system, and just one small serving of mushrooms contains more than 20% of the copper
we need daily.
With our fast-paced lifestyles and the highly processed foods, we may frequently find
ourselves eating in haste; the magnesium, potassium, phosphorus, and selenium nutrients
found in a single dish of mushrooms can really make up for some of the deficiencies we
struggle to combat in our diets.
13.4 Bioactive compounds of edible mushrooms

Both the fruit bodies and the mycelia of M. esculenta contain an uncommon amino acid,
cis-3-amino-l-proline; this amino acid does not appear to be protein bound. In addition to
M. esculenta, the amino acid is known to exist only in M. conica and M. crassipes (Moriguchi
et al., 1979).
Laboratory experiments using rodent models suggest that the polysaccharides from
M. esculenta fruit bodies have several medicinal properties, including antitumor effects,
immunoregulatory properties, fatigue resistance, and antiviral effects. Extracts from the
fruit bodies have antioxidant properties. It also has been shown that the polysaccharides
from M. esculenta mycelia have antioxidant activity. The fungus is listed in the IUCN
National Register of medicinal plants in Nepal. M. esculenta is also used in traditional
Chinese medicine to treat indigestion, excessive phlegm, and shortness of breath (Rotzoll
et al., 2005; Nitha and Janardhanan, 2008).
Secondary metabolites were either absent or present in very different ratios and in
general showed significantly less potency in cultivated Chaga. Cultivated Chaga further-
more results in a reduced diversity of phytosterols, particularly lanosterol, an intermedi-
ate in the synthesis of ergosterol and lanostane-type triterpenes. This effect was partially
reversed by the addition of silver ion, an inhibitor of ergosterol biosynthesis.
The major active compounds found are unsaturated fatty acids such as linoleic acid
and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid
with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty
acids inhibit aromatase with similar potency and that mutations at the active site regions
affect its interaction with these two fatty acids.
Due to their natural polyisoprene content (1.1%–7.7% by dry weight of fruit bodies),
L. volemus fruit bodies can also be used to produce rubber. The chemical structure of rubber
from the mushroom consists of a high-molecular-mass homologue of polyprenol, arranged
as a dimethylallyl group, two trans isoprene units, and a long sequence of cis isoprenes
(between 260 and 300 units), terminated by a hydroxyl or fatty acid ester. Biosynthetically,
the creation of the polyisoprene begins with the compound trans, trans-farnesyl pyrophos-
phate, and is thought to terminate by esterification of polyisoprenyl pyrophosphate. The
enzyme isopentenyl-diphosphate delta-isomerase has been identified as required for the
initiation of rubber synthesis in L. volemus and several other Lactarius species.
13.4.1 Alkaloids
G. lucidum contains other compounds often found in fungal materials, including poly-
saccharides (such as beta-glucan), coumarin, mannitol, and alkaloids (Paterson, 2006).
Ergotamine is the secondary metabolite and the principal alkaloid produced by the ergot
fungus Claviceps purpurea and related family in the fungi Clavicipitaceae. It possesses
structural similarity to several neurotransmitters and has biological activity as vasocon-
strictor. It is used medicinally for the treatment of acute migraine attacks. Its medicinal
usage began in the sixteenth century to induce childbirth. It has been used to prevent
postpartum hemorrhage (bleeding after birth). The mechanism of action of ergotamine is
complex. The molecules share structural similarity with neurotransmitters such as sero-
tonin, dopamine, and epinephrine and can thus bind to several receptors (Schardl, 2000).
13.4.1.1 Psilocybin mushrooms

Psilocybin (also known as psilocybine) is a psychedelic alkaloid of the tryptamine family,
found in psilocybin mushrooms. It is present in hundreds of species of fungi, including
those of the genus Psilocybe, such as Psilocybe cubensis and Psilocybe semilanceata. But it is
also reportedly isolated from a dozen or so other genera. Psilocybin mushrooms are com-
monly called “magic mushrooms” or more simply “shrooms.” The psilocybin content of
psychoactive mushrooms varies and depends on species, growth, drying conditions, and
mushroom sizes. The intensity and duration of recreational and entheogenic use of psilo-
cybin mushrooms vary depending on species of mushrooms, dosage, individual physiol-
ogy, set, and setting.
13.4.1.2 Psilocybin biochemistry

Psilocybin is a product that is converted into the pharmacologically active compound psi-
locin in the body by dephosphorylation. This chemical reaction takes place under strongly
acidic conditions or enzymatically by phosphatases in the body. Psilocybin is a zwitter-
ionic alkaloid that is soluble in water, moderately soluble in methanol and ethanol, and
insoluble in most organic solvents. Mature mycelium contains some amount of psilocy-
bin, which can be extracted with an acidic solution, usually of citric acid or ascorbic acid
(vitamin C). Young mycelium (recently germinated from spores) does not contain substan-
tial amounts of alkaloids. It is also known to mimic the effects of serotonin.
13.4.1.3 Psilocybin and medicine

In a current study of psilocybin led by Charles Grob, 12 subjects are being administered
with either the hallucinogen or a placebo in two separate sessions. Grob hopes to reduce
the psychological distress (e.g., obsessive–compulsive behavior) that is associated with
death by treating patients with psilocybin.
13.4.1.4 Psilocybin effects

The effects of psilocybin are often pleasant, even ecstatic, including a deep sense of con-
nection to others, confusion, hilarity, and a general feeling of connection to nature and the
universe. Difficult trips may occur when psychedelic compounds are taken in a nonsup-
portive or inadequate environment, by an inexperienced person, in an unexpectedly high
dose, or when the substance triggers difficult areas of one’s psyche.
At low doses, hallucinatory effects occur, including walls that seem to breathe, a vivid
enhancement of colors, and the animation of organic shapes. At higher doses, experiences
tend to be less social and more entheogenic, often catalyzing intense spiritual experiences.
A very small number of people are unusually sensitive to psilocybin’s effects, where
doses as little as 0.25 g of dried Psilocybe cubensis mushrooms (normally a threshold dose
of around 2 mg psilocybin) can result in effects usually associated with medium and high
doses. Likewise, there are some people who require relatively high doses of psilocybin
to gain low-dose effects. Individual brain chemistry and metabolism play a major role in
determining a person’s response to psilocybin. Psilocybin is metabolized mostly in the
liver where it becomes psilocin. It is broken down by the enzyme monoamine oxidase
(MAO). MAO inhibitors have been known to sustain the effects of psilocybin for longer
periods of time; people who are taking an MAOI for a medical condition (or are seeking to
potentiate the mushroom experience) should be careful.
Mental and physical tolerances to psilocybin build and dissipate quickly. Taking psi-
locybin more than three or four times in a week (especially 2 days in a row) can result
in diminished effects. Tolerance dissipates after a few days, so frequent users often keep
doses spaced 5–7 days apart to avoid the effect.
13.4.1.5 Adverse effects to psilocybin

Individuals that have relatives with schizophrenia should be very careful about consum-
ing psilocybin or any hallucinogenic drug at all due to the risk of triggering a psychosis.
Because of the ease of cultivating psilocybin mushrooms or gathering wild species, puri-
fied psilocybin is practically nonexistent on the illegal drug market.
The psychoactive alkaloid in the teonanacatl mushrooms is psilocybin, a potent indole
alkaloid. Psilocin, a dephosphorylated version of psilocybin, is about 10 times stronger.
After ingestion by humans, psilocybin is automatically converted into psilocin. Most
psilocybin-containing mushrooms have only a trace of psilocin. According to David Arora
(1986), the common psilocybin mushroom of the Pacific coast of North America, Psilocybe
cyanescens, has a higher concentration of natural psilocin and is appropriately named
“potent psilocybe.” Although two of the most famous species of psilocybin mushrooms
are Psilocybe mexicana and Stropharia cubensis, there are literally dozens of other species
in the four aforementioned genera with similar hallucinogenic properties. In fact, Paul
Stamets (Psilocybin Mushrooms of the World, 1996) describes all of the species and includes
color photographs. Like so many little brown mushrooms (LBMs), they are difficult to
identify unless you are familiar with mushroom structure and spore taxonomy and have a
good compound microscope at your disposal. In fact, two deadly look-alike LBMs (Galerina
autumnalis and Pholiotina filaris) resemble certain species of Psilocybe. The small ring on
their stems (called an annulus) and rusty brown spores (rather than black spores) are dead
giveaways to avoid these potentially lethal mushrooms (Figure 13.2).
Indole alkaloids contain the indole carbon–nitrogen ring that is also found in the
fungal alkaloids ergine and psilocybin, the neurotransmitter serotonin, and the mind-
altering drug LSD. These alkaloids may interfere or compete with the action of serotonin
in the brain.
OPO3H2
HO
CH2CH2NH2 CH2CH2N(CH3)2
N N
H H
Serotonin Psilocybin
Figure 13.2 Structure of serotonin and psilocybin.

13.4.2 Flavonoids
L. deliciosus, Sarcodon imbricatus, and Tricholoma portentosum contain more flavonoids. The
total phenols and flavonoids were the major components found in the mushroom extracts;
ascorbic acid was found in small amounts (0.18–0.52 mg/g), and β-carotene and lycopene
were only found in vestigial amounts (<91 μg/g). L. deliciosus contains 8.14 ± 0.81 (mg/g) of
flavonoids, S. imbricatus 2.82 ± 0.09 (mg/g), and T. portentosum 0.40 ± 0.02 (mg/g).
The extracts with the entire mushroom showed higher phenolic and flavonoid con-
tents than either the cap or the stipe. Also, the amount of phenolic and flavonoid com-
pounds in the cap methanolic extracts was higher than the amount found in stipe extracts.
The antimicrobial screening of phenolic compounds is extracted from L. deliciosus,
S. imbricatus, and T. portentosum against Bacillus cereus, Bacillus subtilis (gram-positive
bacteria), E. coli, and Pseudomonas aeruginosa (gram-negative bacteria) and Candida
albicans and Cryptococcus neoformans (fungi). The minimal inhibitory concentrations
(MICs) for bacteria and fungi were determined as an evaluation of the antimicrobial
activity of the tested mushrooms. The diameters of the inhibition zones correspond-
ing to the MICs are also presented. All the mushrooms revealed antimicrobial activity
showing different selectivities and MICs for each microorganism. L. deliciosus showed
better results than T. portentosum and S. imbricatus (lower MICs), which is in agreement
with the higher content of phenols and flavonoids found in the first species.
The entire and the cap mushroom extracts inhibited B. cereus, B. subtilis, P. aeruginosa,
C. albicans, and C. neoformans, while the stipe mushroom extract only inhibited B. cereus,
P. aeruginosa, and C. neoformans. Only this mushroom revealed activity against P. aeruginosa
(gram-negative bacteria) and C. albicans (fungi) and for the gram-positive bacteria showed
lower MICs. The T. portentosum extract was effective only against gram-positive bacteria
(B. cereus, B. subtilis) and C. neoformans. When the cap was used, the same selectivity was
obtained, but with higher MICs; the stipe extract was only effective against B. cereus. In
fact, the content in total phenols and flavonoids for the stipe extracts was always lower
than in the other extracts. As expected due to its lower content in bioactive compounds,
S. imbricatus was the less effective (higher MICs) mushroom, showing activity only against
B. cereus and C. neoformans. The cap extract was selective for B. cereus, while the stripe
extract was not effective against the tested microorganisms (Turkoglu et al., 2007).
13.4.3 Phenols
Phenolic acids can be found in mushroom basidiomycete species. For example, protocat-
echuic acid and pyrocatechol are found in Agaricus bisporus as well as other phenylated
substances like phenylacetic and phenylpyruvic acids. Other compounds like atromentin
and thelephoric acid can also be isolated from fungi in the Agaricomycetes class. Orobol,
an isoflavone, can be isolated from Aspergillus niger (Table 13.3).
Omphalotus nidiformis is not edible. Although reputedly mild tasting, eating it will
result in vomiting, which generally occurs 30 min to 2 h after consumption and lasts for
several hours. There is no diarrhea, and the patients recover without lasting ill effects. Its
toxicity was first mentioned by Anthony M. Youngin in his 1982 guidebook Common
Australian Fungi. The toxic ingredient of many species of Omphalotus is a sesquiterpene
compound known as illudin S. This, along with illudin M and a cometabolite illudosin, has
been identified in O. nidiformis. The two illudins are common to the genus Omphalotus and
not found in any other basidiomycete mushroom. Additional three compounds unique to
O. nidiformis have been identified and named illudins F, G, and H.
Table 13.3 Mushroom metabolites

Mushroom Metabolite
Pleurotus ostreatus Lovastatin
Hypholoma sublateritium Clavaric acid
Polyozellus multiplex Polyozellin, thelephoric acid, kynapcins
Lentinus edodes Eritadenine
Coprinopsis atramentaria 1-Aminocyclopropanol
Pholiota squarrosa, Daedalea quercina Extract (Pholiota squarrosa), quercinol
(Daedalea quercina)
Phellinus linteus Phellinstatin
Phellinus linteus Hispidin and hypholomine B
Daedalea quercina Quercinol
Extracts of several species of Australian mushrooms have been investigated for cyto-
toxicity to cancer cells; material from O. nidiformis showed marked toxicity to gastric (AGS),
colon (HT-29), and estrogen-independent breast cancer (MDA-MB-231) cell lines. Irofulven,
a compound derived from illudin S, is undergoing phase II clinical trials as a possible
therapy for various types of cancers. Fruit body extracts have antioxidant and free radical
scavenging properties, which may be attributed to the presence of phenolic compounds
(Ribéreau-Gayon, 1972).
13.4.4 Sterols
G. lucidum produces a group of triterpenes, called ganoderic acids, which have a molecular
structure similar to that of steroid hormones. Sterols isolated from the mushroom include
ganoderol, ganoderic acid, ganoderiol, ganodermanontriol, lucidadiol, and ganodermadiol.
Lactarius volemus fruit bodies contain a unique sterol molecule called volemolide,
a derivative of the common fungal sterol ergosterol that may have application in fun-
gal chemotaxonomy. A 2001 study identified further nine sterols, three of which were
previously unknown to science. According to the authors, these types of highly oxy-
genated compounds—similar to sterols found in marine soft corals and sponges—are
rare in fungi. The mushroom also contains volemitol (d-glycero-d-mannoheptitol), a
seven-carbon sugar alcohol first isolated from the species by the French scientist Émile
Bourquelot in 1889. Volemitol occurs as a free sugar in many plant and brown algal
species.
13.4.5 Terpenes
G. lucidum contains terpenoids that have been on the less volatile triterpenoid (triterpene)
and sterol-type compounds. The triterpene chemical structure is based on the ground
structure of lanosterol, which is an important intermediate in the biosynthetic pathway
for steroids and triterpenes in microorganisms and animals. Sterols, compounds closely
related to triterpenoids, are also found in Ganoderma. They have been isolated from the
fruiting body and mycelium and have also been shown to exhibit potent cytotoxic activity.
A specific sterol, ergosterol peroxide, was isolated from G. lucidum and has been shown
to enhance the inhibitory effect of linoleic acid on the inhibition of mammalian DNA
polymerase-β (Hseu et al., 1996).
13.4.6 Triterpenes
G. lucidum produces a group of triterpenes, called ganoderic acids, which have a molecular
structure similar to that of steroid hormones. It also contains other compounds often found
in fungal materials, including polysaccharides (such as beta-glucan), coumarin, mannitol,
and alkaloids. Sterols isolated from the mushroom include ganoderol, ganoderenic acid,
ganoderiol, ganodermanontriol, lucidadiol, and ganodermadiol (Shiao et al., 1988).
13.4.7 Saponins
Saponins are toxic to some microorganisms and to animals, which includes the growth
of mycelium of Pleurotus sapidus, G. lucidum, Cantharellus cibarius, Laccaria amethystina,
Clitocybe odora, Lepista nuda, Lepista saeva, L. deliciosus, Laccaria laccata, Pleurotus ostreatus,
and Hericium erinaceus having the saponins with antioxidant activities.
13.4.8 Tannins
Wild edible Nigerian mushrooms including Cantharellus cibarius, L. amethystina, Clitocybe
odora, L. nuda, Macrolepiota procera, Lepista saeva, L. deliciosus, Laccaria laccata, Pleurotus ostrea-
tus, and Hericium erinaceus were investigated. The mushrooms were harvested fresh, sun
dried, pulverized, and analyzed according to standard procedures. Proximate analysis
showed high level of proteins (14.03%–60.38%), crude fibers (3.94%–20.36%), carbohydrates
(4.17%–32.50%), ashes (17.44%–33.60%), fats (1.29%–14.29%), and folic acids (4.75–5.51 g/g)
in all species. Mineral analysis of all species indicated the presence of potassium, sodium,
magnesium, manganese, calcium, copper, and iron. Potassium is of the highest amount
in all species of plant (1370–5710 g/100 g). High antioxidant activity was also observed
in these mushrooms with the species L. amethystina and L. nuda exhibiting the strongest
antioxidant activity with values as high as 53.64 and 53.65 nm, respectively. Phytochemical
screening revealed that above 10 species have the presence of varying quantities of alka-
loids, flavonoids, saponins, and tannins (Lattif et al., 1996).
13.4.9 Anthraquinones
13.4.9.1 Red from mushrooms
The Dermocybe family of mushroom produces oranges and reds. The addition of an iron
mordant gives darker shades and almost black ones. With older mushrooms, longer cook-
ing times or the addition of ammonia can give lilac shades. Low heat or the addition of
acid or vinegar gives warm reds, for example, Dictyophora cinnabarina and Cortinarius
semisanguineus.
13.4.9.2 Blue from mushrooms

Blue was the most used color in many of the fabrics from the grave sites. Blue may have
come from dyers.
Woad, Isatis tinctoria: Blue from the mushrooms Thelephora, with iron or tin mordants,
that yields greens and blues.
Hydnellum suaveolens, S. imbricatus: Yields blues if it is old and its top has darkened.
Hapalopilus rutilans: With ammonia, yields strong, colorfast violet-blue shades.
Cortinarius violaceus: Produces violet-blue shades, with an iron mordant and dark greys.
13.5 Antimicrobial compounds and their applications

Most studies on mushrooms with antibacterial activity describe the action of its extracts
without identifying the compounds responsible for this activity. However, some com-
pounds have been described as active against gram-positive bacteria. Five of these com-
pounds are terpenes. Confluentin (1A), grifolin (1B), and neogrifolin (1C) from Albatrellus
fletti showed activity against B. cereus and Enterococcus faecalis. The best result was for
Enterococcus faecalis (MIC 0.5–1.0 mg/mL) (Liu et al., 2010). Ganomycins A and B iso-
lated from Ganoderma pfeifferi showed activity against B. subtilis, Micrococcus flavus, and
Staphylococcus aureus (15–25 mm zones of inhibition at a concentration of 250 µg/mL)
(Mothana et al., 2000).
A steroid, 3,11-dioxolanosta-8,24(Z)-diene-26-oic acid (2), was isolated from the
Jahnoporus hirtus mushroom and revealed activity against B. cereus and Enterococcus faecalis
(Liu et al., 2010).
Four sesquiterpenes with antimicrobial activity were described. The enokipodins
A, B, C, and D, isolated from the mycelium of Flammulina velutipes, with activity against
B. subtilis, but only enokipodins A and C, showed activity against S. aureus (Ishikawa
et al., 2001).
Oxalic acid (3), an organic acid isolated from the mycelium of Lentinus edodes, showed
activity against B. cereus, S. aureus, and Streptococcus faecalis (Bender et al., 2003).
Coloratin A, a benzoic acid derivative isolated from Xylaria intracolorata, inhibited
S. aureus (Quang et al., 2006).
Eight compounds of anthraquinone derivatives were also reported due to their anti-
bacterial activities. 6-Methylxanthopurpurin-3-O-methyl ether, austrocortilutein, austro-
cortilutein, austrocortirubin, and torosachrysone, isolated from the mushroom Cortinarius
basirubencens, and physcion, erythroglaucin, and emodin isolated from other species of
Cortinarius, were all effective against S. aureus (Beattie et al., 2010).
CSAP (Cordyceps sinensis antibacterial protein-N-terminal sequence ALATQHGAP),
isolated from Cordyceps sinensis, showed strong activity against S. aureus and poor activ-
ity against B. subtilis. However, the antibacterial action of this protein was bacteriostatic
(Zheng et al., 2006).
The ribonuclease, isolated from Pleurotus sajor-caju, showed activity against S. aureus,
acting on RNA (Ngai and Ng, 2004).
The peptide plectasin, isolated from Pseudoplectania nigrella, is a macromolecule belong-
ing to the class of defensins, present in animals and plants, which act at the cell wall,
more specifically in the synthesis of peptidoglycan. This peptide showed activity against
B. cereus, Bacillus thuringiensis, Corynebacterium diphtheriae, Enterococcus faecalis, E. faecium,
(VREF), S. aureus, (MRSA), Staphylococcus epidermidis (MRSE), Streptococcus pneumoniae,
(PRSP), and Streptoccus pyogenes.
The in vitro action of plectasin against Streptococcus pneumoniae is comparable to the
action of penicillin and vancomycin (Mygind et al., 2005).
The peptides peptaibol boletusin, peptaibol chrysospermin 3, and peptaibol chryso-
spermin 5 (isolated from Boletus spp.) allow for the opening of pores for ion transport
and showed activity against B. subtilis, Corynebacterium lilium, and S. aureus. The peptaibol
chrysospermin 3 also showed activity against Streptococcus sp. (Lee et al., 1999).
Fraction B from Pycnoporus sanguineus, whose main constituent is a phenoxazin-3-one-
type pigment, showed activity against S. aureus and Streptococcus A, B, C, and G. Lower
values of MIC were obtained against Streptococcus strains (Smania et al., 1995).
13.6 Conclusion
Antibiotic resistance among microbes urgently necessitates the development of novel anti-
microbial agents such as alternate therapies using natural products. Many pharmaceutical
substances with potent and unique health enhancing properties have been isolated from
medicinal mushrooms and distributed worldwide. Mushroom-based products either from
the mycelia or fruiting bodies are consumed in the form of capsules, tablets, or extracts.
It has been reported by many workers that fruit bodies of different mushrooms like Lactarius
sp., Fomitopsis sp., Boletus sp., Pleurotus tuber-regium, L. deliciosus, S. imbricatus and T. portentosum,
Russula delica, Pleurotus eryngii var. ferulae, Infundibulicybe geotropa, L. controversus, L. delicious
and Phellinus hartigii, Lactarius indigo, and Stereum ostrea contain a wide range of antimicrobial
activity. With an increasing number of bacteria developing resistance to commercial antibiot-
ics, such as MSRA (methicillin-resistant S. aureus and Pseudomonas), extracts and derivatives
from mushrooms hold great promise for novel medicines in modern times.
Acknowledgment
The authors are grateful to the secretary and correspondent, the principal, the dean faculty
of sciences, and the staff members of the Department of Botany and Microbiology, AVVM
Sri Pushpam College, Poondi, Thanjavur, Tamil Nadu, India.
References
Allen, J. W. and Merlin, M. D. 1992. Psychoactive fungi use in Koh Samui and Koh Pha-Ngan,
Thailand. J Ethnopharmacol 35(3): 205–228.
Arora, D. 1986. Mushrooms Demystified, 2nd ed. Ten Speed Press, Berkeley, CA.
Atli, B., Yamac, M., and Yildiz, Z. 2013. Optimization of submerged fermentation conditions for
lovastatin production by the culinary-medicinal oyster mushroom, Pleurotus ostreatus (Higher
Basidiomycetes). Int J Med Mushrooms 15(5): 487–495.
Beattie, K. D., Rouf, R., Gander, L., May, T. W., Ratkowsky, D., Donner, C. D., Gill, M., Grice, I. D.,
and Tiralongo, E. 2010. Antibacterial metabolites from Australian macrofungi from the genus
Cortinarius. Phytochemistry 71: 948–955.
Bender, S., Dumitrache, C. N., Backhaus, J., Christie, G., Cross, R. F., Lonergan, G. T., and Baker, W. L.
2003. A case for caution in assessing the antibiotic activity of extracts of culinary-medicinal
Shiitake mushroom [Lentinus edodes (Berk.) Singer] (Agaricomycetideae). Int J Med Mushrooms
5: 31–35.
Borchers, A. T., Krishnamurthy, A., Keen, C. L., Meyers, F. J., and Gershwin, M. E. 2008. The immu-
nobiology of mushrooms. Exp Biol Med 233(3): 259–276.
Dickinson, C. and Lucas, J. 1982. VNR Color Dictionary of Mushrooms. Van Nostrand Reinhold,
New York, pp. 9–11.
Engler, M., Anke, T., and Sterner, O. 1998. Production of antibiotics by Collybia nivalis, Omphalotus ole-
arius, a Favolaschia and a Pterula species on natural substrates. Z Naturforsch C 53(5–6): 318–324.
Gao, Y., Xu, H., Lu, Z., and Xu, Z. 2009. Quantitative determination of steroids in the fruiting bodies
and submerged-cultured mycelia of Inonotus obliquus. Se Pu 27(6): 745–749.
Han, F., Kang, L. Z., Zeng, X. L., Ye, Z. W., Guo, L. Q., and Lin, J. F. 2014. Bioproduction of bacca-
tin III, an advanced precursor of paclitaxol with transgenic Flammulina velutipes expressing
10-Deacetylbaccatin III-10-O-acetyl transferase gene. J Sci Food Agric 94(12): 2376–2383.
Hseu, R. S., Wang, H. H., Wang, H. F., and Moncalvo, J. M. 1996. Differentiation and grouping of
isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR
compared with grouping on the basis of internal transcribed spacer sequences. Appl Environ
Microbiol 62(4): 1354–1363.
Ina, K., Kataoka, T., and Ando, T. 2013. The use of lentinan for treating gastric cancer. Anticancer
Agents Med Chem 13(5): 681–688.
Ishikawa, N. K., Fukushi, Y., Yamaji, K., Tahara, S., and Takahashi, K. 2001. Antimicrobial cuparene-
type sesquiterpenes, enokipodins C and D, from a mycelial culture of Flammulina velutipes.
J Nat Prod 64: 932–934.
Keegan, R. J., Lu, Z., Bogusz, J. M., Williams, J. E., and Holick, M. F. 2013. Photobiology of vitamin D
in mushrooms and its bioavailability in humans. Dermatoendocrinol 5(1): 165–176.
Koyyalamudi, S. R., Jeong, S. C., Song, C. H., Cho, K. Y., and Pang, G. 2009. Vitamin D2 formation and
bioavailability from Agaricus bisporus button mushrooms treated with ultraviolet irradiation.
J Agric Food Chem 57(8): 3351–3355.
Lattif, L. A., Daran, A. B. M., and Mohammed, A. B. 1996. Relative distribution of minerals in the
pileus and stalk of some selected edible mushroom. Food Chem 56: 155–121.
Lee, S. J., Yeo, W. H., Yun, S., and Yoo, D. 1999. Isolation and sequence analysis of new peptaibol,
boletusin, from Boletus spp. J Pept Sci 5: 374–378.
Liu, X. T., Winkler, A. L., Schwan, W. R., Volk, T. J., Rott, M. A., and Monte, A. 2010. Antibacterial
compounds from mushrooms I: A lanostane-type triterpene and prenylphenol derivates from
Jahnoporus hiritus and Albatrellus fletti and their activities against Bacillus cereus and Enterococcus
faecalis. Planta Med 76: 182–185.
Lo, H. C. and Wasser, S. P. 2011. Medicinal mushrooms for glycemic control in diabetes melli-
tus: History, current status, future perspectives, and unsolved problems (review). Int J Med
Mushrooms 13(5): 401–426.
Moriguchi, M., Sada, S.-I., and Hatanaka, S.-I. 1979. Isolation of cis-3-amino-l-proline from cultured
mycelia or Morchella esculenta. Appl Environ Microbiol 38(5): 1018–1019.
Mothana, R. A. A., Jansen, R., Julich, W. D., and Lindequist, U. 2000. Ganomycins A and B, new
antimicrobial Farnesyl hydroquinones from the Basidiomycete Ganoderma pfeifferi. J Nat Prod
63: 416–418.
Mygind, P. H., Fischer, R. L., Schnorr, K. M., Hansen, M. T., Sonksen, C. P., Ludvigsen, S., Raventos,
D. et al. 2005. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic
fungus. Nature 437: 975–980.
Ngai, P. H. K. and Ng, T. B. 2004. A ribonuclease with antimicrobial, antimitogenic and antiprolifera-
tive activities from the edible mushroom Pleurotus sajor-caju. Peptides 25: 11–17.
Nitha, B. and Janardhanan, K. K. 2008. Aqueous-ethanolic extract of morel mushroom mycelium
Morchella esculenta, protects cisplatin and gentamicin induced nephrotoxicity in mice. Food
Chem Toxicol 46(9): 3193–3199.
Paterson, R. R. 2006. Ganoderma—A therapeutic fungal biofactory. Phytochemistry 67(18): 1985–2001.
Postemsky, P. and Curvetto, N. 2014. Enhancement of wheat grain antioxidant activity by solid state
fermentation with Grifola spp. J Med Food 17(5): 543–549.
Quang, D. N., Bach, D. D., Hashimoto, T., and Asakawa, Y. 2006. Chemical constituents of the
Vietnamese inedible mushroom Xylaria intracolorata. Nat Prod Res 20: 317–321.
Ribéreau-Gayon, P. 1972. Plant Phenolics. Oliver and Boyd, Edinburgh.
Rotzoll, N., Dunkel, A., and Hofmann, T. 2005. Activity-guided identification of (S)-malic acid 1-O-d-
glucopyranoside (morelid) and gamma-aminobutyric acid as contributors to umami taste and
mouth-drying oral sensation of morel mushrooms (Morchella deliciosa Fr.). J Agric Food Chem
53(10): 4149–4156.
Rubel, W. and Arora, D. 2008. A study of cultural bias in field guide determinations of mushroom
edibility using the iconic mushroom, Amanita muscaria, as an example. Econ Bot 62(3): 223–243.
Schardl, C. L. 2000. Symbiotic parasites and mutualistic pathogens: Clavicipitaceous symbionts
of grasses. In: Fungal Pathology, Kronstad, J. W. (ed.). Dordrecht, the Netherlands: Kluwer
Academic Publishers, pp. 307–345.
Shiao, M. S., Lin, L. J., and Yeh, S. F. 1988. Triterpenes in Ganoderma lucidum. Phytochemistry 27:
873–875.
Smania, A., Monache, F. D., Smania, E. F. A., Gil, M. L., Benchetrit, L. C., and Cruz, F. S. 1995.
Antibacterial activity of a substance produced by the fungus Pycnoporus sanguineus (Fr.) Murr.
J Ethnopharmacol 45: 177–181.
Smith, J. E., Rowan, N. J., and Sullivan, R. 2002. Medicinal Mushrooms for Cancer. Cancer Research UK,
London, U.K.
Turkoglu, A., Duru, M. E., Mercan, N., Kivrak, I., and Gezer, K. 2007. Antioxidant and antimicrobial
activities of Laetiporus sulphureus (Bull.) Murrill. Food Chem 101: 267–273.
Ulziijargal, E., Yang, J. H., Lin, L. Y., Chen, C. P., and Mau, J. L. 2013. Quality of bread supplemented
with mushroom mycelia. Food Chem 138(1): 70–76.
Weigand-Heller, A. J., Kris-Etherton, P. M., and Beelman, R. B. 2012. The bioavailability of ergothio-
neine from mushrooms (Agaricus bisporus) and the acute effects on antioxidant capacity and
biomarkers of inflammation. Prev Med 54(Suppl): S75–S78.
Zheng, H., Maoqing, Y., Liqiu, X., Wenjuan, T., Lian, L., and Guolin, Z. 2006. Purification and char-
acterization of an antibacterial protein from the cultured mycelia of Cordyceps sinensis. Wuhan
Univ J Nat Sci 11: 709–714.
chapter fourteen
Aspergillosis and its resistance

Marine natural products as future treatment
Kumar Saurav, Subhasish Saha, Manoj Singh, Soumik Sarkar,
Dharumadurai Dhanasekaran, and K. Kannabiran
Contents
14.1 Introduction......................................................................................................................... 255
14.1.1 Aspergilloma........................................................................................................... 257
14.1.2 Chronic necrotizing aspergillosis (CNA)............................................................ 257
14.1.3 Invasive pulmonary aspergillosis (IPA).............................................................. 257
14.1.4 Allergic bronchopulmonary aspergillosis.......................................................... 257
14.2 Drug resistance................................................................................................................... 258
14.2.1 Resistance to amphotericin B................................................................................ 258
14.2.2 Resistance to azoles................................................................................................ 259
14.2.3 Resistance to echinocandins................................................................................. 260
14.2.4 Resistance to allylamine........................................................................................ 261
14.3 Antifungal therapy............................................................................................................. 261
14.3.1 Current antifungal agents..................................................................................... 261
14.3.2 Antifungal agents under clinical trial................................................................. 263
14.3.3 Natural products as antifungal agents................................................................ 264
14.3.4 Microbial natural products as a source of antifungals..................................... 265
14.3.5 Marine microbial natural products as a source of antifungals........................ 267
14.4 Antifungal compounds from marine actinomycetes.................................................... 271
References...................................................................................................................................... 272
14.1 Introduction
Over the past two decades, fungal infections have increased significantly and have been
associated with increased morbidity and mortality. As advances in medical care have
improved the survival of patients with severe and life-threatening illnesses, the more
aggressive nature of such care has led to a rapid increase in the number of immunosup-
pressed populations. These changes have been correlated with a substantial increase in
the rate of invasive fungal infections, mainly resulting from the rapid increase in the num-
ber of at-risk patients. Despite the remarkable progress in antifungal drug research in
the past decade, difficulty in prompt treatment along with the complexity of the clinical
characteristics of at-risk patients continues to make it a great challenge for the health-
care workers and researchers. The most commonly recognized causes of o pportunistic
invasive fungal infections traditionally are Candida albicans, Cryptococcus neoformans,
255
and Aspergillus fumigatus. Along with the widespread use of antifungal prophylaxis, the

epidemiology of infection has shifted toward nonalbicans Candida, nonfumigatus Aspergillus,
opportunistic yeastlike fungi (e.g., Trichosporon and Rhodotorula spp.), Zygomycetes, and
hyaline molds (e.g., Fusarium and Scedosporium spp.). These new and emerging fungi are
characterized, and they exhibit greater resistance to standard antifungal drugs. Invasive
fungal infections due to these previously rare fungi are also more difficult to diagnose and
are associated with even higher mortality rates.
Aspergillus is a ubiquitous soil-dwelling organism found in organic debris, dust, com-
post, foods, spices, and rotted plants. The genus Aspergillus includes over 185 species.
Around 20 species have so far been reported as causative agents of o pportunistic infec-
tions in humans. Among these, Aspergillus fumigatus is the most commonly isolated spe-
cies, followed by Aspergillus flavus and Aspergillus niger. Aspergillus oryzae, Aspergillus
terreus, Aspergillus ustus, and Aspergillus versicolor are among the other s pecies less com-
monly isolated as opportunistic pathogens (Rinaldi, 1983). Aspergillus spp. are well known
to play a role in three different clinical settings in humans: (1) opportunistic infections, (2)
allergic states, and (3) toxicoses. Immunosuppression is the major factor predisposing to
development of opportunistic infections (Ho and Yuen, 2000). Aspergillus, like other fila-
mentous fungi, is primarily acquired from an inanimate reservoir, usually by the inhala-
tion of airborne spores. The organism grows best at 37°C, and the small spores (2–3 µm)
are easily inhaled and deposited deep in the lungs, leading to a variety of clinical syn-
dromes (Figure 14.1). These infections may present in a wide spectrum, varying from local
involvement to dissemination and as a whole called aspergillosis. Among all filamentous
fungi, Aspergillus is in general the most commonly isolated one in invasive infections.
It is the second most commonly recovered fungus in opportunistic mycoses following
Candida. Although these are distinct pulmonary entities, on rare occasions, one condition
may change to another; for example, an aspergilloma may change to invasive pulmonary
aspergillosis (IPA) (Tomee et al., 1995).
Inhalation of Aspergillus spores
Colonization
Cavitary lung Chronic lung disease or ICH Asthma

Normal host
disease mild ICH
No sequel Aspergilloma Chronic necrotizing IPA ABPA

aspergillosis
Figure 14.1 The clinical spectrum of conditions associated with inhalation of Aspergillus spores.
ICH, immunocompromised host; IPA, invasive pulmonary aspergillosis; ABPA, allergic broncho-
pulmonary aspergillosis.
Chapter fourteen: Aspergillosis and its resistance 257
14.1.1 Aspergilloma
This is the most common and best-recognized form of pulmonary involvement due to
Aspergillus. The aspergilloma (fungal ball) consists of masses of fungal mycelia, inflam-
matory cells, fibrin, mucus, and tissue debris, usually developing in a preformed lung
cavity. Although other fungi such as Zygomycetes and Fusarium may cause the formation
of a fungal ball, Aspergillus sp., specifically A. fumigatus, are by far the most common etio-
logic agents.
14.1.2 Chronic necrotizing aspergillosis (CNA)

Also called semi-invasive aspergillosis, this entity was first described in two reports in
1981 and 1982 (Gefter et al., 1981; Binder et al., 1982). Chronic necrotizing aspergillosis
(CNA) is an indolent, destructive process of the lung due to the invasion by Aspergillus spe-
cies (usually A. fumigatus). This entity is different from aspergilloma in that there is local
invasion of the lung tissue, and a preexisting cavity is not needed, although a cavity with
a fungal ball may develop in the lung as a secondary phenomenon due to destruction by
the fungus.
14.1.3 Invasive pulmonary aspergillosis (IPA)

The vast majority of IPA cases are seen in immunocompromised patients (Hibberd and
Rubin, 1994). Neutropenia is the most important risk factor, and it is estimated that IPA
accounts for 7.5% of all infections in neutropenic patients following induction therapy
for acute myelogenous leukemia. The risk of IPA increases with the duration of neutro-
penia (i.e., neutrophil count, <500 cells/µL) and is estimated to be 1% per day for the first
3 weeks, after which time it increases to 4% per day. Transplantation, especially lung
and bone marrow transplantation (BMT), is another increasingly significant risk factor
for IPA (Soubani et al., 1996). It is estimated that 5% of BMT recipients develop IPA with
mortality rates ranging between 30% and 80% (Wald et al., 1997). In BMT recipients, IPA
may be seen in the first few weeks after the procedure with delayed engraftment or graft
failure and more commonly in the setting of treatment with corticosteroids for graft-
versus-host disease.
14.1.4 Allergic bronchopulmonary aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity reaction to
Aspergillus antigens, mostly due to A. fumigatus. It is typically seen in patients with
long-standing asthma or cystic fibrosis, and it is estimated that 7%–14% of corticosteroid-
dependent asthma patients and 6% of patients with cystic fibrosis meet the diagnostic
criteria for ABPA (Basich et al., 1981; Mroueh and Spock, 1994). The factors leading to
ABPA are not clearly understood. It is believed that Aspergillus-specific, IgE-mediated
type I hypersensitivity reactions and Aspergillus-specific, IgG-mediated type III hyper-
sensitivity reactions play a central role in the pathogenesis of ABPA.
14.2 Drug resistance

Empirical antifungal therapy using parenteral amphotericin B deoxycholate (AMB),
fi rst-generation azoles (e.g., ketoconazole), and 5-fluocytosine (5-FC) were the only
available therapeutic options for fungal infections. However, AMB has significant
dose-limiting nephrotoxicity and infusion-related toxicity, while the first-generation
azoles and 5-FC have a relatively limited therapeutic spectrum and potency. Thus, the
introduction of potent, broad-spectrum oral and parenteral second-generation triazoles
such as fluconazole and itraconazole since the early 1990s has been a welcomed addi-
tion to our antifungal armamentarium. Fluconazole, a fungistatic triazole, has been
shown to be clinically effective against yeasts, dermatophytes, and dimorphic fungi
(Galgiani, 1990; Grant and Clissold, 1990), while itraconazole, which has a broader spec-
trum of antifungal activity, is effective against moulds, including Aspergillus species
and various pheohyphomycetes as well as endemic fungi (Boogaerts and Maertens,
2001). Both have limitations, such as a relatively narrow antifungal spectrum, pro-
pensity for selection of resistant strains (fluconazole) (Kontoyiannis and Bodey, 2002),
erratic bioavailability of the capsule form, adverse gastrointestinal side effects to the
oral solution form (itraconazole), and many other clinically important drug–drug
interactions (itraconazole).
The development of lipid formulations of AMB that lack much of the parent
compound’s nephrotoxicity, new extended spectrum third-generation triazoles and the
echinocandins, and a new class of cell wall acting parenteral antifungals are some of
the major recent advancements in antifungal therapy (Kontoyiannis, 2001). Three lipid
formulations of AMB are commercially available in the United States and most of the
western European countries: liposomal AMB (L-AMB), AMB lipid complex (ABLC), and
AMB colloidal dispersion (ABCD). Although these lipid products represent attractive
alternatives to delivery of AMB from a toxicity standpoint, their use should be limited
to patients who cannot tolerate, or whose infection does not respond to, AMB, because
they are all substantially more expensive than the parent drug, with L-AMB being the
most expensive.
14.2.1 Resistance to amphotericin B

The mode of action and the known mechanisms of resistance of A. fumigatus against
current antifungal agents are summarized in Table 14.1. It is suggested that the mecha-
nisms of action of amphotericin are mainly by promotion of oxidative damage of cell
membranes through the generation of reactive oxygen species (Sterling and Merz, 1998;
Moore et al., 2000). Clinical resistance of IA to AMB-based therapy is observed fre-
quently in clinical practice (Kontoyiannis and Bodey, 2002; Patterson, 2002). Typically,
such infections with unfavorable outcome occur in severely immunocompromised
patients with multiple underlying host factors. Several preclinical studies have docu-
mented the resistance of A. terreus to AMB (Walsh et al., 2003; Lionakis et al., 2005).
Although the mechanisms of resistance of A. terreus to AMB have not been elucidated,
it appears that this fungus has much less ergosterol (the target molecule of AMB action)
in its fungal membrane than the more AMB-susceptible A. fumigates (Walsh et al., 2003).
It is true that both primary and secondary in vitro resistance of A. fumigatus to AMB
have been reported (Verweij et al., 1998; Manavathu et al., 2000), but such resistance
is generally considered a rare phenomenon (Moosa et al., 2002; Paterson et al., 2003;
Dannaoui et al., 2004).
Table 14.1 Mode of action and the known mechanisms of resistance of A. fumigatus
against current antifungal agents
Antifungal agent Mechanisms of action Mechanisms of resistance
Polyenes Fungicidal Decreased access of AMB to drug
Amphotericin B Interaction with ergosterol, target in fungal membrane
Lipid formulations intercalation of fungal membrane (a) Altered membrane ergosterol
Liposomal nystatin that leads to increased content and reduced intercalation
Azoles permeability to univalent and (b) Increased cell wall rigidity
Itraconazole divalent cations and cell death (c) Sequestration of fungi to
Voriconazole Alternative mechanism of action lysosomes
Posaconazole through oxidation of fungal Decreased oxidative damage
Echinocandins membrane (a) Overexpression of catalases and
Caspofungin Inhibition of P-450 14-a-DM superoxide dismutases of
Micafungin (ERG 11), accumulation of A. fumigatus
Anidulafungin lanosterol leading to perturbation (b) Anaerobic environment
Allylamines of fungal cell membrane Increased drug efflux
Terbinafine Inhibition of cell wall glucan Overexpression of target enzyme
synthesis, leading to susceptibility Decreased affinity to the binding
of the fungal cell to osmotic lysis site
Fungistatic Upregulation of homeostatic
Inhibition of squalene epoxidase stress response pathways (HSP 90;
(Erg 1), with subsequent calcineurin)
ergosterol depletion and Altered drug uptake
accumulation of toxic sterol Exogenous cholesterol import
intermediates (rescue ergosterol depletion)
Upregulation of homeostatic stress
response pathways (HSP 90;
calcineurin)
Overexpression of target site
Upregulation of genes encoding
for β-glucan synthetase
Overexpression of genes related to
transport of cell wall components
Increased drug efflux
Overexpression of target site
(ERG1)
Overexpression of salicylate
monooxygenase (drug
degradation)
Source: Chamilos, G. and Kontoyiannis, D.P., Drug Resist. Update, 8(6), 344, 2005.
Manavathu et al. (2005) have reported recently on in vivo/in vitro correlation for
AMB in a mouse model of A. fumigatus infection, but their results were from tests on a
laboratory-generated AMB-resistant A. fumigatus isolate. Other investigators were unable
to correlate the in vitro susceptibility of A. fumigatus to AMB with the outcome in animal
model studies (Johnson et al., 2000).
14.2.2 Resistance to azoles

The triazoles are antifungal agents that act by blocking the ergosterol biosynthetic
pathway at the C-14 demethylation stage. Triazoles bind to lanosterol 14-α demethylase
(14α-DM, or Cyp51), a cytochromes P450 enzyme that is encoded by the ERG 11 gene
(Lamb et al., 1999; Odds et al., 2003). This leads to ergosterol depletion and accumulation
of lanosterol and other toxic 14-α methylated sterols.
Cowen and Lindquist (2005) showed a novel molecular mechanism of resistance of
Aspergillus and other opportunistic fungi that involves HSP90, a molecular chaperone with
an essential role in folding, transport, and maturation of key regulatory proteins under
stress-induced conditions. Using S. cerevisiae mutants that expressed high or low levels of
HSP90, they demonstrated that HSP90 is required both for the emergence of drug resistance
to azoles and echinocandins and for the continued drug resistance once it has occurred.
Furthermore, the investigators found that HSP90 function in drug resistance is mediated
by the calcineurin pathway, which is known to be implicated in virulence of several patho-
genic fungi and also enables fungal cells to tolerate drugs that block ergosterol biosynthesis.
Liu et al. (2003) have recently reported that cross-resistance or tolerance mechanisms
between different classes of azoles in A. fumigatus strains sequentially exposed to azoles.
Serial passages of 10 A. fumigatus spp. in plates containing FLU, an azole with minimal
activity against Aspergillus spp., resulted in attenuation of in vivo fungicidal activity of ITC
and VRC against all isolates tested. The degree of azole cross-resistance is azole specific,
and its pattern might reflect the similarities in molecular structures among different tri-
azoles (Xiao et al., 2004) (Figure 14.2). Similarly, in a large surveillance study in Sweden that
included 400 clinical and 150 environmental A. fumigatus isolates, there was no evidence
of cross-resistance between ITC and VRC in 10 (2.5%) clinical and 36 (24%) environmental
isolates that displayed high ITC MICs (≥2 µg/mL). In a recent study of 596 clinical and
environmental isolates of A. fumigatus, although there was no evidence of azole resistance,
there was a trend toward extended loss of susceptibility across VRC and ITC, POS and
ITC, and VRC and POS (Guinea et al., 2005). Broad-spectrum cross-resistance among all
the azoles has been shown in A. fumigatus causing IA in a patient who was on prolonged
ITC secondary prophylaxis (Warris et al., 2002).
Balajee et al. (2004) have identified 10 variants of multidrug-resistant A. fumigatus clini-
cal isolates, all of which had an unusual sporulation pattern and a unique mitochondrial
cytochrome b sequence. These isolates exhibited increased MICs not only against all the
triazoles tested but also against AMB.
14.2.3 Resistance to echinocandins

Echinocandins are a unique class of antifungals that target the fungal cell wall by
noncompetitive inhibition of 1,3-β-d-glucan synthetase, a fungus-specific enzyme target.
Cross-resistance between echinocandins and the other classes of antifungals has not
been described thus far. Warn et al. (2003) have shown a murine model of disseminated
KET ITR
AMB
FLU
Figure 14.2 Drug-resistant pattern of Aspergillus isolate to various standard drugs by disc diffusion
assay. (From Kumar, S. and Kannabiran, K., J. Med. Mycol., 20(2), 101, 2010.)
a spergillosis; the echinocandin micafungin has significant activity against both an ITC-
resistant A. fumigatus isolate and an AMB-resistant A. terreus isolate.
14.2.4 Resistance to allylamine

Terbinafine is an oral allylamine that blocks ergosterol biosynthesis by inhibiting a
membrane-bound squalene epoxidase encoded by the ERG1 gene (Ryder, 1992). Inhibition of
squalene epoxidase leads to ergosterol deficiency along with accumulation of the toxic squa-
lene, which probably accounts for the in vivo reported fungicidal activity of TRB. Although
TRB is indicated exclusively for skin and nail infections, it is suggested that it might have a
role in the treatment of IA, alone or in combination with triazoles (Schiraldi et al., 1996).
The decreased sensitivity of Aspergillus sp. against available drugs and lack of its
effectiveness to combat such pathogens continued to be the major challenges for health-
care-providers. Hence, there is an urgent need to search for novel bioactive compounds
from natural sources with having a broad spectrum of activity with less side effects against
drug-resistant Aspergillus strains for effective control and management of aspergillosis.
14.3 Antifungal therapy

Aspergillus sp. are commonly found in soil, water, and decaying material all over the
world. Unlike invasive candidiasis, invasive aspergillosis (IA) occurs predominantly in
highly immunocompromised patients (Baddley et al., 2001; Marr et al., 2002). Aspergillus
fumigatus is the most common causative species of IA, but nonfumigatus Aspergillus
such as Aspergillus niger, Aspergillus flavus, and Aspergillus terreus have been increasingly
reported (Baddley et al., 2001; Marr et al., 2002). IA is an emerging condition among
patients with other causes of immunosuppression, such as organ transplantation, in
patients with advanced acquired immunodeficiency syndrome (AIDS), and patients
treated with newer immunosuppressive agents such as infliximab (Patterson, 2005;
Pfaller et al., 2006). The underlying disease condition greatly determines the level of
risk for patients to become infected by Aspergillus species (Table 14.2). However, the
frequency of cases observed by individual physicians may vary greatly for different
patient groups, depending on the risk period. For some patients, this is lifelong, such as
patients with acquired immunodeficiency syndrome (AIDS) or chronic granulomatous
disease, whereas in other patient groups, such as those treated for acute myeloid leuke-
mia, the risk period is the highest.
Since 1970, a number of autopsy studies have been published that indicate that the
epidemiology of invasive fungal infections in immunocompromised patients is chang-
ing. Two decades ago, autopsy studies found Candida to be the predominant pathogen.
However, studies of autopsy cases from Europe, the United States, and Japan show that the
number of patients dying from invasive aspergillosis has increased significantly over the
past two decades.
14.3.1 Current antifungal agents

The antifungal agents currently available for the treatment of systemic fungal infections
are amphotericin B, lipid formulations of amphotericin B, 5-fluorocytosine, and the azoles,
miconazole, ketoconazole, fluconazole, and itraconazole. Currently, the criteria used for
the selection of optimal new drug candidates include inhibitors of fungal cell wall bio-
synthesis, potency comparable to amphotericin B, safety comparable to fluconazole, fungi-
cidal activity in vitro, and fungicidal activity in vivo.
Table 14.2 Spectrum of patient groups or conditions with increased risk of invasive
Aspergillus infection categorized by infection risk
High (>10%) Moderate (1%–10%) Low (<1%) Negligible
Chronic AIDS patients Systemic lupus Normal healthy
granulomatous Liver, heart, or pancreas erythematosus on persons
disease transplant recipients prednisolone Hospitalized
Lung transplant Acute myeloid leukemia Diabetes mellitus patients without
recipients Allogeneic BMT Alcoholism neutropenia or
Acute myeloid recipients without Corticosteroid treatment with
leukemia GVHD of grade I treatment corticosteroids or
Allogeneic BMT Autologous BMT Patients treated for other risk
recipients with recipients, intensive solid tumors conditions
GVHD > grade II care patients on Intensive care patients
steroids, severe Agammaglobulinemia
combined Kidney transplant
immunodeficiency recipients
syndrome Influenza
Lymphoma Surgery in
Major burn (e.g., >30%) contaminated
operating room air
Major trauma
Source: Shao, P.L. et al., Int. J. Antimicrob. Ag., 30(6), 487, 2007.
BMT, bone marrow transplantation; GVHD, graft-versus-host disease.
Amphotericin B (AMB) is a polyene antifungal agent first isolated from Streptomyces

nodosus in 1955. AMB is an elongated cyclic molecule consisting of hydrophilic polyhy-
droxyl and hydrophobic polyene domains (Figure 14.3).
Amphotericin B complexed with ergosterol disrupts the fungal plasma membrane,
which leads to increased membrane permeability, leakage of the cytoplasmic contents,
and finally death to the fungal cell. Liposomal preparations of amphotericin B have
been used to reduce the nephrotoxicity of conventional amphotericin B. The polyenes
are fungicidal and have the broadest spectrum of antifungal activity among others
(Hiemenz and Walsh, 1996; Andriole, 1999). They have excellent in vitro activity against
Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsu-
latum, Paracoccidioides brasiliensis, Sporotrichum species, and Torulopsis (Candida) glabrata.
It also has excellent activity against Candida albicans and most other Candida species
OH
H 3C OH
O OH
HO O OH OH OH OH O O
CH3
H OH
H3C
H3C O
O
HO
H
H2N OH
Figure 14.3 Chemical structure of amphotericin B. (From National Library of Medicine ChemIDPlus.)
CI CI
N O O
N H
N
N O
(a) CH3
CI CI
N
N
N O O
O
N
N
N
N
N CH3
O
(b) H3C
Figure 14.4 Chemical structure of (a) ketoconazole and (b) itraconazole.
except for Candida lusitaniae. It has variable activity against Aspergillus species and
Zygomycetes (Mucor) species, whereas Fusarium, Trichosporon species, and Pseudallescheria
boydii are often resistant (Andriole, 1999).
Initially different azole compounds, imidazoles, clotrimazole, miconazole, and keto-
conazole (Figure 14.4), followed by the triazoles, fluconazole, and itraconazole inhibit
fungal cytochrome P450 3A-dependent C 14-demethylase, which is responsible for the
conversion of lanosterol to ergosterol, thereby depleting ergosterol in the fungal cell mem-
brane (Andriole, 1999). Azole antifungal activity varies with each compound, and clini-
cal efficacy may not coincide with in vitro activity. Azoles are active against C. albicans,
C. neoformans, C. immitis, H. capsulatum, B. dermatitidis, P. brasiliensis, and C. glabrata, and some
Aspergillus spp., Fusarium spp., and Zygomycetes are resistant to currently available azoles
(Groll et al., 1998).
14.3.2 Antifungal agents under clinical trial

Isavuconazole is a new triazole currently undergoing phase III clinical trials. This compound
has shown in vitro activity against a large number of clinically important yeasts and moulds
including Aspergillus spp., Fusarium spp., Scedosporium spp., Candida spp., the Zygomycetes, and
Cryptococcus spp. Similar to voriconazole, reduced in vitro activity is seen against Histoplasma
capsulatum. In vivo efficacy has been demonstrated in murine models of invasive aspergillosis
and candidiasis. Additionally, there are several potential pharmacokinetic and drug–drug
interaction advantages of this compound over existing antifungal agents.
OH
H 3C HO O
OH
H HN
OH NH H
H 3C N
O
H HN O HN O
H
O O CH3
OH O
H
N OH O
N
H H H
OH OH
HO
H3C
Figure 14.5 Chemical structure of anidulafungin. (From National Library of Medicine ChemIDPlus.)
Posaconazole works by disrupting the close packing of acyl chains of phospholipids,

impairing the functions of certain membrane-bound enzyme systems such as ATPase
and enzymes of the electron transport system and thus inhibiting growth of the fungi.
It does this by blocking the synthesis of ergosterol by inhibiting the enzyme lanosterol
14-α-demethylase and accumulation of methylated sterol precursors. Posaconazole is sig-
nificantly more potent at inhibiting 14-α demethylase than itraconazole. Recent advance-
ment in the clinical effectiveness of posaconazole prophylaxis was made in a trial for
patients with acute myelogenous leukemia (AML), where no significant differences were
observed for persistent fever, pneumonia, lung infiltrates indicative of invasive pulmonary
aspergillosis, or attributable and overall mortality (Vehreschild et al., 2010).
There are two echinocandins, anidulafungin (Figure 14.5) and aminocandin, currently
undergoing clinical evaluation. Anidulafungin was developed by Eli Lilly and licensed to
Vicuron Pharmaceuticals in May 1999 and is a semisynthetic derivative of echinocandin B,
a fungal metabolite originally isolated from Aspergillus rugulovalvus (formerly Aspergillus
rugulosus). Vicuron has completed a phase III trial of anidulafungin for the treatment of
esophageal candidiasis, and phase III trials are in progress for the treatment of invasive
aspergillosis and candidiasis/candidemia. Recently, phase IV trial of the drug to evaluate a
short course of anidulafungin, followed optionally by oral voriconazole, for the treatment
of candidemia and invasive candidiasis has completed in October 2009 (USA clinicaltrials.
gov, National Institute of Health).
Indevus licensed the echinocandin, aminocandin (HMR- 3270), from Novexel, France
(Originally Sanofi-Aventis antiinfective group) in April 2003 and initiated phase I clini-
cal trials against systemic fungal infections in February 2004. Although the structure of
aminocandin is not yet in the public domain, it is known to be a semisynthetic derivative
of deoxymulundocandin, a natural product originally isolated from the fungus Aspergillus
sydowii by Hoechst India in 1992.
14.3.3 Natural products as antifungal agents

Pharmacological screening and usage of natural products for the treatment of human
diseases have had a long history from Ayurvedic medicine to modern drugs (Swerdlow,
2000). The majority of modern drugs are reported to be mostly from natural products
(Newman et al., 2003). In the past few decades, a worldwide increase in the incidence of
fungal infections has been observed as well as a rise in the resistance of some species
of fungus to different fungicidals used in medicinal practice. The majority of clinically
used antifungals have various drawbacks in terms of toxicity, efficacy, and cost, and their
frequent use has led to the emergence of resistant strains. Hence, there is a great demand
for novel antifungals belonging to a wide range of structural classes, selectively acting on
new targets with fewer side effects. One approach might be the testing of natural prod-
ucts for their antifungal activities as potential sources for drug development. As reported
by Butler (2005), about 70 natural products or natural product derivatives are currently
undergoing clinical trials in different parts of the world including United States, Europe,
Japan, and Korea, out of which 30 were derived from microorganism and 12 were marine
derived.
14.3.4 Microbial natural products as a source of antifungals

Microbial natural products are the important source of both existing and new drugs.
The exploration of microorganisms as a source of therapeutically useful compounds
is one of the recent developments and less well-known history than the use of plants
and plant extracts in human medicine. Secondary metabolites are defined as naturally
produced substances that do not play an explicit role in the organisms that produce
them. These microorganisms may have evolved the ability to produce such compounds
because of the selection advantages conferred upon them as a result of the interactions
of the compounds with specific receptors in other organisms (Demain, 1983; Omura
1986). One of the targets for novel antifungals under active investigation is the fun-
gal cell wall. Antifungal agents acting on this target are inherently selective. Fungal
cell wall composition varies among species, but it generally has three polymeric com-
ponents: glucan, chitin, and mannoproteins. Inhibitors of glucan synthesis have been
shown to possess antifungal activity in vitro as well as in vivo in many different animal
models. Some of the microbial natural products that are inhibitors of glucan synthesis
are listed in Table 14.3.
The classical inhibitors of chitin synthesis are nikkomycins and polyoxins. The
substrate analogues of UDP-N-acetylglucosamine (building block for chitin biosynthe-
sis) were isolated from two different Streptomyces species: S. tendae (nikkomycin) and
S. cacaoi var. asoensis (polyoxin) (Cabib, 1991). Nikkomycins exhibit activity against
dimorphic fungi but low activity against yeast and filamentous fungi. Nikkomycins
and polyoxins are currently used exclusively as agricultural fungicides, due to their
modest activity against human pathogens (Cohen, 1993). Recently, two novel antifun-
gal compounds were found: phellinsin A and arthrichtin. Phellinsin A is a phenolic
compound exhibiting antifungal activity against human pathogens such as Trichophyton
mentagrophytes and A. fumigatus and very weak activity against other human pathogens
such as C. neoformans and Coccidioides immitis. Arthrichitin is a cyclic depsipeptide iso-
lated from Arthrinium phaeospermum and from the marine fungus Hypoxylon oceanicum
(Vijayakumar et al., 1996) with moderate activity against Candida spp., Trichophyton spp.,
and several other phytopathogens.
Mannoproteins are the third main component of the fungal cell wall. They form the
outer layer of the cell wall and contain as much as 50% carbohydrate. The majority of
the cell wall mannoproteins are anchored by β-(1,6)- and β-(1,3)-glucan and play several
important functions of fungal membrane. Inhibitors of the mannoproteins function are the
Table 14.3 Microbial natural product inhibitors of glucan synthesis

Compounds Producing species
Lipopeptides
Echinocandin B Aspergillus nidulans
Aculeacin Aspergillus rugulosus
Mulundocandin Aspergillus aculeatus
Sporiofungins Aspergillus sydowii
Pneumocandins Penicillium arenicola
Cryptocandin Cryptosporiopsis sp.
WFI 1899 and related sulfate derivatives Glarea lozoyensis
FR901469 Pezicula sp.
Arborcandins Cryptosporiopsis sp.
Clavariopsins Cryptosporiopsis quercina
Glycolipids Coleophoma empetri
Papulacandins Coleophoma crateriformis
Corynecandin Tolypocladium parasiticum
Mer-WF 3010 Chalaria sp.
Fusacandin Unidentified fungus
BU-4794F Unidentified fungus
L-687781 Clavariopsis aquatic
Acidic terpenoids Papularia sphaerosperma
Enfumafungin Coryneum modonium
Arundifungin Phialophora cyclaminis
Ascoteroside Fusarium sambucinum
Ergokonin A Gilmaniella sp.
Dictyochaeta simplex
Hormonema sp.
Arthrinium arundinis
A. phaeospermum
Leotiales anamorphs
Coelomycete undetermined
Ascotricha amphitricha
Mycoleptodiscus
atromaculans
Trichoderma
longibrachiatum
T. koningii
T. viride
Source: Vicente, M.F. et al., Clin. Microbiol. Infect., 9(1), 15, 2003.
pradimicin/benanomycin family, whose chemical structure possesses a benzo [a] naph-

thacenequinone skeleton (Fromtling, 1998). The free carboxyl group of these compounds
interacts with the saccharide portion of cell surface mannoprotein, which is followed by
disruption of the plasma membrane and leakage of intracellular potassium. These anti-
fungal (and antiviral) agents were produced by Actinomadura sp. These antibiotics exhib-
ited remarkable in vivo activity against systemic fungal infections caused by C. albicans,
A. fumigatus, and C. neoformans in mice.
Table 14.4 Microbial natural products as inhibitors of sphingolipid biosynthesis

and protein synthesis
Compounds Producing species
Sphingolipid biosynthesis Aspergillus fumigatus Paecilomyces variotii Streptomyces sp.
Sphingofungins Trichoderma viride
Lipoxamycin Isaria sinclairii
Viridiofungins Fusarium moniliforme
Myriocin Sporormiella australis
Fumonisin B1 Aureobasidium pullulans
Australifungin Unidentified sterile fungus
Aureobasidin A Micromonospora chalcea
Khafrefungin Streptomyces galbus Micromonospora sp.
Rustmicin Micromonospora sp.
Galbonolide B Sporomiella minimoides
Minimoidin Sordaria araneosa
Protein synthesis Zopfiella marina
Sordarin Penicillium minioluteum
Zofimarin Unidentified sterile fungus
BE31405 Xylaria sp.
SCH57404 Hypoxylon croceum
Xylarin Graphium putredinis
Hypoxysordarin
GR135402
Source: Adapted from Vicente, M.F. et al., Clin. Microbiol. Infect., 9(1), 15, 2003.
Sphingolipids, although present in relatively small proportion in the fungal cytoplas-

mic membrane, are essential for cellular functions (Wells and Lester, 1983), and inhibition
of sphingolipid synthesis results in growth inhibition and cell death (Zweerink et al., 1992;
Mandala et al., 1995). A list of few compounds, which are inhibitors of sphingolipid bio-
synthesis and protein synthesis, is provided in Table 14.4.
14.3.5 Marine microbial natural products as a source of antifungals

Microorganisms from extreme environments have gained considerable attention in recent
years because of their diversity and biological activities, mainly due to their ability to pro-
duce novel chemical compounds of high commercial value. Microbial sources serve as a
template for the isolation of many bioactive anticancer compounds. Several earlier studies
reported on the antifungal activity of several novel marine natural products isolated from
marine algae, fungi, bacteria, sponges, and sea stars (Mayer and Lehmann, 2000; Mayer
and Hamann, 2002, 2005; Mayer et al., 2007). A list of antifungal compounds isolated from
marine microorganisms is provided in Table 14.5.
Six new bengazole derivatives and a new bengamide L were reported from the sponge
Pachastrissa sp. (Fernández et al., 1999). Although no studies on mechanism of action of
the compound, the bengazole derivatives were observed to be active against Candida albi-
cans with a minimum inhibitory concentration (MIC) of 0.8–1.5 µg/mL. Oceanapiside, a
Table 14.5 List of antifungal compounds isolated from marine microorganisms

Compounds Organism Pharmacological activity
Bengazole, bengamide Sponges C. albicans inhibition
Oceanapiside Sponges C. glabrata inhibition
Spongistatin I Sponges A. nidulans inhibition
Tanikolide Bacterium C. albicans inhibition
Theopederins F–J Sponges S. cerevisiae inhibition
Basiliskamides A and B Bacterium C. albicans and A. fumigatus inhibition
Corticatic acids A and E Sponges C. albicans and A. fumigatus inhibition
Swinhoeiamide A Sponges C. albicans and A. fumigatus inhibition
Patagonicoside A Sea cucumber Cladosporium cucumerinum inhibition
Oxybis methyl phenol Fungus C. albicans, T. rubrum, and A. niger
inhibition
Polyester 15G256h Fungus Cell wall biosynthesis inhibition
Astroscleridae sterol Sponges S. cerevisiae inhibition
Dysidea arenaria sterol Sponges Fluconazole resistance reversal in
C. albicans
Massadine Sponges Geranylgeranyltransferase I inhibition
Naamine G Sponges C. herbarum inhibition
Utenospongin B Sponges C. tropicales and F. oxysporum inhibition
(2S,3R)-2-Aminododecan-3-ol Ascidian C. albicans inhibition
Capisterones A and B Algae Enhancement of fluconazole activity
Dysidea herbacea phenol Sponges C. albicans and A. niger inhibition
Spongistatin Sponges Broad panel of yeasts and filamentous
fungi
Halocidin Ascidian C. albicans inhibition
Hassallidin A Bacterium C. albicans and A. fumigates inhibition
Latrunculins Sponges C. albicans inhibition comparable to
clotrimazole
Majusculoic acid Bacterium C. albicans inhibition, less potent than
fluconazole
Sources: Mayer, A.M. and Lehmann, V.K., Pharmacologist, 42, 62, 2000; Mayer, A.M. and Hamann, M.T., Comp.
Biochem. Physiol., 132(3), 315, 2002.
new glycosidicyamino alcohol lipid from the sponge Oceanapia philippensis, demonstrated
antifungal activity against the fluconazole-resistant yeast Candida glabrata (MIC = 10 µg/mL)
(Nicholas et al., 1999). This is the most noteworthy study with a marine-derived antifungal
compound having broad-spectrum activity against pathogens. Ovechkina et al. (1999)
demonstrated potent microtubule-severing activity with spongistatin 1, a macrocyclic
lactone isolated from the sponge Hyrtios erecta. Tsukamoto et al. (1999) isolated five new
bioactive metabolites, theopederins F–J, from the sponge Theonella swinhoei.
Laboratory cultures of the marine bacterium Bacillus laterosporus produced the novel
polyketides basiliskamides A and B (Figure 14.6) (Barsby et al., 2002). Both compounds
showed potent activity against Candida albicans (MIC = 1.0 and 3.1 µg/mL, respectively)
and Aspergillus fumigatus (MIC = 2.5 and 5.0 µg/mL, respectively), which was comparable
to amphotericin B.
O
O 13 14
H2N
7 9 11
H2N 1
3 5 O OH
20
OH O 16 18 O
(a) O (b)
Figure 14.6 Chemical structure of novel polyketides basiliskamides A (a) and basiliskamides B

(b) isolated from marine bacterium Bacillus laterosporus. (From National Library of Medicine
ChemIDPlus.)
O
O OH
OH
OH
OH
(a) (b)
Figure 14.7 Two novel polyacetylenic acids, corticatic acids A (a) and corticatic acids E (b) isolated
from the marine sponge Petrosia corticata. (Source: National Library of Medicine ChemIDPlus.)
O
HO
P
HO O
O NH2 O
OCH3
O
OH OH OCH3
Figure 14.8 Chemical structure of a novel antifungal calyculin derivative swinhoeiamide A, isolated
from the marine sponge Theonella swinhoei. (Source: National Library of Medicine ChemIDPlus.)
Two novel polyacetylenic acids, corticatic acids A and E (Figure 14.7) isolated from the
marine sponge Petrosia corticata (Nishimura et al., 2002), were shown to inhibit geranyl-
geranyltransferase type I (GGTase I), an enzyme involved in fungal cell wall biosynthesis.
Interestingly, while corticatic acids A and E inhibited C. albicans with IC50 values of 3.3 and
7.3 µM, the fact that there is little sequence identity between human and Candida GGTase I
suggested that these marine compounds may become leads for novel and “selective anti-
fungal agents.”
Swinhoeiamide A (Figure 14.8), a novel antifungal calyculin derivative, was isolated
from the marine sponge Theonella swinhoei (Edrada et al., 2002). Swinhoeiamide A showed
strong antifungal activity toward C. albicans and A. fumigatus (MIC = 1.2 and 1.0 µg/mL,
respectively).
NH
H
N N OMe
MeO OH
OMe
Figure 14.9 Chemical structure of novel imidazole alkaloid, naamine G, isolated from marine
sponge Leucetta chagosensis. (From National Library of Medicine ChemIDPlus.)
Yang et al. (2003) reported a new sterol sulfate isolated from a deep water marine
sponge of the family Astroscleridae, which exhibited antifungal activity against “super-
sensitive” Saccharomyces cerevisiae (MIC = 15 μg/mL). Jacob et al. (2003) investigated the
antifungal properties of a previously described sterol isolated from the marine sponge
Dysidea arenaria. Interestingly, they observed a reversal of fluconazole resistance from 300
to 8.5 μM when combined with 3.8 μM of the Dysidea arenaria sterol, putatively as a result
of inhibition of the MDR1-type efflux pump in multidrug-resistant C. albicans.
A novel imidazole alkaloid, naamine G (Figure 14.9), was reported from the Indonesian
marine sponge Leucetta chagosensis that exhibited strong antifungal activity against the
phytopathogenic fungus Cladosporium herbarum (Hassan et al., 2004).
It remains to be determined if this compound will also be effective against fungi that
infect mammalian hosts. Rifai et al. (2004) reported that untenospongin B (Figure 14.10a),
isolated from the Moroccan marine sponge Hippospongia communis, was more potent than
amphotericin B, a clinically used antifungal agent, against Candida tropicalis (MIC = 4–8 μg/
mL) and Fusarium oxysporum (MIC = 2–4 μg/mL). Kossuga et al. (2004) reported a new anti-
fungal agent polyketide, (2S,3R)-2-aminododecan-3-ol (Figure 14.10b), isolated from the
Brazilian ascidian Clavelina oblonga, which was very active against C. albicans (MIC = 0.7 ±
0.05 μg/mL). Although the mechanism of action of this compound remains undetermined,
its bioactivity was comparable to the clinically used antifungal agents nystatin (MIC = 1–4
μg/mL) and ketoconazole (MIC = 1–4 μg/mL).
Sionov et al. (2005) observed that a phenol compound (Figure 14.11) from the marine
sponge Dysidea herbacea had significant activity against the human fungal pathogens
C. albicans and Aspergillus fumigatus (MIC = 1.95–7.8 μg/mL), and the activity is comparable
with the clinically used antifungal amphotericin B (MIC = 1–2 μg/mL).
Pettit et al. (2005) extended the in vitro and in vivo pharmacology of the marine spon-
gistatin 1 (Figure 14.12) isolated from the marine sponge Hyrtios erecta, a previously
described anticancer agent. The macrocyclic lactone polyether was shown to be fungi-
cidal to 74 reference strains and clinical isolates (MIC = 1–32 μg/mL), including several
fungal strains resistant to the clinically used drugs flucytosine, ketoconazole, and fluco-
nazole. Furthermore, mechanism of action studies revealed that spongistatin disrupted
NH2
O OH O
OH
(a) (b)
Figure 14.10 Chemical structure of (a) untenospongin B, isolated from the Moroccan marine
sponge Hippospongia communis. (b) (2S,3R)-2-aminododecan-3-ol, isolated from the Brazilian ascid-
ian Clavelina oblonga. (From National Library of Medicine ChemIDPlus.)
OCH3 OH
Br O
Br Br
Br
Figure 14.11 Chemical structure of a phenol compound extracted from the marine sponge Dysidea
herbacea. (From National Library of Medicine ChemIDPlus.)
OH
HO
HO
O
H OH H
H O O
OMe
O HO
OH O
O H H O
OH O H
CI O
AcO
OAc
OH
Figure 14.12 Chemical structure of spongistatin 1 isolated from the marine sponge Hyrtios erecta.
(From National Library of Medicine ChemIDPlus.)
cytoplasmatic and spindle microtubules in Cryptococcus neoformans in a time- and

concentration-dependent manner, preventing nuclear migration and both nuclear and cel-
lular cell division.
14.4 Antifungal compounds from marine actinomycetes

Actinomycetes are a group of prokaryotic organisms, are gram-positive bacteria that grew
extensively in soils rich with organic matter, and are capable of producing several second-
ary metabolites (Demain and Sanchez, 2009). Majority of the studies on extremophilic
organisms, however, have been confined to the isolation and characterization of extremo-
philic bacteria, but in general, marine actinomycetes are relatively less explored for novel
bioactive secondary metabolites till date. The marine environment remains as a virtually
untapped source for novel actinomycetes. The distribution and abundance of actinomycetes
generally depend on various ecological habitats, which include beach sand and seawater.
An important reason for discovering novel secondary metabolites is to circumvent the
problem of resistant pathogens, which are no longer susceptible to the currently used drugs.
The number of deaths due to these clever pathogenic organisms is on the rise, which needs
to be controlled. Secondary metabolites from marine actinomycetes may form the basis for
the synthesis of novel therapeutic drugs, which may be efficient to combat a range of resis-
tant microbes. The exploitation of marine actinomycetes as a source for discovery of novel
secondary metabolites yielded numerous novel antifungal metabolites in the past decade.
Bonactin (Figure 14.13), an antimicrobial ester, was isolated from the liquid culture of a
Streptomyces sp. BD21–2 obtained from a shallow-water sediment sample collected at Kailua
Beach, Oahu, Hawaii, (USA) (Schumacher et al., 2003). Bonactin displays antimicrobial
activity against gram-positive and gram-negative bacteria as well as against several fungi.
O 8΄ O OH
1 3 O 6 8 O 9 11 O 14 16
HO
2΄ 10΄
Figure 14.13 Chemical structure of bonactin—an ester compound isolated from Streptomyces sp.
BD21–2. (From National Library of Medicine ChemIDPlus.)
O H O H O H
1
N N N
7΄
5 3΄ 5΄
3 O O O
HO
HO 7 HO HO
9 O O O 3
H2N H2N H2N
(a) (b) (c)
Figure 14.14 Daryamide A (a), daryamide B (b), and daryamide C (c), a polyketides isolated from
Streptomyces sp. CNQ-085.
Daryamides (Figure 14.14) are cytotoxic and antifungal polyketides isolated from cul-
ture broth of a Streptomyces strain, CNQ-085. These bioactive compounds have been shown
to exhibit moderate cytotoxicity against the human colon carcinoma cell line HCT-116 and
moderate antifungal activities against Candida albicans (Asolkar et al., 2006). Similarly,
chandrananimycins, isolated from marine Actinomadura sp. MO48, have been shown to
exhibit antibacterial, anticancer, and antifungal activities (Maskey et al., 2003).
References
Andriole, V. T. 1999. Current and future antifungal therapy: New targets for antifungal agents.
J Antimicrob Chemother, 44(2), 151–162.
Asolkar, R. N., Jensen, P. R., Kauffman, C. A., and Fenical, W. 2006. Daryamides AC, weakly cyto-
toxic polyketides from a marine-derived actinomycete of the genus Streptomyces strain CNQ-
085. J Nat Prod, 69(12), 1756–1759.
Baddley, J. W., Stroud, T. P., Salzman, D., and Pappas, P. G. 2001. Invasive mold infections in alloge-
neic bone marrow transplant recipients. Clin Infect Dis, 32(9), 1319–1324.
Balajee, S. A., Weaver, M., Imhof, A., Gribskov, J., and Marr, K. A. 2004. Aspergillus fumigatus variant with
decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother, 48(4), 1197–1203.
Barsby, T., Kelly, M. T., and Andersen, R. J. 2002. Tupuseleiamides and basiliskamides, new acyl-
dipeptides and antifungal polyketides produced in culture by a Bacillus laterosporus isolate
obtained from a tropical marine habitat. J Nat Prod, 65(10), 1447–1451.
Basich, J. E., Graves, T. S., Baz, M. N., Scanlon, G., Hoffmann, R. G., Patterson, R., and Fink, J. N. 1981.
Allergic bronchopulmonary aspergillosis in corticosteroid-dependent asthmatics. J Allergy
Clin Immunol, 68(2), 98–102.
Binder, R. E., Faling, L. J., Pugatch, R. D., Mahasaen, C., and Snider, G. L. 1982. Chronic necrotizing
pulmonary aspergillosis: A discrete clinical entity. Medicine, 61(2), 109.
Boogaerts, M. and Maertens, J. 2001. Clinical experience with itraconazole in systemic fungal infec-
tions. Drugs, 61(1), 39–47.
Butler, M. S. 2005. Natural products to drugs: Natural product derived compounds in clinical trials.
Nat Prod Rep, 22(2), 162–195.
Cabib, E. 1991. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by
polyoxin D and nikkomycins. Antimicrob Agents Chemother, 35(1), 170–173.
Chamilos, G. and Kontoyiannis, D. P. 2005. Update on antifungal drug resistance mechanisms of
Aspergillus fumigatus. Drug Resist Update, 8(6), 344–358.
Cohen, E. 1993. Chitin synthesis and degradation as targets for pesticide action. Arch Insect Biochem
Physiol, 22(1–2), 245–261.
Cowen, L. E. and Lindquist, S. 2005. Hsp90 potentiates the rapid evolution of new traits: Drug resis-
tance in diverse fungi. Science, 309(5744), 2185–2189.
Dannaoui, E., Meletiadis, J., Tortorano, A. M., Symoens, F., Nolard, N., Viviani, M. A., and Grillot, R.
2004. Susceptibility testing of sequential isolates of Aspergillus fumigatus recovered from treated
patients. J Med Microbiol, 53(2), 129–134.
Demain, A. L. 1983. New applications of microbial products. Science, 219(4585), 709–714.
Demain, A. L. and Sanchez, S. 2009. Microbial drug discovery: 80 years of progress. J Antibiot,
62(1), 5–16.
Edrada, R. A., Ebel, R., Supriyono, A., Wray, V., Schupp, P., Steube, K., van Soest, R., and Proksch, P.
2002. Swinhoeiamide A, a new highly active calyculin derivative from the marine sponge
Theonella swinhoei. J Nat Prod, 65(8), 1168–1172.
Fernández, R., Dherbomez, M., Letourneux, Y., Nabil, M., Verbist, J. F., and Biard, J. F. 1999. Antifungal
metabolites from the marine sponge Pachastrissa sp.: New bengamide and bengazole deriva-
tives. J Nat Pro, 62(5), 678–680.
Fromtling, R. A. 1998. Human mycoses and current antifungal therapy. Drug News Perspect,
11(3), 185.
Galgiani, J. N. 1990. Fluconazole, a new antifungal agent. Ann Intern Med, 113(3), 177–179.
Gefter, W. B., Weingrad, T. R., Epstein, D. M., Ochs, R. H., and Miller, W. T. 1981. “Semi-invasive”
pulmonary aspergillosis: A new look at the spectrum of Aspergillus infections of the lung.
Radiology, 140(2), 313–321.
Grant, S. M. and Clissold, S. P. 1990. Fluconazole. A review of its pharmacodynamic and pharma-
cokinetic properties, and therapeutic potential in superficial and systemic mycoses. Drugs, 39,
877–916.
Groll, A. H., Piscitelli, S. C., and Walsh, T. J. 1998. Clinical pharmacology of systemic antifungal
agents: A comprehensive review of agents in clinical use, current investigational compounds,
and putative targets for antifungal drug development. Adv Pharmacol, 44, 343–500.
Guinea, J., Recio, S., Peláez, T., Torres-Narbona, M., and Bouza, E. 2008. Clinical isolates of Aspergillus
species remain fully susceptible to voriconazole in the post-voriconazole era. Antimicrob Agents
Chemother, 52, 3444–3446.
Hassan, W., Edrada, R., Ebel, R., Wray, V., Berg, A., van Soest, R., Wiryowidagdo, S., and Proksch, P.
2004. New imidazole alkaloids from the Indonesian sponge Leucetta chagosensis. J Nat Prod,
67(5), 817–822.
Hibberd, P. L. and Rubin, R. H. 1994. Clinical aspects of fungal infection in organ transplant recipi-
ents. Clin Infect Dis, 19(Supplement 1), S33–S40.
Hiemenz, J. W. and Walsh, T. J. 1996. Lipid formulations of amphotericin B: Recent progress and
future directions. Clin Infect Dis, 22(Supplement 2), S133–S144.
Ho, P. L. and Yuen, K. Y. 2000. Aspergillosis in bone marrow transplant recipients. Crit Rev Oncol
Hematol, 34(1), 55–69.
Jacob, M. R., Hossain, C. F., Mohammed, K. A., Smillie, T. J., Clark, A. M., Walker, L. A., and
Nagle, D. G. 2003. Reversal of fluconazole resistance in multidrug efflux-resistant fungi by the
dysidea a renaria sponge sterol 9α, 11α-epoxycholest-7-ene-3β, 5α, 6α, 19-tetrol 6-acetate. J Nat
Prod, 66(12), 1618–1622.
Johnson, E. M., Oakley, K. L., Radford, S. A., Moore, C. B., Warn, P., Warnock, D. W., and Denning, D.
W. 2000. Lack of correlation of in vitro amphotericin B susceptibility testing with outcome in a
murine model of Aspergillus infection. J Antimicrob Chemother, 45(1), 85–93.
Kontoyiannis, D. P. 2001. A clinical perspective for the management of invasive fungal infections:
Focus on IDSA guidelines. Pharmacotherapy, 21(8P2), 175S–187S.
Kontoyiannis, D. P. and Bodey, G. 2002. Invasive aspergillosis in 2002: An update. Eur J Clin Microbiol
Infect Dis, 21(3), 161–172.
Kossuga, M. H., MacMillan, J. B., Rogers, E. W., Molinski, T. F., Nascimento, G. G., Rocha, R. M., and
Berlinck, R. G. 2004. (2S,3R)-2-aminododecan-3-ol, a new antifungal agent from the ascidian
Clavelina oblonga. J Nat Prod, 67(11), 1879–1881.
Kumar, S. and Kannabiran, K. 2010. Antifungal activity of Streptomyces VITSVK5 spp. against drug
resistant Aspergillus clinical isolates from pulmonary tuberculosis patients. J Med Mycol, 20(2),
101–107.
Lamb, D., Kelly, D., and Kelly, S. 1999. Molecular aspects of azole antifungal action and resistance.
Drug Resist Update, 2(6), 390–402.
Lionakis, M. S., Lewis, R. E., Torres, H. A., Albert, N. D., Raad, I. I., and Kontoyiannis, D. P.
2005. Increased frequency of non-fumigatus Aspergillus species in amphotericin B- or
triazole–pre-exposed cancer patients with positive cultures for Aspergilli. Diagn Microbiol Infect
Dis, 52(1), 15–20.
Liu, W., Lionakis, M. S., Lewis, R. E., Wiederhold, N., May, G. S., and Kontoyiannis, D. P. 2003.
Attenuation of itraconazole fungicidal activity following preexposure of Aspergillus fumigatus
to fluconazole. Antimicrob Agents Chemother, 47(11), 3592–3597.
Manavathu, E. K., Cutright, J. L., Loebenberg, D., and Chandrasekar, P. H. 2000. A comparative
study of the in vitro susceptibilities of clinical and laboratory-selected resistant isolates of
Aspergillus spp. to amphotericin B, itraconazole, voriconazole and posaconazole (SCH 56592).
J Antimicrob Chemother, 46(2), 229–234.
Manavathu, E. K., Cutright, J. L., and Chandrasekar, P. H. 2003. In vivo resistance of a laboratory-
selected Aspergillus fumigatus isolate to Amphotericin B. Antimicrob Agents Chemother, 49(1),
428–430.
Mandala, S. M., Thornton, R. A., Frommer, B. R., Curotto, J. E., Rozdilsky, W., Kurtz, M. B.,
Giacobbe, R. A. et al. 1995. The discovery of australifungin, a novel inhibitor of sphinganine
N-acyltransferase from Sporormiella australis. Producing organism, fermentation, isolation, and
biological activity. J Antibiotics, 48(5), 349–356.
Marr, K. A., Carter, R. A., Crippa, F., Wald, A., and Corey, L. 2002. Epidemiology and outcome of
mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis, 34(7), 909–917.
Maskey, R. P., Li, F. C. S., Qin, H. H., Fiebig, and Laatsch, H. 2003. Chandrananimycins A–C:
Production of novel anticancer antibiotics from a marine Actinomadura sp. isolate M048 by
variation of medium composition and growth conditions. J Antibiot, 56, 622–629.
Mayer, A. M. and Hamann, M. T. 2002. Marine pharmacology in 1999: Compounds with antibac-
terial, anticoagulant, antifungal, anthelmintic, anti-inflammatory, antiplatelet, antiprotozoal
and antiviral activities affecting the cardiovascular, endocrine, immune and nervous systems,
and other miscellaneous mechanisms of action. Comp Biochem Physiol, 132(3), 315–339.
Mayer, A. M. and Hamann, M. T. 2005. Marine pharmacology in 2001–2002: Marine compounds
with anthelmintic, antibacterial, anticoagulant, antidiabetic, antifungal, anti-inflammatory,
antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the
cardiovascular, immune and nervous systems and other miscellaneous mechanisms of action.
Comp Biochem Physiol C Pharmacol 140(3), 265–286.
Mayer, A. M. and Lehmann, V. K. 2000. Marine pharmacology in 1998: Marine compounds with
antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antipro-
tozoal, and antiviral activities; with actions on the cardiovascular, endocrine, immune, and
nervous systems; and other miscellaneous mechanisms of action. Pharmacologist, 42, 62–69.
Mayer, A. M., Rodríguez, A. D., Berlinck, R. G., and Hamann, M. T. 2007. Marine pharmacology in
2003–4: Marine compounds with anthelmintic antibacterial, anticoagulant, antifungal, anti-
inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activi-
ties; affecting the cardiovascular, immune and nervous systems, and other miscellaneous
mechanisms of action. Comp Biochem Physiol C Pharmacol, 145(4), 553–581.
Moore, C. B., Sayers, N., Mosquera, J., Slaven, J., and Denning, D. W. 2000. Antifungal drug resistance
in Aspergillus. J Infect, 41(3), 203–220.
Moosa, M. Y. S., Alangaden, G. J., Manavathu, E., and Chandrasekar, P. H. 2002. Resistance to ampho-
tericin B does not emerge during treatment for invasive aspergillosis. J Antimicrob Chemother,
49(1), 209–213.
Mroueh, S. and Spock, A. 1994. Allergic bronchopulmonary aspergillosis in patients with cystic
fibrosis. Chest, 105(1), 32–36.
the period 1981–2002. J Nat Prod, 66(7), 1022–1037.
Nicholas, G. M., Hong, T. W., Molinski, T. F., Lerch, M. L., Cancilla, M. T., and Lebrilla, C. B.
1999. Oceanapiside, an antifungal bis-α,ω-amino alcohol glycoside from the marine sponge
Oceanapia phillipensis. J Nat Prod, 62(12), 1678–1681.
Nishimura, S., Matsunaga, S., Shibazaki, M., Suzuki, K., Harada, N., Naoki, H., and Fusetani, N.
2002. Corticatic acids D and E, polyacetylenic geranylgeranyltransferase type I inhibitors, from
the marine sponge Petrosia corticata. J Nat Prod, 65(9), 1353–1356.
Odds, F. C., Brown, A. J., and Gow, N. A. 2003. Antifungal agents: Mechanisms of action. Trends
Microbiol, 11(6), 272–279.
Omura, S. 1986. Philosophy of new drug discovery. Microbiol Rev, 50(3), 259.
Ovechkina, Y. Y., Pettit, R. K., Cichacz, Z. A., Pettit, G. R., and Oakley, B. R. 1999. Unusual antimi-
crotubule activity of the antifungal agent spongistatin 1. Antimicrob Agents Chemother, 43(8),
1993–1999.
Paterson, P. J., Seaton, S., Prentice, H. G., and Kibbler, C. C. 2003. Treatment failure in inva-
sive aspergillosis: Susceptibility of deep tissue isolates following treatment with ampho-
tericin B. J Antimicrob Chemother, 52(5), 873–876.
Patterson, T. F. 2002. New agents for treatment of invasive aspergillosis. Clin Infect Dis, 35(4), 367–369.
Patterson, T. F. 2005. Advances and challenges in management of invasive mycoses. Lancet, 366(9490),
1013–1025.
Pettit, R. K., Woyke, T., Pon, S., Cichacz, Z. A., Pettit, G. R., and Herald, C. L. 2005. In vitro and
in vivo antifungal activities of the marine sponge constituent spongistatin. Med Mycol,
43(5), 453–463.
Pfaller, M. A., Pappas, P. G., and Wingard, J. R. 2006. Invasive fungal pathogens: Current epidemio-
logical trends. Clin Infect Dis, 43(Supplement 1), S3–S14.
Rifai, S., Fassouane, A. F., Kijjoa, A., and Van Soest, R. 2004. Antimicrobial activity of untenospongin B,
a metabolite from the marine sponge Hippospongia communis collected from the Atlantic coast
of Morocco. Mar Drugs, 2(3), 147–153.
Rinaldi, M. 1983. Invasive aspergillosis. Rev Infect Dis, 5, 1061–1077.
Ryder, N. S. 1992. Terbinafine: Mode of action and properties of the squalene epoxidase inhibition.
Br J Derm, 126, 2–7.
Schiraldi, G. F., Lo Cicero, S., Colombo, M. D., Rossato, D., Ferrarese, M., and Soresi, E. 1996. Refractory
pulmonary aspergillosis: Compassionate trial with terbinafine. Br J Derm, 134(s46), 25–29.
Schumacher, R. W., Talmage, S. C., Miller, S. A., Sarris, K. E., Davidson, B. S., and Goldberg, A.
2003. Isolation and structure determination of an antimicrobial ester from a marine sediment-
derived bacterium. J Nat Prod, 66(9), 1291–1293.
Shao, P. L., Huang, L. M., and Hsueh, P. R. 2007. Recent advances and challenges in the treatment of
invasive fungal infections. Int J Antimicrob Agents, 30(6), 487–495.
Sionov, E., Roth, D., Sandovsky-Losica, H., Kashman, Y., Rudi, A., Chill, L., Berdicevsky, I., and
Segal, E. 2005. Antifungal effect and possible mode of activity of a compound from the marine
sponge Dysidea herbacea. J Infect, 50(5), 453–460.
Soubani, A. O., Miller, K. B., and Hassoun, P. M. 1996. Pulmonary complications of bone marrow
transplantation. Chest, 109(4), 1066–1077.
Sterling, T. R. and Merz, W. G. 1998. Resistance to amphotericin B: Emerging clinical and microbio-
logical patterns. Drug Resist Update, 1(3), 161–165.
Swerdlow, J. L. 2000. Nature’s Medicine—Plants that Heal. Random House, Inc., New York, p. 56.
Tomee, J. F., van der Werf, T. S., Latge, J. P., Koeter, G. H., Dubois, A. E., and Kauffman, H. F. 1995.
Serologic monitoring of disease and treatment in a patient with pulmonary aspergilloma. Am
J Respir Crit Care Med, 151(1), 199–204.
Tsukamoto, S., Matsunaga, S., Fusetani, N., and Toh-e, A. 1999. Theopederins FJ: Five new antifun-
gal and cytotoxic metabolites from the marine sponge, Theonella swinhoei. Tetrahedron, 55(48),
13697–13702.
Vehreschild, J. J., Rüping, M. J. G. T., Wisplinghoff, H., Farowski, F., Steinbach, A., Sims, R., and
Cornely, O. A. 2010. Clinical effectiveness of posaconazole prophylaxis in patients with acute
myelogenous leukaemia (AML): A 6 year experience of the Cologne AML cohort. J Antimicrob
Chemother, 65(7), 1466–1471.
Verweij, P. E., Oakley, K. L., Morrissey, J., Morrissey, G., and Denning, D. W. 1998. Efficacy of LY303366
against amphotericin B-susceptible and-resistant Aspergillus fumigatus in a murine model of
invasive aspergillosis. Antimicrob Agents Chemother, 42(4), 873–878.
Vicente, M. F., Basilio, A., Cabello, A., and Pelaez, F. 2003. Microbial natural products as a source of
antifungals. Clin Microbiol Infect, 9(1), 15–32.
Vijayakumar, E. K. S., Roy, K., Chatterjee, S., Deshmukh, S. K., Ganguli, B. N., Fehlhaber, H. W., and
Kogler, H. 1996. Arthrichitin. A new cell wall active metabolite from Arthrinium phaeospermum.
J Org Chem, 61(19), 6591–6593.
Wald, A., Leisenring, W., van Burik, J. A., and Bowden, R. A. 1997. Epidemiology of Aspergillus infec-
tions in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis, 175(6),
1459–1466.
Walsh, T. J., Petraitis, V., Petraitiene, R., Field-Ridley, A., Sutton, D., Ghannoum, M. et al. 2003.
Experimental pulmonary aspergillosis due to Aspergillus terreus: Pathogenesis and treatment
of an emerging fungal pathogen resistant to amphotericin B. J Infect Dis, 188(2), 305–319.
Warn, P. A., Morrissey, G., Morrissey, J., and Denning, D. W. 2003. Activity of micafungin (FK463)
against an itraconazole-resistant strain of Aspergillus fumigatus and a strain of Aspergillus ter-
reus demonstrating in vivo resistance to amphotericin B. J Antimicrob Chemother, 51(4), 913–919.
Warris, A., Weemaes, C. M., and Verweij, P. E. 2002. Multidrug resistance in Aspergillus fumigatus.
N Engl J Med, 347(26), 2173–2174.
Wells, G. B. and Lester, R. L. 1983. The isolation and characterization of a mutant strain of
Saccharomyces cerevisiae that requires a long chain base for growth and for synthesis of phos-
phosphingolipids. J Biol Chem, 258(17), 10200–10203.
Xiao, L., Madison, V., Chau, A. S., Loebenberg, D., Palermo, R. E., and McNicholas, P. M. 2004. Three-
dimensional models of wild-type and mutated forms of cytochrome P450 14α-sterol demeth-
ylases from Aspergillus fumigatus and Candida albicans provide insights into posaconazole
binding. Antimicrob Agents Chemother, 48(2), 568–574.
Yang, S. W., Chan, T. M., Pomponi, S. A., Chen, G., Loebenberg, D., Wright, A., Patel, M., Gullo,
V., Pramanik, B., and Chu, M. 2003. Structure elucidation of a new antifungal sterol sulfate,
Sch 575867, from a deep-water marine sponge (Family: Astroscleridae). J Antibiot (Tokyo) 56(2),
186–189.
Zweerink, M. M., Edison, A. M., Wells, G. B., Pinto, W., and Lester, R. L. 1992. Characterization
of a novel, potent, and specific inhibitor of serine palmitoyltransferase. J Biol Chem, 267(35),
25032–25038.
section two

compounds from animals
chapter fifteen
Secondary metabolites from

microorganisms isolated from
marine sponges from 2000 to 2012
Mohammad F. Mehbub, Christopher M.M. Franco, and Wei Zhang
Contents
15.1 Introduction......................................................................................................................... 279
15.2 Microbial sources................................................................................................................ 281
15.2.1 Marine fungi............................................................................................................ 281
15.2.2 Actinobacteria......................................................................................................... 302
15.2.3 Bacteria..................................................................................................................... 302
15.3 Sponge sources.................................................................................................................... 302
15.4 Chemical diversity among the microbial metabolites and their activities................. 303
15.5 Identification of microbial diversity.................................................................................304
15.6 Isolation techniques for bacteria, actinobacteria, and fungi from sponges...............304
15.6.1 Sponge sampling and isolation of microorganisms..........................................304
15.6.2 Pretreatment of sponges for selective isolation of actinobacteria....................306
15.7 Concluding remarks........................................................................................................... 307
References...................................................................................................................................... 307
15.1 Introduction
The continuously developing resistance of pathogenic bacteria, the reemergence of viral
diseases, and cancers that are still incurable make us redouble our efforts to find cures to
alleviate human vulnerabilities. The introduction of new drugs is crucial, as older antibi-
otics and drugs begin to lose their efficacy. Rationally designed drugs have made inroads
into the pharmaceutical industry, but natural products continue to introduce the chemi-
cal diversity required to maintain a contribution of around 60% of the drugs, directly or
after chemical modification, which are available in the market (Newman and Cragg, 2007).
Microbial natural products contribute more than 40% of new chemical entities reported
between 1981 and 2010 (Newman et al., 2003; Baltz et al., 2005; Koehn and Carter, 2005;
Fisher, 2014).
Whereas the majority of microorganisms that produce valuable products have been
obtained predominantly from terrestrial sources, there has been a recent trend to collect
microorganisms that are associated with other life forms such as endophytes of plants
(Govindasamy et al., 2014). There has also been an evolving realization that the oceans,
which cover a larger proportion of the earth’s surface, have a different microbial diversity,
279
and here too there are associations with marine life forms such as sponges. It was noted
that due to the aqueous milieu, their metabolites have more potent activities and are usu-
ally structurally very different from those found from terrestrial-based samples. Recent
studies have borne out this hypothesis, and the marine environment is proving to be an
eclectic source of novel chemical diversity that is contributing to drug discovery. Many
bioactive substances have been isolated from a variety of marine organisms, includ-
ing phytoplankton, bryozoans, algae, sponges, tunicates, and molluscs (Faulkner, 2002;
Proksch et al., 2002; Zhang et al., 2005; Mehbub et al., 2014). Microorganisms associated
with these animals have shown the ability to adapt, and this adaptation capacity may be
the key reason for their secondary metabolite production capacity (Piel, 2004, 2009; König
et al., 2006; Brady et al., 2009; Valliappan et al., 2014).
Marine sponges (phylum Porifera) are of particular interest because they are remark-
able filter feeders; some can filter 24 m3 kg−1 sponge day−1 (Vogel, 1977). During the filtra-
tion process, they concentrate bacterial cells that are otherwise diluted in seawater. They
harbor dense and diverse microbial consortia, which comprise as much as 40% of sponge
tissue volume and span all three domains of life (Taylor et al., 2007). This makes sponges
excellent models for the study of marine host-associated bacteria as they represent a sub-
stantial reservoir of novel microbial diversity (Taylor et al., 2004). Therefore, in recent
years, the search has intensified for microorganisms from sponges with the expectation
that novel compounds will result from their screening.
There are more than 8500 sponge species (Van Soest et al., 2012) that contain very
diverse microbial consortia (Taylor et al., 2004); and, it has been reported that individual
species from sponges during the past decade contains at least a few different types of
bioactive natural products (Mehbub et al., 2014). Therefore, sponges could be termed the
“drugstore of the sea” (Blunt et al., 2009). Many of these compounds likely serve as agents
of defense that protect the immobile animals from being overgrown or ingested (Pawlik,
1992; Paul and Ritson-Williams, 2008), but for most substances, an ecological function
has not been experimentally demonstrated. Likewise, the often-stated question whether
sponge-derived natural products are biosynthesized by sponges or by associated microor-
ganisms remains largely unanswered (Faulkner et al., 1993; Piel et al., 2004). Insights into
this issue could have a significant impact on marine pharmacology. For most compounds,
drug development is currently not possible due to a limited access to the biological mate-
rial. If the actual source is a bacterium, supply could be ensured by cultivating the pro-
ducer or by isolating the biosynthetic genes and expressing the pathway in culturable
bacteria (Piel, 2006; Schmitt et al., 2008). The advantage of the latter approach is that it
should be generally applicable to a wide range of compounds independent of cultivation.
Although the genetic tools to express bacterial pathways are in principle available (Fujii,
2009), the application of this strategy to sponge symbionts is currently highly challenging
for several reasons.
This review focuses on microorganisms from sponges that have been reported to pro-
duce secondary metabolites or bioactive compounds from 2000 to 2012. The microorgan-
isms reported here are the bacteria, with actinobacteria looked at separately due to their
recognized ability to produce a wide range of secondary metabolites, and fungi, includ-
ing yeast. Data have been compiled from the published literature and data reviewed by
Faulkner (2002) and Blunt et al. (2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012,
2013, 2014) from Natural Product Reports.
Species isolated previously from marine sediments but subsequently reported from
sponges, for example, Salinispora strains isolated from the Great Barrier Reef sponge
Pseudoceratina clavata (Kim et al., 2005), will not be included.
Chapter fifteen: Secondary metabolites from microorganisms 281
15.2 Microbial sources

The microbial populations include archaea and bacteria (Webster et al., 2001; Hentschel
et al., 2003), fungi (Höller et al., 2000), cyanobacteria (Thacker and Starnes, 2003), unicellu-
lar algae (Vacelet, 1981), dinoflagellates (Garson et al., 1998), and actinobacteria (Maldonado
et al., 2005b), which make up at least half the tissue volume in some sponge species (Vacelet
and Donadey, 1977; Hentschel et al., 2003).
Members of the phylum Actinobacteria and specifically the order Actinomycetales
have been identified as abundant members of sponge-associated microbial communities
(Hentschel et al., 2002; Zhang et al., 2006). However, in this survey, it is evident that there
has been a shift from actinobacteria to fungi as the main microorganisms. The number of
fungi from sponge samples reported as producers of new compounds is now at least three
times higher compared to the reports for actinobacteria from the same source.
Over the 2000–2012 period, a total of 269 new compounds were isolated from sponge-
associated microbes (Figure 15.1). Of these, 186 new compounds were isolated from 27
genera of fungi including two compounds from one yeast genus, compared to just 56 new
compounds from seven actinobacterial genera and 27 new compounds from seven bacte-
rial genera (Table 15.1). Confirming the switch to fungal sources are the 69 publications
on fungal metabolites compared to 20 for actinobacteria and 15 for bacteria during this
review period. However, our understanding of the sponge-associated fungal, actinobacte-
rial, and bacterial communities and their structures is still inadequate.
15.2.1 Marine fungi

Fungi from marine sponges are now the primary source of novel metabolites from micro-
bial sources that have industrial as well as medicinal values. By 1998, more than 100 metab-
olites from marine-derived fungi had been described by different researchers (Biabani
and Laatsch, 1998). However, the majority of the reports focused on natural product
70
Actinobacteria Bacteria Fungi Yeast
60
Number of new compounds
50
40
30
20
10
0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Year
Figure 15.1 The total number of new compounds isolated from sponge-associated microorganisms
from 2000 to 2012.
Table 15.1 New compounds isolated from microorganisms from marine sponges from 2000 to 2012: their chemical class,
282
country of collection, and reported activity

Country
of Name of Type of Name of Reported
Compound name Chemical class collection microorganism microorganism sponge activity References
GGL.1 1,2-O-diacyl-3-[α- Glycoglycerolipid Croatia Microbacterium sp. Actinobacterium Halichondria Anticancer Wicke et al. (2000)
glucopyranosyl-(1–6)-α- panicea
glucopyranosyl)]glycerol
GGL.2 1-O-acyl-3-[R- Glycoglycerolipid Croatia Microbacterium sp. Actinobacterium Halichondria Anticancer Wicke et al. (2000)
glucopyranosyl-(1–3)-(6-O- panicea
acyl-R-mannopyranosyl)]
glycerol
GGL.3 1-O-acyl-3-[6-O-acetyl- Glycoglycerolipid Croatia Microbacterium sp. Actinobacterium Halichondria Anticancer Wicke et al. (2000)
R-glucopyranosyl-(1–3)-(6- panicea
O-acyl-R-mannopyranosyl)]
glycerol
GGL.4 1,2-O-diacyl-3-[α- Glycoglycerolipid Croatia Microbacterium sp. Actinobacterium Halichondria Anticancer Wicke et al. (2000)
galactofuranosyl)]glycerol panicea
DPG tetraacyldiphosphatidyl Diphosphatidylglycerol Croatia Microbacterium sp. Actinobacterium Halichondria Anticancer Wicke et al. (2000)
glycerol panicea
Compound 1 (C25H31N3O6)/β- Tripeptide Bulgaria Pseudomonas/ Bacterium Dysidea fragilis Antiviral De Rosa et al.
aminopimelic acid Alteromonas (2000)
4′-N-Methyl-5′- Indolocarbazole Spain Micromonospora sp. Actinobacterium Clathrina Cytotoxic Hernández et al.
hydroxystaurosporine alkaloid coriacea (2000)
5′-Hydroxystaurosporine Indolocarbazole Spain Micromonospora sp. Actinobacterium Clathrina Cytotoxic Hernández et al.
alkaloid coriacea (2000)
Iso-cladospolide B Hexaketide Indonesia Not identified Fungus Not identified NR Smith et al. (2000)
Seco-patulolide C Hexaketide Indonesia Not identified Fungus Not identified NR Smith et al. (2000)
Pandangolide 1 Polyketide Indonesia Not identified Fungus Not identified NR Smith et al. (2000)
Pandangolide 2 Polyketide Indonesia Not identified Fungus Not identified NR Smith et al. (2000)
Spiciferone A Spiciferone derivative Indonesia Drechslera Fungus Callyspongia NR Edrada et al.
hawaiiensis aerizusa (2000)
Spiciferone B Spiciferone derivative Indonesia Drechslera Fungus Callyspongia NR Edrada et al.
(Continued)
Table 15.1 (Continued) New compounds isolated from microorganisms from marine sponges from 2000 to 2012: their chemical class,
Country
Spiciferol A Spiciferone derivative Indonesia Drechslera Fungus Callyspongia NR Edrada et al.
Spiciferone A (1) Spiciferone derivative Indonesia Drechslera Fungus Callyspongia NR Edrada et al.
Butoxyl-spiciferin Spiciferone derivative Indonesia Drechslera Fungus Callyspongia NR Edrada et al.
Asperic acid Pyran derivative United Aspergillus niger Fungus Hyrtios proteus NR Varoglu and
States Crews (2000)
Nafuredin Lactone Palau Aspergillus niger Fungus Not identified Cytotoxic Takano et al.
(2001)
2-Acetamido-2-deoxy-d- Amino sugar Russia Pseudoalteromonas Bacterium Not identified NR Muldoon et al.
galacturonic acid distincta (2001)
5-Acetamido-3,5,7,9- Amino sugar Russia Pseudoalteromonas Bacterium Not identified NR Muldoon et al.
tetradeoxy-7-formamido-l- distincta (2001)
glycero-l-manno-
nonulosonic acid
Aspergione A Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
Chapter fifteen: Secondary metabolites from microorganisms
versicolor exigua
Aspergione B Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
versicolor exigua
Aspergione C Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
versicolor exigua
Aspergione D Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
versicolor exigua
Aspergione E Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
versicolor exigua
Aspergione F Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001a)
versicolor exigua
Aspergillone Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001b)
versicolor exigua
(Continued)
283
Table 15.1 (Continued) New compounds isolated from microorganisms from marine sponges from 2000 to 2012: Their chemical class,
284

Country
Aspergillodiol Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001b)
versicolor exigua
Aspergillol Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001b)
versicolor exigua
12-Acetyl-aspergillol Chromone Indonesia Aspergillus Fungus Xestospongia NR Lin et al. (2001b)
versicolor exigua
Lunatin Anthraquinone Indonesia Curvularia lunata Fungus Niphates olmeda Antibacterial Jadulco et al.
(2002)
Herbarin A α-Pyrone France Cladosporium Fungus Aplysina Miscellaneous Jadulco et al.
herbarum aerophoba (2002)
Herbarin B α-Pyrone France Cladosporium Fungus Aplysina Miscellaneous Jadulco et al.
herbarum aerophoba (2002)
Herbaric acid Phthalide Indonesia Cladosporium Fungus Callyspongia NR Jadulco et al.
herbarum aerizusa (2002)
Varitriol Macrotetrolide Venezuela Emericella Fungus Not identified Anticancer Malmstrøm et al.
variecolor (2002)
Varioxirane NR Venezuela Emericella Fungus Not identified NR Malmstrøm et al.
variecolor (2002)
Dihydroterrein NR Venezuela Emericella Fungus Not identified NR Malmstrøm et al.
variecolor (2002)
Varixanthone NR Venezuela Emericella Fungus Not identified Antimicrobial Malmstrøm et al.
variecolor (2002)
Xestodecalactone B Decalactone Indonesia Penicillium cf. Fungus Xestospongia Antiyeast Edrada et al.
montanense exigua (2002)
Microsphaerone A ɣ-Pyrone France Microsphaeropsis sp. Fungus Aplysina Anticancer Wang et al. (2002)
aerophoba
Microsphaerone B ɣ-Pyrone France Microsphaeropsis sp. Fungus Aplysina Anticancer Wang et al. (2002)
aerophoba
Xestodecalactone A Decalactone Indonesia Penicillium cf. Fungus Xestospongia NR Edrada et al.
(Continued)
Country
Xestodecalactone C Decalactone Indonesia Penicillium cf. Fungus Xestospongia NR Edrada et al.
YM-266183 Thiopeptide Japan Bacillus cereus Bacterium Halichondria Antibacterial Nagai et al. (2003)
japonica
YM-266184 Thiopeptide Japan Bacillus cereus Bacterium Halichondria Antibacterial Nagai et al. (2003)
japonica
Pseudoalterobactin A Siderophore Palau Pseudoalteromonas Bacterium Cinachyrella NR Kanoh et al.
sp. australiensis (2003)
Pseudoalterobactin B Siderophore Palau Pseudoalteromonas Bacterium Cinachyrella NR Kanoh et al.
sp. australiensis (2003)
Cyclo-(glycyl-l-seryl-l-prolyl- Cyclic peptide Italy Ruegeria sp. Bacterium Suberites Antibacterial Mitova et al.
l-glutamyl) domuncula (2004)
Cyclo-(glycyl-l-prolyl-l- Cyclic peptide Italy Ruegeria sp. Bacterium Suberites Antibacterial Mitova et al.
glutamyl) domuncula (2004)
Communesin C Indole alkaloid Italy Penicillium sp. Fungus Axinella Anticancer Jadulco et al.
(communesin verrucosa (2004)
derivative)
Communesin D Indole alkaloid Italy Penicillium sp. Fungus Axinella Anticancer Jadulco et al.
(communesin verrucosa (2004)
derivative)
Petrosifungins A Cyclic peptide Italy Penicillium Fungus Petrosia NR Bringmann et al.
brevicompactum ficiformis (2004)
Petrosifungins B Cyclic peptide Italy Penicillium Fungus Petrosia NR Bringmann et al.
brevicompactum ficiformis (2004)
Sorbicillactone A Sorbicillin alkaloid Italy Penicillium Fungus Ircinia Anti-HIV Bringmann et al.
chrysogenum fasciculata (2005)
Sorbicillactone B Sorbicillin alkaloid Italy Penicillium Fungus Ircinia Anti-HIV Bringmann et al.
Sorbivinetone Sorbicillin alkaloid Italy Penicillium Fungus Ircinia NR Bringmann et al.
(Continued)
285
286

Country
(S)-2,4-Dihydroxy-1-butyl(4- Benzoate China Penicillium Fungus Mycale plumose Cytotoxic Xin et al. (2005)
hydroxy)benzoate aurantiogriseum
Dehydroxynocardamine Cyclic peptide Korea Streptomyces sp. Actinobacterium Not identified Miscellaneous Lee et al. (2005)
Desmethylenylnocardamine Cyclic peptide Korea Streptomyces sp. Actinobacterium Not identified Miscellaneous Lee et al. (2005)
Clonostachysin A Cyclic peptide Japan Clonostachys Fungus Halichondria Miscellaneous Adachi et al.
rogersoniana japonica (2005)
Clonostachysin B Cyclic peptide Japan Clonostachys Fungus Halichondria Miscellaneous Adachi et al.
rogersoniana japonica (2005)
Guangomide A Cyclic depsipeptide Papua Not identified Fungus Ianthella sp. Antibacterial Amagata et al.
New fungus (2006)
Guinea
Guangomide B Cyclic depsipeptide Papua Not identified Fungus Ianthella sp. Antibacterial Amagata et al.
New fungus (2006)
Guinea
Homodestcardin Cyclic depsipeptide Papua Not identified Fungus Ianthella sp. NR Amagata et al.
New fungus (2006)
Guinea
RHM1 Peptide Papua Acremonium sp. Fungus Teichaxinella sp. Cytotoxic Boot et al. (2006)
New
Guinea
RHM2 Octapeptide Papua Acremonium sp. Fungus Teichaxinella sp. Cytotoxic Boot et al. (2006)
New
Guinea
Tropolactone A Meroterpene United Aspergillus sp. Fungus Not identified Cytotoxic Cueto et al. (2006)
States
Tropolactone B Meroterpene United Aspergillus sp. Fungus Not identified Cytotoxic Cueto et al. (2006)
States
Tropolactone C Meroterpene United Aspergillus sp. Fungus Not identified Cytotoxic Cueto et al. (2006)
States
(Continued)
Country
Tropolactone D Meroterpene United Aspergillus sp. Fungus Not identified Cytotoxic Cueto et al. (2006)
States
IB-01212 Cyclic depsipeptide Japan Clonostachys sp. Fungus Not identified Cytotoxic Cruz et al. (2006)
Roridin R Macrocyclic Indonesia Myrothecium sp. Fungus Not identified Cytotoxic Xu et al. (2006)
trichothecene
12-Hydroxyroridin E Macrocyclic Indonesia Myrothecium sp. Fungus Not identified Cytotoxic Xu et al. (2006)
trichothecene
Roridin Q Macrocyclic Indonesia Myrothecium sp. Fungus Not identified Cytotoxic Xu et al. (2006)
trichothecene
2′,3′-Deoxyroritoxin D Macrocyclic Indonesia Myrothecium Fungus Not identified Cytotoxic, Xu et al. (2006)
trichothecene roridum antiyeast
5-cis-3-Oxo-C12-HSL N-Acyl-l-homoserine Norway Mesorhizobium sp. Bacterium Phakellia Antibacterial, Krick et al. (2007)
(compound 1) lactone ventilabrum cytotoxic
5-cis-3-Oxo-C12-homoserine N-Acyl-l-homoserine Norway Mesorhizobium sp. Bacterium Phakellia NR Krick et al. (2007)
lactone lactone ventilabrum
Bromoalterochromide A Brominated Australia Pseudoalteromonas Bacterium Fascaplysinopsis Cytotoxic Speitling et al.
chromopeptide maricaloris reticulata (2007)
Bromoalterochromide A′ Brominated Australia Pseudoalteromonas Bacterium Fascaplysinopsis Cytotoxic Speitling et al.

chromopeptide maricaloris reticulata (2007)
Aurantiomide B Quinazoline alkaloid China Penicillium Fungus Mycale plumose Cytotoxic Xin et al. (2007)
aurantiogriseum
Aurantiomide C Quinazoline alkaloid China Penicillium Fungus Mycale plumose Cytotoxic Xin et al. (2007)
aurantiogriseum
Glyco-C30-carotenoic acid Diapolycopenedioic NR Rubritalea Bacterium Halichondria Miscellaneous Shindo et al.
acid squalenifaciens okadai (2007)
Trichodermanone A Sorbicillin–polyketide Dominica Trichoderma sp. Fungus Agelas dispar Miscellaneous Neumann et al.
(2007)
Trichodermanone B Sorbicillin–polyketide Dominica Trichoderma sp. Fungus Agelas dispar Miscellaneous Neumann et al.
(2007)
(Continued)
287
288

Country
Trichodermanone C Sorbicillin–polyketide Dominica Trichoderma sp. Fungus Agelas dispar Miscellaneous Neumann et al.
(2007)
Trichodermanone D Sorbicillin–polyketide Dominica Trichoderma sp. Fungus Agelas dispar Miscellaneous Neumann et al.
(2007)
Aurantiomide A Quinazoline alkaloid China Penicillium Fungus Mycale plumose NR Xin et al. (2007)
aurantiogriseum
Aspinotriol A Pentaketide Micronesia Aspergillus ostianus Fungus Not identified NR Kito et al. (2007)
sponge
Aspinotriol B Pentaketide Micronesia Aspergillus ostianus Fungus Not identified NR Kito et al. (2007)
sponge
Aspinonediol Pentaketide Micronesia Aspergillus ostianus Fungus Not identified NR Kito et al. (2007)
sponge
Streptophenazine A Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine B Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine C Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine D Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine E Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine F Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine G Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Streptophenazine H Phenazine Germany Streptomyces sp. Actinobacterium Halichondria Antibacterial Mitova et al.
panicea (2008)
Chlorohydroaspyrone A Aspyrone derivative Korea Exophiala sp. Fungus Halichondria Antibacterial Zhang et al.
(polyketide) panicea (2008a)
(Continued)
Country
Chlorohydroaspyrone B Aspyrone derivative Korea Exophiala sp. Fungus Halichondria Antibacterial Zhang et al.
(polyketide) panicea (2008a)
Scopularide A Cyclic depsipeptide Croatia Scopulariopsis Fungus Tethya Antibacterial, Yu et al. (2008)
brevicaulis aurantium cytotoxic
Scopularide B Cyclic depsipeptide Croatia Scopulariopsis Fungus Tethya Antibacterial, Yu et al. (2008)
brevicaulis aurantium cytotoxic
Gymnastatin Q NR Japan Gymnascella Fungus Halichondria Anticancer Amagata et al.
dankaliensis japonica (2008)
Dihydroinfectopyrone Pyrone Thailand Order Fungus Not identified Anticancer Proksch et al.
pleosporales (2008)
Aspergillide A Polyketide Micronesia Aspergillus ostianus Fungus Not identified Cytotoxic Kito et al. (2008)
Aspergillide B Polyketide Micronesia Aspergillus ostianus Fungus Not identified Cytotoxic Kito et al. (2008)
Aspergillide C Polyketide Micronesia Aspergillus ostianus Fungus Not identified Cytotoxic Kito et al. (2008)
Gymnastatin R NR Japan Gymnascella Fungus Halichondria Cytotoxic Amagata et al.
Dankastatin A NR Japan Gymnascella Fungus Halichondria Cytotoxic Amagata et al.
Dankastatin B NR Japan Gymnascella Fungus Halichondria Cytotoxic Amagata et al.

(Z)-6-Benzylidene-3- Diketopiperazine Thailand Strain CRIF2 Fungus Not identified Cytotoxic Prachyawarakorn
hydroxymethyl-1,4- (order et al. (2008)
dimethyl-3- Pleosporales)
methylsulfanylpiperazine-
2,5-dione
(3S,3′R)-3-(3′-Hydroxybutyl)- Diketopiperazine Thailand Strain CRIF2 Fungus Not identified Cytotoxic Prachyawarakorn
7-methoxyphthalide (order et al. (2008)
Pleosporales)
Diapolycopenedioic acid Glyco-C30-carotenoic Japan Rubritalea Bacterium Halichondria Miscellaneous Shindo et al.
xylosyl ester A acid squalenifaciens okadai (2008)
(Continued)
289
290

Country
xylosyl ester B acid squalenifaciens okadai (2008)
xylosyl ester C acid squalenifaciens okadai (2008)
Compound 3 (C10H11NO4) Siderophore Indonesia Pseudoalteromonas Bacterium Halisarca Miscellaneous You et al. (2008)
sp. ectofibrosa
Cebulactam A1 Macrolactam Philippines Saccharopolyspora Actinobacterium Haliclona sp. NR Pimentel-Elardo
cebuensis et al. (2008)
Cebulactam B1 Macrolactam Philippines Saccharopolyspora Actinobacterium Haliclona sp. NR Pimentel-Elardo
cebuensis et al. (2008)
Cyclo-[phenylalanyl-leucyl]2 Peptide Thailand Pseudoalteromonas Bacterium Halisarca NR Rungprom et al.
sp. ectofibrosa (2008)
Cyclo-[leucyl-isoleucyl]2 Peptide Thailand Pseudoalteromonas Bacterium Halisarca NR Rungprom et al.
sp. ectofibrosa (2008)
1-(2,8-Dihydroxy-1,2,6- Polyketide NR Mycelia sterilia Fungus Not identified NR Hao et al. (2008)
trimethyl-
1,2,6,7,8,8ahexahydro-
naphthalen-1-yl)-3-methoxy-
propan-1-one
4,8-Dihydroxy-7-(2-hydroxy- Polyketide NR Mycelia sterilia Fungus Not identified NR Hao et al. (2008)
ethyl)-6-methoxy-3,4-
dihydro-2-naphthalen-1-one
1-Methyl-naphthalene-2,6- Polyketide NR Mycelia sterilia Fungus Not identified NR Hao et al. (2008)
dicarboxylic acid
Circumdatin J Alkaloid Micronesia Aspergillus ostianus Fungus Not identified NR Ookura et al.
(2008)
6-Hydroxymethyl-1- Phenazine Korea Brevibacterium sp. Bacterium Callyspongia Antibacterial Choi et al. (2009)
phenazine-carboxamide KMD 003 sp.
1,6-Phenazinedimethanol Phenazine Korea Brevibacterium sp. Bacterium Callyspongia Antibacterial Choi et al. (2009)
KMD 003 sp.
(Continued)
Country
Angucyclinone (PM070747) Benz[α]anthraquinone Tanzania Saccharopolyspora Actinobacterium Unidentified Anticancer Pérez et al. (2009)
taberi
(PEM-06-F23–
019B)
Hydroxydecylparaben, Paraben France Microbulbifer sp. Bacterium Leuconia nivea Antimicrobial Quévrain et al.
4-hydroxybenzoic acid (2009)
3-hydroxy-decyl ester
Methyldecylparaben, Paraben France Microbulbifer sp. Bacterium Leuconia nivea Antimicrobial Quévrain et al.
methyl-decyl ester
Hydroxymethyldecylparaben, Paraben France Microbulbifer sp. Bacterium Leuconia nivea Antimicrobial Quévrain et al.
3-hydroxy-methyl-decyl
ester
Dodec-5-enylparaben, Paraben France Microbulbifer sp. Bacterium Leuconia nivea Antimicrobial Quévrain et al.
dodec-5-enyl ester
Aspergillusol A Tyrosine Thailand Aspergillus Fungus Xestospongia Antiyeast, Ingavat et al.
aculeatus testudinaria cytotoxic (2009)

Beauversetin Tetramic acid Germany Beauveria bassiana Fungus Myxilla Cytotoxic Neumann et al.
incrustans (2009)
Epoxyphomalin A Prenylated polyketide Dominica Phoma sp. Fungus Ectyplasia perox Cytotoxic Mohamed et al.
(2009)
Epoxyphomalin B Prenylated polyketide Dominica Phoma sp. Fungus Ectyplasia perox Cytotoxic Mohamed et al.
(2009)
2-(1H-Indol-3-yl)ethyl Indole Japan Pichia Yeast Halichondria Miscellaneous Sugiyama et al.
2-hydroxypropanoate membranifaciens okadai (2009)
2-(1H-Indol-3-yl)ethyl Indole Japan Pichia Yeast Halichondria Miscellaneous Sugiyama et al.
5-hydroxypentanoate membranifaciens okadai (2009)
JBIR-37 Glycosyl benzenediol Japan Acremonium sp. Fungus Not identified NR Izumikawa et al.
(2009)
291
(Continued)
292

Country
JBIR-38 Glycosyl benzenediol Japan Acremonium sp. Fungus Not identified NR Izumikawa et al.
(2009)
Paecilopyrone A α-Pyrone Korea Paecilomyces Fungus Petrosia sp. NR Elbandy et al.
lilacinus (2009)
Paecilopyrone B α-Pyrone Korea Paecilomyces Fungus Petrosia sp. NR Elbandy et al.
lilacinus (2009)
Phomaligol B Cyclohexenone Korea Paecilomyces Fungus Petrosia sp. NR Elbandy et al.
lilacinus (2009)
Phomaligol C Cyclohexenone Korea Paecilomyces Fungus Petrosia sp. NR Elbandy et al.
lilacinus (2009)
Neobacillamide A Alkaloid China Bacillus Bacterium Dysidea avara NR Yu et al. (2009)
vallismortis C89
Chlorocylindrocarpol Sesquiterpene Korea Acremonium sp. Fungus Stelletta sp. NR Zhang et al.
(2009)
Acremofuranone A Sesquiterpene Korea Acremonium sp. Fungus Stelletta sp. NR Zhang et al.
(2009)
Acremofuranone B Sesquiterpene Korea Acremonium sp. Fungus Stelletta sp. NR Zhang et al.
(2009)
Dihydroxybergamotene Sesquiterpene Korea Acremonium sp. Fungus Stelletta sp. NR Zhang et al.
(2009)
Trichopyrone [6-(4-hydroxy- Pyranone Dominica Trichoderma viride Fungus Agelas dispar NR Abdel-Lateffa
1-pentenyl)-4-meth et al. (2009)
oxy-3-methyl-2H-pyran-2-
one]
JBIR-15 Aspochracin derivative Japan Aspergillus Fungus Mycale sp. NR Motohashi et al.
sclerotiorum (2009)
Mayamycin Polyketide Germany Nocardiopsis sp. Actinobacterium Halichondria Antibacterial, Schneemann et al.
panicea cytotoxic (2010a)
JBIR-58 Salicylamide Japan Streptomyces sp. Actinobacterium Not identified Cytotoxic Ueda et al.
SpD081030ME-02 (2010a)
(Continued)
Country
Fellutamide C Lipopeptide Japan Aspergillus Fungus Petrosia sp. Cytotoxic Lee et al. (2010)
versicolor
Epoxyphomalin D Prenylated polyketide Dominica Paraconiothyrium Fungus Ectyplasia perox Cytotoxic Mohamed et al.
sporulosum (2010)
JBIR-97 NR Japan Tritirachium sp. Fungus Pseudoceratina Cytotoxic Ueda et al.
purpurea (2010b)
purpurea (2010b)
purpurea (2010b)
Trichoderin A Aminolipopeptide NR Trichoderma sp. Fungus Not identified Antituberculosis Pruksakorn et al.
(2010)
Trichoderin A1 Aminolipopeptide NR Trichoderma sp. Fungus Not identified Antituberculosis Pruksakorn et al.
(2010)
Trichoderin B Aminolipopeptide NR Trichoderma sp. Fungus Not identified Antituberculosis Pruksakorn et al.
(2010)
JBIR-65 Diterpene Japan Actinomadura sp. Actinobacterium Not identified Miscellaneous Takagi et al.
SpB081030SC-15 (2010a)
JBIR-34 Indole-containing Japan Streptomyces sp. Actinobacterium Haliclona sp. Miscellaneous Motohashi et al.
peptide Sp080513GE-23 (2010)
JBIR-35 Indole-containing Japan Streptomyces sp. Actinobacterium Haliclona sp. Miscellaneous Motohashi et al.
peptide Sp080513GE-23 (2010)
Nocapyrone A ɣ-Pyrone Germany Nocardiopsis sp. Actinobacterium Halichondria NR Schneemann et al.
panicea (2010b)
Nocapyrone B ɣ-Pyrone Germany Nocardiopsis sp. Actinobacterium Halichondria NR Schneemann et al.
panicea (2010b)
Nocapyrone C ɣ-Pyrone Germany Nocardiopsis sp. Actinobacterium Halichondria NR Schneemann et al.
panicea (2010b)
Nocapyrone D ɣ-Pyrone Germany Nocardiopsis sp. Actinobacterium Halichondria NR Schneemann et al.
panicea (2010b)
(Continued)
293
294

Country
JBIR-74 Roquefortine C analog Japan Aspergillus sp. Fungus Not identified NR Takagi et al.
(mycotoxin) (2010b)
JBIR-75 Roquefortine C analog Japan Aspergillus sp. Fungus Not identified NR Takagi et al.
(mycotoxin) (2010b)
Epoxyphomalin C Prenylated polyketide Dominica Paraconiothyrium Fungus Ectyplasia perox NR Mohamed et al.
sporulosum (2010)
Epoxyphomalin E Prenylated polyketide Dominica Paraconiothyrium Fungus Ectyplasia perox NR Mohamed et al.
sporulosum (2010)
Sorbifuranone A Sorbicillin Italy Penicillium Fungus Ircinia NR Bringmann et al.
Sorbifuranone B Sorbicillin Italy Penicillium Fungus Ircinia NR Bringmann et al.
Sorbifuranone A Sorbicillin Italy Penicillium Fungus Ircinia NR Bringmann et al.
Marilone B Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia Antagonistic of Almeida et al.
sp. cf. C. serotonin (2011)
flammea receptor
Butylrolactone-VI Dibenzylbutyrolactone Chile Aspergillus sp. Fungus Cliona chilensis Antibacterial San-Martin et al.
(2P-22) (2011)
Cillifuranone Intermediate in Croatia Penicillium Fungus Tethya Antibiotic Wiese et al. (2011)
sorbifuranone chrysogenum aurantium
strain LF066
Insuetolide A Meroterpene Israel Aspergillus Fungus Psammocinia Antifungal Cohen et al.
aculeatus sp. (2011)
Bendigole D 3-Keto sterol NR Actinomadura sp. Actinobacterium Suberites Anti- Simmons et al.
japonicus inflammatory, (2011)
cytotoxic
Bendigole F 3-Keto sterol NR Actinomadura sp. Actinobacterium Suberites Anti- Simmons et al.
japonicus inflammatory (2011)
(Continued)
Country
(3S,8R)-Methyl Hexylitaconic acid Korea Penicillium sp. Fungus Stelletta sp. Anti- Li et al. (2011b)
8-hydroxy-3- inflammatory
methoxycarbonyl-2-
methylenenonanoate
(3S)-Methyl-9-hydroxy-3- Hexylitaconic acid Korea Penicillium sp. Fungus Stelletta sp. Anti- Li et al. (2011b)
methoxycarbonyl-2- inflammatory
methylenenonanoate
Marilone A Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia Antimalarial and Almeida et al.
sp. cf. C. anticancer (2011)
flammea
Marilone C Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia Antimalarial and Almeida et al.
sp. cf. C. Anticancer (2011)
flammea
Myrocin D Diterpene Italy Arthrinium sp. Fungus Geodia Anticancer Ebada et al. (2011)
cydonium
22-Deoxythiocoraline Thiocoraline analog United Verrucosispora sp. Actinobacterium Chondrilla Cytotoxic Wyche et al.
States caribensis f. (2011)
caribensis
Thiochondrilline C Thiocoraline analog United Verrucosispora sp. Actinobacterium Chondrilla Cytotoxic Wyche et al.
caribensis
12-Sulfoxythiocoraline Thiocoraline analog United Verrucosispora sp. Actinobacterium Chondrilla Cytotoxic Wyche et al.
caribensis
Acremostrictin Tricyclic lactone Korea Acremonium Fungus Not identified Antibacterial, Julianti et al.
strictum miscellaneous (2011)
Insuetolide C Meroterpene Israel Aspergillus Fungus Psammocinia sp. Cytotoxic Cohen et al.
aculeatus (2011)
(E)-6-(4′-Hydroxy-2′- Sesquiterpene Israel Aspergillus Fungus Psammocinia sp. Cytotoxic Cohen et al.
butenoyl)-strobilactone A aculeatus (2011)
(Continued)
295
296

Country
Fellutamide F Lipopeptide Korea Aspergillus Fungus Petrosia sp. Cytotoxic Lee et al. (2011)
versicolor
Dihydrotrichodermolide Polyketide (sorbicillin NR (East Phialocephala sp. Fungus Stelletta sp. Cytotoxic Li et al. (2011a)
dimer) Pacific)
Dihydrodemethylsorbicillin Polyketide (sorbicillin NR (East Phialocephala sp. Fungus Stelletta sp. Cytotoxic Li et al. (2011a)
monomer) Pacific)
Phialofurone Benzofuranone NR (East Phialocephala sp. Fungus Stelletta sp. Cytotoxic Li et al. (2011a)
Pacific)
Bendigole E 3-Keto sterol NR Actinomadura sp. Actinobacterium Suberites NR Simmons et al.
japonicus (2011)
JBIR-56 Peptide Japan Streptomyces sp. Actinobacterium Not identified NR Motohashi et al.
(2011)
JBIR-57 Peptide Japan Streptomyces sp. Actinobacterium Not identified NR Motohashi et al.
(2011)
Streptomycindole Indole alkaloid China Streptomyces sp. Actinobacterium Craniella NR Huang et al.
australiensis (2011)
Thiochondrilline A Thiocoraline analog United Verrucosispora sp. Actinobacterium Chondrilla NR Wyche et al.
caribensis
Thiochondrilline B Thiocoraline analog United Verrucosispora sp. Actinobacterium Chondrilla NR Wyche et al.
caribensis
Arthrinin A Diterpene Italy Arthrinium sp. Fungus G. cydonium NR Ebada et al. (2011)
Arthrinin B Diterpene Italy Arthrinium sp. Fungus G. cydonium NR Ebada et al. (2011)
Arthrinin C Diterpene Italy Arthrinium sp. Fungus G. cydonium NR Ebada et al. (2011)
Arthrinin D Diterpene Italy Arthrinium sp. Fungus G. cydonium NR Ebada et al. (2011)
Asperaculin A Sesquiterpene Thailand Aspergillus Fungus Xestospongia NR Ingavat et al.
aculeatus testudinaria (2011)
Pre-aurantiamine Diketopiperazine Thailand Aspergillus Fungus Stylissa NR Antia et al. (2011)
aculeatus flabelliformis
(Continued)
Country
(−)-9-Hydroxyhexylitaconic Diketopiperazine Thailand Aspergillus Fungus S. flabelliformis NR Antia et al. (2011)
acid aculeatus
(−)-9-Hydroxyhexylitaconic Diketopiperazine Thailand Aspergillus Fungus S. flabelliformis NR Antia et al. (2011)
acid-4-methyl ester aculeatus
Insuetolide B Meroterpene Israel Aspergillus Fungus Psammocinia NR Cohen et al.
aculeatus sp. (2011)
Austalide M Meroterpene Italy Aspergillus sp. Fungus Tethya NR Zhou et al. (2011)
aurantium
Austalide N Meroterpene Italy Aspergillus sp. Fungus Tethya NR Zhou et al. (2011)
aurantium
Austalide O Meroterpene Italy Aspergillus sp. Fungus Tethya Cytotoxic Zhou et al. (2011)
aurantium
Austalide P Meroterpene Italy Aspergillus sp. Fungus Tethya Cytotoxic Zhou et al. (2011)
aurantium
Austalide Q Meroterpene Italy Aspergillus sp. Fungus Tethya Cytotoxic Zhou et al. (2011)
aurantium
(3S,8R)-8-Hydroxy-3-carboxy- Hexylitaconic acid Korea Penicillium sp. Fungus Stelletta sp. NR Li et al. (2011b)
2-methylenenonanoic acid
(3S)-9-Hydroxy-3-carboxy-2- Hexylitaconic acid Korea Penicillium sp. Fungus Stelletta sp. NR Li et al. (2011b)
methylenenonanoic acid
Stachyline A Tyrosine-derived Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
metabolite sp. cf. C. (2010)
flammea
Stachyline B Tyrosine-derived Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
flammea
Stachyline C Tyrosine-derived Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
metabolite sp. cf. C. flC. (2010)
fla
(Continued)
297
298

Country
Stachyline D Tyrosine-derived Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
flammea
Compound 1, new (5a,6a)- Sesterterpene NR Aspergillus ustus Fungus Suberites No significant Liu et al. (2011)
isomer of ophiobolin H (Adriatic domuncula cytotoxicity
Sea) found
Compound 2, (5a,6a)-5-O- Sesterterpene NR Aspergillus ustus Fungus Suberites No significant Liu et al. (2011)
methylophiobolin H (Adriatic domuncula cytotoxicity
Sea) found
Compound 3, Sesterterpene NR Aspergillus ustus Fungus Suberites No significant Liu et al. (2011)
5-O-methylophiobolin H (Adriatic domuncula cytotoxicity
Sea) found
Compound 4, s(6a)-21,21-O- Sesterterpene NR Aspergillus ustus Fungus Suberites No significant Liu et al. (2011)
dihydroophiobolin G (Adriatic domuncula cytotoxicity
Sea) found
Compound 5, (6a)-18,19,21,21- Sesterterpene NR Aspergillus ustus Fungus Suberites No significant Liu et al. (2011)
Otetrahydro-18,19- (Adriatic domuncula cytotoxicity
dihydroxyophiobolin G Sea) found
Tetromycin 1 Tetromycin France Streptomyces Actinobacterium Axinella Antiparasitic, Pimentel-Elardo
axinellae polypoides inhibition of et al. (2011)
cathepsin L-like
proteases
cathepsin L-like
proteases
cathepsin L-like
proteases
(Continued)
Country
cathepsin L-like
proteases
Lobophorin C Kijanimicin China Streptomyces Actinobacterium Hymeniacidon Cytotxic Wei et al. (2011)
microflavus perlevis
Lobophorin D Kijanimicin China Streptomyces Actinobacterium Hymeniacidon Anticancer Wei et al. (2011)
microflavus perlevis
JBIR-109 Trichostatin analog Japan Streptomyces sp. Actinobacterium Not identified Anticancer Hosoya et al.
strain RM72 (2012)
strain RM72 (2012)
strain RM72 (2012)
Urdamycinone E Glycosylated benz[α] Thailand Streptomycessp. Actinobacterium Xestospongia sp. Antimalarial, Supong et al.
anthraquinone BCC45596 antituberculosis (2012)
Urdamycinone G Glycosylated benz[α] Thailand Streptomyces sp. Actinobacterium Xestospongia sp. Antimalarial, Supong et al.
anthraquinone BCC45596 antituberculosis (2012)
Dehydroxyaquayamycin Glycosylated benz[α] Thailand Streptomyces sp. Actinobacterium Xestospongia Antimalarial, Supong et al.
anthraquinone BCC45596 sp. antituberculosis (2012)
Acremolin Methyl guanine Korea Acremonium Fungus Not identified Cytotoxic Julianti et al.
strictum Choristida (2012)
sponge
Disydonol A Sesquiterpene China Aspergillus sp. Fungus Xestospongia Cytotoxic Sun et al. (2012)
testudinaria
Disydonol C Sesquiterpene China Aspergillus sp. Fungus Xestospongia Cytotoxic Sun et al. (2012)
testudinaria
Aspergilusidone A Depsidone Thailand Aspergillus unguis Fungus Not identified Cytotoxic Sureram et al.
CRI282–03 (2012)
Aspergilusidone B Depsidone Thailand Aspergillus unguis Fungus Not identified Cytotoxic Sureram et al.
CRI282–03 (2012)
(Continued)
299
300

Country
Aspergilusidone C Depsidone Thailand Aspergillus unguis Fungus Not identified Cytotoxic Sureram et al.
CRI282–03 (2012)
New compound Diaryl ether Thailand Aspergillus unguis Fungus Not identified Cytotoxic Sureram et al.
CRI282–03 (2012)
Aspergiterpenoid A Sesquiterpene China Aspergillus sp. Fungus Xestospongia Antibacterial, Li et al. (2012)
testudinaria cytotoxic
(−)-Sydonol Sesquiterpene China Aspergillus sp. Fungus Xestospongia Antibacterial, Li et al. (2012)
(−)-Sydonic acid Sesquiterpene China Aspergillus sp. Fungus Xestospongia Antibacterial, Li et al. (2012)
(−)-5-(hydroxymethyl)-2- Sesquiterpene China Aspergillus sp. Fungus Xestospongia Antibacterial, Li et al. (2012)
(2′,6′,6′-trimethyltetrahydro- testudinaria cytotoxic
2H-pyran-2-yl)phenol
JBIR 124 Sorbicillin Japan Penicillium Fungus Not identified Miscellaneous Kawahara et al.
citrinum sp. (2012)
I080624G1f01
Cyclodysidin A Cyclic lipopeptide Croatia Streptomyces strain Actinobacterium Dysidea tupha NR Abdelmohsen
RV15 et al. (2012)
Cyclodysidin B Cyclic lipopeptide Croatia Streptomyces strain Actinobacterium Dysidea tupha NR Abdelmohsen
RV15 et al. (2012)
Cyclodysidin C Cyclic lipopeptide Croatia Streptomyces strain Actinobacterium Dysidea tupha NR Abdelmohsen
RV15 et al. (2012)
Cyclodysidin D Cyclic lipopeptide Thailand Streptomyces strain Actinobacterium Dysidea tupha NR Abdelmohsen
RV15 et al. (2012)
Disydonol B Sesquiterpene China Aspergillus sp. Fungus Xestospongia None Sun et al. (2012)
testudinaria
1-Hydroxy-10-methoxy- Dibenz[b,e]oxepine Japan Beauveria bassiana Fungus Not identified None Yamazaki et al.
dibenz[b,e]oxepin-6,11-dione TPU942 (2012)
(5E)-2-methyl-5-[(1′R*, Sesquiterpene Thailand Emericellopsis Fungus Hyrtios erecta None Pinheiro et al.
5′R*)-2-methylidene-7- minima (2012)
oxobicyclo[3.2.1]oct-6-
ylidene]-4-oxopentanoic acid
(Continued)
Country
Eurocristatine Diketopiperazine Thailand Eurotium cristatum Fungus Not identified None Gomes et al.
(2012)
JBIR-113 Depsipeptide Japan Penicillium sp. Fungus Not identified NR Kawahara et al.
(2012)
(2012)
(2012)
Cyclomarinone Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
Maristachone A Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
Maristachone B Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
Maristachone C Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)

flammea
Maristachone D Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
Maristachone E Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
Marilactone Phthalide (polyketide) Australia Stachylidium sp. Fungus Callyspongia NR Almeida et al.
sp. cf. C. (2012)
flammea
NR, not reported.
301
chemistry rather than describing the biological aspects of fungi associated with sponges
(Höller et al., 2000). Fungal associations with sponges have been well established (Yarden,
2014) though not well characterized (Suryanarayanan, 2012) and require further in-depth
study (Webster and Taylor, 2012). In this report, the fungi isolated from sponges between
2000 and 2012 that were reported to produce novel compounds belong to the following
genera: Acremonium, Arthrinium, Aspergillus, Beauveria, Cladosporium, Clonostachys, Curvularia,
Drechslera, Emericella, Emericellopsis, Eurotium, Exophiala, Gymnascella, Microsphaeropsis,
Myrothecium, Paecilomyces, Paraconiothyrium, Penicillium, Phialocephala, Phoma, Scopulariopsis,
Stachylidium, Trichoderma, and the yeast Pichia (Table 15.1). Aspergillus spp. produced 59
new compounds followed by Penicillium spp. that produced 26. Of a total of 272 natural
products that were isolated from fungi during the period of 1970–2002, 28% came from
sponges (Bugni and Ireland, 2004). During the current review period, 69% of the 269 new
compounds were produced by fungi from sponges.
As the ecology of fungi in the marine environment is revealed through molecular
ecology methods (Gao et al., 2008), the relationships between fungi and their hosts in the
marine environment will help our understanding of the marine ecosystem and could lead
to improved collection and isolation methods and the identification of chemically unex-
plored species (Bugni and Ireland, 2004; Abdelmohsen et al., 2014).
15.2.2 Actinobacteria
Actinobacterial associations have been found in reef and deepwater sponges, and evidence
for sponge-specific symbioses exists (Hentschel et al., 2006). Moreover, marine actinobacteria
in particular have yielded a higher percentage of novel secondary metabolites as compared
to fungi (Lam, 2006) in the past. In this review, we report on only seven genera of actino-
bacteria isolated from sponges that have been reported to produce 57 new compounds. The
genera are Actinomadura, Microbacterium, Micromonospora, Nocardiopsis, Saccharopolyspora, and
Verrucosispora, with Streptomyces spp. producing the highest number of compounds (32).
In at least one case, actinobacterial symbionts such as a Micromonospora sp. have been
shown to produce bioactive compounds (manzamines) that have no terrestrial equivalents
(Montalvo et al., 2005) and are of considerable interest. Sponges in the South China Sea har-
bor a large diversity of actinobacteria and show evidence of host specificity (Li et al., 2006).
15.2.3 Bacteria
Bacteria associated with sponges that were reported to produce new metabolites were
from four classes and seven genera only—Alphaproteobacteria (Mesorhizobium, Ruegeria),
Firmicutes (Bacillus), Gammaproteobacteria (Microbulbifer, Pseudoalteromonas, Pseudomonas),
and Verrucomicrobia (e.g., Rubritalea). However, no new compound has been reported
from sponge-associated cyanobacteria since 2000, which revealed that these photosyn-
thetic microorganisms were given less attention.
15.3 Sponge sources

Over the 13-year period (2000–2012), from a total of 104 reported studies, which would
have used at least one sample per study, 35 sponge genera and 30 unidentified sponges
were obtained from 23 countries or regions around the world. The sponge genera with the
highest number of reported compounds are Halichondria with 34 compounds from three
species from 12 separate studies. All three groups of microorganisms were isolated, but
from different studies. This was a common feature of the studies, where each publication
reported on compounds from only one type of microbe. Xestospongia with 25 compounds
from fungi and actinobacteria were reported in eight separate studies, Callyspongia spp.
with 22 compounds from fungi and bacteria in six separate studies, Stelletta spp. with 11
compounds from fungi only in four separate studies, and Suberites spp. with 10 compounds
from all three types of microorganism in three studies. The rest of the sponge samples pro-
duced between 1 and 8 compounds were reported in 1–3 studies for each genus.
15.4 Chemical diversity among the microbial

metabolites and their activities
A diverse array of compounds has been discovered from sponges such as terpenoids, ste-
roids, phenolic compounds, alkaloids, polysaccharides, peptides, polyketides, and fatty
acids, which all showed potential biological activity (Mehbub et al., 2014). Among the 269
new compounds isolated from sponge-associated microbes, only 159 compounds showed
bioactivity that includes antibacterial, anticancer/cytotoxicity, antifungal, anti-HIV,
anti-inflammatory, antiparasitic, antimalarial, antituberculosis, antiviral, and antiyeast
(Figure 15.2). It was noted that the fungi associated with sponges in particular produce sim-
ilar classes of compounds as sponges. Of particular note are the multiple congeners, with
about two-thirds of the compounds produced as a set of three or more congeners, which
is common in secondary metabolism. At least 40% of compounds had no activity reported
when the publication of the compounds first appeared in the literature. Subsequent test-
ing is likely to have taken place as it is unlikely that these new resources would not be
assessed for their bioactive potential. However, it was noted that there were few screening
assays reported that were based on receptor-binding activity or mode of action studies.
90
80
Number of new bioactive compounds
70
60
50
40
30
20
10
0
al l l st us
ity ng IV or
y
iti
c
ria sis ira ea
xic u i-H at ras ala ulo tiv iy neo
to tif nt m pa rc An nt lla
to An A m ti tim e A e
/cy fla An An t ub isc
er tii
n ti- M
a nc An An
tic
An
Bioactivity
Figure 15.2 The reported bioactivity of compounds isolated from microorganisms associated with
marine sponges from 2000 to 2012.
15.5 Identification of microbial diversity

Identification of microorganisms is important because biotechnology relies on defin-
ing their activities (Hugenholtz and Pace, 1996). Microbial species identified rapidly by
sequencing their signature ribosomal genes; 16S rRNA for bacteria and actinobacteria and
28S rRNA for fungi are the most common. Full characterization is achieved using polypha-
sic methodology that employs both phenotypic and genotypic characteristics (Vandamme
et al., 1996; Achtman and Wagner, 2008). Phenotypic methods include classical taxonomy,
colony characteristics, biochemical and physiological studies, numerical taxonomy, cell
wall composition, fatty acid analyses, isoprenoid quinones and whole-cell protein analy-
ses, polyamines, cytochromes, and in some cases advanced spectroscopic methods (FTIR,
pyrolysis mass spectrometry, UV resonance Raman spectroscopy). The genotypic methods
include 16S and 28S rRNA gene sequencing and, increasingly, multilocus gene sequencing,
DNA base content, and DNA–DNA hybridization (Sarethy et al., 2014).
15.6 Isolation techniques for bacteria,

actinobacteria, and fungi from sponges
The use of improved cultivation approaches for the discovery of marine natural products
from marine microorganisms is of paramount importance for the development of new
pharmaceuticals (Bull et al., 2005). Conventional isolation methods are used to cultivate
the most abundant microorganisms, but more selective methods are required to culture
the uncommon and new microbial species and genera that have not been isolated previ-
ously (Ferguson et al., 1984; Eilers et al., 2000). It has been shown recently that previously
uncultivable microorganisms could be grown in pure culture if provided with the chemi-
cal components of their natural environment (Kaeberlein et al., 2002; Rappe et al., 2002).
The main parameters that have influenced the isolation of novel strains include enrich-
ment, pretreatment of samples, choice of nutrients in isolation, application of appropriate
antibiotics, use of seawater and salt concentrations, inclusion of sponge extracts, and above
all cultivation techniques that mimic nature (Hameş-kocabaş and Uzel, 2012). Moreover,
temperature, pH, culture conditions, incubation time, and removal of emerging isolates
are also very important (Kaewkla and Franco, 2013).
Using specific enrichment techniques, primarily by varying the media, hundreds of
different organisms from the family of Streptosporangiacae were isolated, and many were
hypothesized to produce novel antibiotics on the basis of observations of other members
of this family (Lazzarini et al., 2001). In a separate example, by changing the media in
combination with using selective agents for motile microorganisms, two new antibiotic-
producing actinobacterial species were discovered, demonstrating the usefulness of a
guided-culturing approach for the isolation of rare strains (Otoguro et al., 2001). Finally, the
culturing of rare organisms has been improved by the development of new techniques. This
is illustrated by the use of microcapsules, derived from a single encapsulated cell, for high-
throughput screening (Zengler et al., 2005). These culturing techniques will undoubtedly
improve access to natural products as new microorganisms are isolated in large numbers.
15.6.1 Sponge sampling and isolation of microorganisms

Through the use of culture-independent molecular techniques, new insights into the struc-
ture of microbial communities have been gained (Vanwonterghem et al., 2014). Molecular tools
have a great potential to assist in developing strategies for identifying previously uncultured
bacteria. Identification of organisms via molecular tools can also help in determining appro-
priate culture media, most effective for isolation and purification of specific microorganisms.
These tools allow us to further investigate or exploit microorganisms (Stewart, 2012).
After deciding on the sponges to be sampled and their location, during the sponge
collection, careful attention is required to minimize contamination. Sterile ziplock bags or
sterile tubes are used accompanied by the rapid transfer of samples to avoid any possible
contamination from runoff (Fenical and Jensen, 2006).
Sterilized seawater is used to remove loosely attached microorganisms, and then the
sponge sample is cut into small pieces (~1 cm3). These parts are then homogenized in a
prechilled sterilized mortar and pestle using sterilized seawater or phosphate-buffered
saline (Zhang et al., 2008b; Abdelmohsen et al., 2010). The process of isolation of actinobac-
teria or fungi is to dilute the crushed sponge samples with sterile seawater or one-quarter
strength Ringer’s solution (Pathom-Aree et al., 2006; Bredholdt et al., 2007). After mixing by
either vortexing or shaking, they are transferred to the isolation agar media in petri dishes
(Jensen et al., 1991; Pathom-Aree et al., 2006).
Isolation media that were used for actinobacteria and for fungi from sponges are pre-
sented in Table 15.2 and Table 15.3, respectively.
Table 15.2 Media used for the successful isolation of actinobacteria from sponges
Medium ingredients References
GA: 6 mL 100% glycerol, 1 g arginine, 1 g K2HPO4, 0.5 g MgSO4 · 7H2O, 18 g Mincer et al. (2002)
agar, and 1 L natural seawater.
Noble: 8 g noble (purified) agar, 500 mg mannitol, 100 mg peptone, 1 L natural Jensen et al. (2005)
seawater, rifampicin (5 µg mL−l), and cycloheximide (100 µg mL−l).
Seawater: Eighteen grams of agar, 1 L natural seawater, rifampicin (5 µg mL−1), Jensen et al. (2005)
and cycloheximide (100 µg mL−l).
PA: 2 g peptone, 0.1 g asparagine, 4 g sodium propionate, 0.5 g K2HPO4, 0.1 g Zhang et al. (2008b)
MgSO4, 0.01 g FeSO4 · 7H2O, 5 g glycerol, 20 g NaCl, 18 g Difco Bacto agar, 1
l distilled water, K2Cr2O7 (50 µg mL–1), and 15 µg nalidixic acid (15 µg mL–1).
Artificial seawater medium (per liter): 23.0 g NaCl, 0.75 g KCl, 1.47 g CaCl2 · Wicke et al. (2000)
2H2O, 5.08 g MgCl2 · 6H2O, 6.16 g MgSO4 · 7H2O, 5 g NH2Cl, 3.5 g yeast
extract, 3.5 g peptone, 0.89 g Na2HPO4 · 2H2O, and 20 g glucose.
Bennett agar (per liter): 1 g yeast extract, 1 g beef extract, 2 g tryptone, 10 g Lee et al. (2005)
dextrose, and 15 g agar.
Bennett agar (as previous) supplemented with nalidixic acid (0.02%) and Pérez et al. (2009)
cycloheximide (0.02%).
Gause’s starch medium: 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g Li et al. (2005)
MgSO4 · 7H2O, 0.5 g NaCl, 0.01 g 7H2O, and 18 g agar 1 l water containing
50% natural seawater, and 0.1 mg mL−1 K2Cr2O7. (The pH of the solution
was set to 7.2 before sterilization.)
RH: 10 g raffinose, 1 g l-histidine, 1 g K2HPO4, 0.5 g MgSO4 · 5H2O, 0.01 g Maldonado et al.
FeSO4, and 15 g agar; pH 7.2. (2009)
Starch casein: 10 g soluble starch, 0.3 g casein, 2 g K2HPO4, 2 g KNO3, 2 g Maldonado et al.
NaCl, 0.05 g MgSO4 · 7H2O, 0.02 g CaCO3, 0.01 g FeSO4 · 7H2O, 15 g agar, (2005b)
and 1 L distilled water.
Salts: solution A (750 mL artificial seawater containing 1 g K2HPO4 and 10 g Magarvey et al.
Bacto Agar) and solution B (250 mL artificial seawater containing 1 g KNO3, (2004)
1 g MgSO4 · 7H2O, 1 g CaCl2 · 2H2O, 0.2 g FeCl3, 0.1 g MnSO4 · 7H2O).
(Solutions A and B are autoclaved separately and mixed and supplemented
with 1 mL trace element solution)
Table 15.3 Media used for isolation of fungi from sponges

Formula References
YMP (per liter): 3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g glucose, Jadulco et al. (2004)
and 24.4 g sea salt. (The pH of the medium was adjusted to 7.2–7.4 using
0.1 N NaOH or HCl prior to inoculation.)
Oatmeal agar: supplemented with 100% seawater. Cruz et al. (2006)
MEA medium (prepared with 75% seawater, obtained locally): 20 g glucose, Lee et al. (2010)
20 g malt extract, 20 g agar, 1 g peptone, penicillin (10,000 units mL−1), and
streptomycin (5 mg mL−1).
GPY agar: based on natural seawater of 30 PSU, containing per liter 1 g Wiese et al. (2011)
glucose, 0.5 g peptone, 01 g yeast extract, and 15 g agar.
PDA: potato dextrose agar (Difco) with 250 mg L−l. (Chloramphenicol was Paz et al. (2010)
also mended with different fungicides.)
Potato carrot agar (KM) per liter: 20 g cooked and mashed potatoes, 20 g Proksch et al. (2008),
cooked and mashed carrots, 20 g agar with and without artificial sea salts, Höller et al. (2000)
and cyclosporine A (0.5 mg L−1).
Modified ISP-4 Huang et al. (2012)
15.6.2 Pretreatment of sponges for selective isolation of actinobacteria

Pretreatment methods have been found successful to isolate rare or selective actinobac-
teria. Physical methods such as drying the sponges in laminar airflow and dilution have
been described (Jensen et al., 2005; Mehbub and Amin, 2012). In addition, dilution and
incubation in water bath at 50°C for 6 min and 40°C for 60 min have been described (Kim
et al., 2005; Selvin et al., 2009).
However, many pretreatment methods that have been described for isolation of acti-
nobacteria from marine sediments can also be applied for sponge samples: mechanical
disruption of sponge tissue using glass beads (Maldonado et al., 2009); physical treatment
such as drying, stamping, and dilution (Mincer et al., 2002; Jensen et al., 2005; Gontang
et al., 2007); heat treatment such as using dry heat and incubation in different temperatures
(Jensen et al., 1991; Mincer et al., 2002, 2005; Bredholt et al., 2008); freezing (Jensen et al.,
2005; Bredholdt et al., 2007); radiation (Bredholdt et al., 2007); heat and radiation (Eccleston
et al., 2008); centrifugation (Maldonado et al., 2005b); and chemical treatment such as
using 1.5% phenol (Bredholt et al., 2008). It has been established that for obligate marine
actinobacteria, sodium or salt is a prerequisite (Maldonado et al., 2005a). And, methods
could be used to isolate actinobacteria according to the desired result. Even careful use
of antibiotics is important for isolating targeted actinobacteria, such as the Salinispora
strain for which cycloheximide and rifampicin are commonly used (Mincer et al., 2005).
Besides, it has been revealed that UV irradiation is another effective method for getting
selective isolates of different actinobacterial genera. For instance, high frequency irradia-
tion helped to isolate Streptosporangium and Rhodococcus, and extremely high frequency
irradiation was favorable for isolating Nocardiopsis, Nocardia, and Streptosporangium spp.,
and UV radiation was effective for isolating Nocardiopsis, Nocardia, and Pseudonocardia
spp. (Bredholdt et al., 2007).
Careful selection of different nutrient sources is very important to isolate novel bacte-
ria or fungi because the microbes residing in the sponges are likely to grow under environ-
mental conditions that mimic their source. Moreover, the use of sponge extracts, sediment
extracts, different salts, and natural seawater has been practiced in the past. In addition,
soluble starch, glycerol, glucose, raffinose, and mannitol were popular as carbon sources,
while peptone, yeast, casein, nitrate, histidine, and l-asparagine were popular as nitrogen
sources (Jensen et al., 2005; Maldonado et al., 2005b, 2009; Mincer et al., 2005; Gontang et al.,
2007; Kennedy et al., 2009).
15.7 Concluding remarks

The discovery of new compounds from the oceans is increasing with over 1000 new com-
pounds per year reported from marine sponges alone. In light of this abundance, the num-
bers of new compounds reported from microorganisms associated with sponges may seem
less significant at around 35 compounds per year in recent years. However, the currently
insurmountable problem of getting a continuous supply of a sponge metabolite for drug
development if chemical synthesis is not economic will lead to a more intensive search
for microbial sources, while efforts are made to culture sponge cells with the capability to
produce the desired compounds on a large scale.
It is not surprising that fungi have overtaken the prokaryotes in the search for new
secondary metabolites due to their ability to synthesize compounds belonging to the
same chemical classes as their sponge hosts. To date, there have been no reports of fungal
symbionts producing a sponge-derived metabolite. While fungi from sponge continue
to be the dominant type of microbial producers of new compounds, it is important that
studies on their ecology and interactions be undertaken to improve our understanding
of their role in this ecosystem. One of the benefits will be to realize new isolation meth-
ods and techniques to reveal a broader diversity than is currently known from sponge
samples. It was noted that a high proportion of the new metabolites are reported without
any reported bioactivity associated with them; when there are reports, the bioactivity is
from a nonspecific evaluation such as antimicrobial activity, rather than a specific mode
of action assay based on a new target site that might reveal a new compound early. Most
reports of anticancer activity are also based on nonspecific cytotoxic activity against one
or more mammalian cell lines without further detailed assessment as to their mecha-
nisms of action.
Further analysis of the reporting of bioactivity also shows that most compounds are
reported to be tested in one assay. Therefore, the challenge is not only to find new com-
pounds but also to develop mechanisms to evaluate their activity in a range of assays. This
will allow the full potential of these varied molecules to be realized in the search for new
and effective therapies for the range of diseases that are still awaiting a cure. Microbial
secondary metabolites are produced by organisms that can be readily scaled up so that the
supply of the compound can meet future demand.
References
Abdel-Lateffa, A., Fischa, K., and Wrightd, A. D. 2009. Trichopyrone and other constituents from the
marine sponge-derived fungus Trichoderma sp. Z. Naturforsch. B. 64:186–192.
Abdelmohsen, U. R., Bayer, K., and Hentschel, U. 2014. Diversity, abundance and natural products of
marine sponge-associated actinomycetes. Nat. Prod. Rep. 31:381–399.
Abdelmohsen, U. R., Pimentel-Elardo, S. M., Hanora, A., Radwan, M., Abou-El-Ela, S. H., Ahmed, S.,
and Hentschel, U. 2010. Isolation, phylogenetic analysis and anti-infective activity screening of
marine sponge-associated actinomycetes. Mar. Drugs 8:399–412.
Abdelmohsen, U. R., Zhang, G., Philippe, A., Schmitz, W., Pimentel-Elardo, S. M., Hertlein-
Amslinger, B., and Bringmann, G. 2012. Cyclodysidins A–D, cyclic lipopeptides from the
marine sponge-derived Streptomyces strain RV15. Tetrahedron Lett. 53:23–29.
Achtman, M. and Wagner, M. 2008. Microbial diversity and the genetic nature of microbial species.
Nat. Rev. Microbiol. 6:431–440.
Adachi, K., Kanoh, K., Wisespongp, P., Nishijima, M., and Shizuri, Y. 2005. Clonostachysins A
and B, new anti-dinoflagellate cyclic peptides from a marine-derived fungus. J. Antibiot.
58:145–150.
Almeida, C., Eguereva, E., Kehraus, S., and Konig, G. M. 2012. Unprecedented Polyketides from a
marine sponge-associated Stachylidium sp. J. Nat. Prod. 76:322–326.
Almeida, C., Kehraus, S., Prudêncio, M., and König, G. M. 2011. Marilones A–C, phthalides from the
sponge-derived fungus Stachylidium sp. Beilstein J. Org. Chem. 7:1636–1642.
Almeida, C., Part, N., Bouhired, S., Kehraus, S., and König, G. M. 2010. Stachylines A−D from the
sponge-derived fungus Stachylidium sp. J. Nat. Prod. 74:21–25.
Amagata, T., Morinaka, B. I., Amagata, A., Tenney, K., Valeriote, F. A., Lobkovsky, E., and Crews, P.
2006. A chemical study of cyclic depsipeptides produced by a sponge-derived fungus. J. Nat.
Prod. 69:1560–1565.
Amagata, T., Tanaka, M., Yamada, T., Minoura, K., and Numata, A. 2008. Gymnastatins and
dankastatins, growth inhibitory metabolites of a Gymnascella species from a Halichondria
sponge. J. Nat. Prod. 71:340–345.
Antia, B. S., Aree, T., Kasettrathat, C., Wiyakrutta, S., Ekpa, O. D., Ekpe, U. J., and Kittakoop, P. 2011.
Itaconic acid derivatives and diketopiperazine from the marine-derived fungus Aspergillus
aculeatus CRI322–03. Phytochemistry. 72:816–820.
Baltz, R. H., Miao, V., and Wrigley, S. K. 2005. Natural products to drugs: Daptomycin and related
lipopeptide antibiotics. Nat. Prod. Rep. 22:717–741.
Biabani, M. A. F. and Laatsch, H. 1998. Advances in chemical studies on low-molecular weight
metabolites of marine fungi. J. Prakt. Chem. 340:589–607.
Blunt, J. W., Copp, B. R., Hu, W.-P., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2007. Marine natu-
ral products. Nat. Prod. Rep. 24:31–86.
Blunt, J. W., Copp, B. R., Hu, W.-P., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2008. Marine natural
products. Nat. Prod. Rep. 25:35–94.
Blunt, J. W., Copp, B. R., Hu, W.-P., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2009. Marine n atural
products. Nat. Prod. Rep. 26:170–244.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H. G., and Prinsep, M. R. 2012. Marine natural p roducts.
Nat. Prod. Rep. 29:144–222.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H. G., and Prinsep, M. R. 2013. Marine natural p roducts.
Nat. Prod. Rep. 30:237–323.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H. G., and Prinsep, M. R. 2014. Marine natural products.
Nat. Prod. Rep. 31:160–258.
Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2003. Marine natural p roducts.
Nat. Prod. Rep. 20:1–48.
Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2006. Marine natural prod-
ucts. Nat. Prod. Rep. 23:26–78.
Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2010. Marine natural products.
Nat. Prod. Rep. 27:165–237.
Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2011. Marine natural prod-
ucts. Nat. Prod. Rep. 28:196–268.
Boot, C. M., Tenney, K., Valeriote, F. A., and Crews, P. 2006. Highly N-methylated linear peptides
produced by an atypical sponge-derived Acremonium sp. J. Nat. Prod. 69:83–92.
Brady, S. F., Simmons, L., Kim, J. H., and Schmidt, E. W. 2009. Metagenomic approaches to natural
products from free-living and symbiotic organisms. Nat. Prod. Rep. 26:1488–1503.
Bredholdt, H., Galatenko, O. A., Engelhardt, K., Fjaervik, E., Terekhova, L. P., and Zotchev, S. B.
2007. Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord,
Norway: Isolation, diversity and biological activity. Environ. Microbiol. 9:2756–2764.
Bredholt, H., Fjaervik, E., Johnsen, G., and Zotchev, S. B. 2008. Actinomycetes from sediments in the
Trondheim fjord, Norway: Diversity and biological activity. Mar. Drugs 6:12–24.
Bringmann, G., Lang, G., Bruhn, T., Schäffler, K., Steffens, S., Schmaljohann, R., and Imhoff, J. F.
2010. Sorbifuranones A–C, sorbicillinoid metabolites from Penicillium strains isolated from
Mediterranean sponges. Tetrahedron 66:9894–9901.
Bringmann, G., Lang, G., Gulder, T. A., Tsuruta, H., Mühlbacher, J., Maksimenka, K., and Müller,
W. E. 2005. The first sorbicillinoid alkaloids, the antileukemic sorbicillactones A and B, from a
sponge-derived Penicillium chrysogenum. Tetrahedron 61:7252–7265.
Bringmann, G., Lang, G., Steffens, S., and Schaumann, K. 2004. Petrosifungins A and B, novel cyclodep-
sipeptides from a sponge-derived strain of Penicillium brevicompactum. J. Nat. Prod. 67:311–315.
Bugni, T. S. and Ireland, C. M. 2004. Marine-derived fungi: A chemically and biologically diverse
group of microorganisms. Nat. Prod. Rep. 21:143–163.
Bull, A. T., Stach, J. E., Ward, A. C., and Goodfellow, M. 2005. Marine actinobacteria: Perspectives,
challenges, future directions. Antonie Van Leeuwenhoek 87:65–79.
Choi, E. J., Kwon, H. C., Ham, J., and Yang, H. O. 2009. 6-Hydroxymethyl-1-phenazine-carboxamide
and 1,6-phenazinedimethanol from a marine bacterium, Brevibacterium sp. KMD 003, associ-
ated with marine purple vase sponge. J. Antibiot. 62:621–624.
Cohen, E., Koch, L., Thu, K. M., Rahamim, Y., Aluma, Y., Ilan, M., and Carmeli, S. 2011. Novel terpe-
noids of the fungus Aspergillus insuetus isolated from the Mediterranean sponge Psammocinia
sp. collected along the coast of Israel. Biorg. Med. Chem. 19:6587–6593.
Cruz, L. J., Insua, M. M., Baz, J. P. et al. 2006. IB-01212, a new cytotoxic cyclodepsipeptide isolated
from the marine fungus Clonostachys sp. ESNA-A009. J. Org. 71:3335–3338.
Cueto, M., MacMillan, J. B., Jensen, P. R., and Fenical, W. 2006. Tropolactones A–D, four meroter-
penoids from a marine-derived fungus of the genus Aspergillus. Phytochemistry 67:1826–1831.
De Rosa, S., De Giulio, A., Tommonaro, G., Popov, S., and Kujumgiev, A. 2000. A β-Amino acid
containing tripeptide from a Pseudomonas−Alteromonas bacterium associated with a Black Sea
sponge. J. Nat. Prod. 63:1454–1455.
Ebada, S. S., Schulz, B., Wray, V., Totzke, F., Kubbutat, M. H., Müller, W. E., and Proksch, P. 2011.
Arthrinins A–D: Novel diterpenoids and further constituents from the sponge derived fungus
Arthrinium sp. Biorg. Med. Chem. 19:4644–4651.
Eccleston, G. P., Brooks, P. R., and Kurtböke, D. I. 2008. The occurrence of bioactive micromonospo-
rae in aquatic habitats of the Sunshine Coast in Australia. Mar. Drugs 6:243–261.
Edrada, R. A., Ebel, R., Supriyono, A., Wray, V., Schupp, P., Steube, K., and Proksch, P. 2002.
Swinhoeiamide A, a new highly active Calyculin derivative from the marine sponge Theonella
swinhoei. J. Nat. Prod. 65:1168–1172.
Edrada, R. A., Wray, V., Berg, A., Grafe, U., Sudarsono, U., Brauers, G., and Proksch, P. 2000. Original
communications-novel spiciferone derivatives from the fungus Drechslera hawaiiensis isolated
from the marine sponge Callyspongia aerizusa. Z. Naturforsch. 55:218–221.
Eilers, H., Pernthaler, J., and Amann, R. 2000. Succession of pelagic marine bacteria during enrich-
ment: A close look at cultivation-induced shifts. Appl. Environ. Microbiol. 66:4634–4640.
Elbandy, M., Shinde, P. B., Hong, J., Bae, K. S., Kim, M. A., Lee, S. M., and Jung, J. H. 2009. α-Pyrones
and yellow pigments from the sponge-derived fungus Paecilomyces lilacinus. Bull. Korean Chem.
Soc. 30:189.
Faulkner, D., Hai-yin, H., Unson, M., Bewley, C., and Garson, M. 1993. New metabolites from marine
sponges: Are symbionts important? Gazz. Chim. Ital. 123:301–307.
Faulkner, D. J. 2002. Marine natural products. Nat. Prod. Rep. 19:1–48.
Fenical, W. and Jensen, P. R. 2006. Developing a new resource for drug discovery: Marine actinomy-
cete bacteria. Nat. Chem. Biol. 2:666–673.
Ferguson, R. L., Buckley, E., and Palumbo, A. 1984. Response of marine bacterioplankton to differ-
ential filtration and confinement. Appl. Environ. Microbiol. 47:49–55.
Fisher, J. 2014. Modern NMR Techniques for Synthetic Chemistry. CRC Press.
Fujii, I. 2009. Heterologous expression systems for polyketide synthases. Nat. Prod. Rep. 26:155–169.
Gao, Z., Li, B., Zheng, C., and Wang, G. 2008. Molecular detection of fungal communities in the
Hawaiian marine sponges Suberites zeteki and Mycale armata. Appl. Environ. Microbiol.
74:6091–6101.
Garson, M., Flowers, A. E., Webb, R. I., Charan, R. D., and McCaffrey, E. J. 1998. A sponge/dinoflagel-
late association in the haplosclerid sponge Haliclona sp.: Cellular origin of cytotoxic alkaloids
by Percoll density gradient fractionation. Cell Tissue Res. 293:365–373.
Gomes, N. M., Dethoup, T., Singburaudom, N., Gales, L., Silva, A., and Kijjoa, A. 2012. Eurocristatine,
a new diketopiperazine dimer from the marine sponge-associated fungus Eurotium cristatum.
Phytochem Lett.
Gontang, E. A., Fenical, W., and Jensen, P. R. 2007. Phylogenetic diversity of gram-positive bacteria
cultured from marine sediments. Appl. Environ. Microbiol. 73:3272–3282.
Govindasamy, V., Franco, C. M., and Gupta, V. V. 2014. Endophytic actinobacteria: Diversity and
ecology. Advances in Endophytic Research. Springer, pp. 27–59.
Hameş-kocabaş, E. and Uzel, A. 2012. Isolation strategies of marine-derived actinomycetes from
sponge and sediment samples. J. Microbiol. Methods. 88:342–347.
Hao, G., Qing-Hua, Z., Miao-Miao, J., Jin-Shan, T., Cheng-Du, M., Kui, H., and Xin-Sheng, Y. 2008.
Polyketides from a marine sponge‐derived fungus Mycelia sterilia and proton–proton long‐
range coupling. Magn. Reson. Chem. 46:1148–1152.
Hentschel, U., Fieseler, L., Wehrl, M. et al. 2003. Microbial diversity of marine sponges. Prog. Mol.
Subcell. Biol. 37:59–88.
Hentschel, U., Hopke, J., Horn, M., Friedrich, A. B., Wagner, M., Hacker, J., and Moore, B. S. 2002.
Molecular evidence for a uniform microbial community in sponges from different oceans.
Appl. Environ. Microbiol. 68:4431–4440.
Hentschel, U., Usher, K. M., and Taylor, M. W. 2006. Marine sponges as microbial fermenters. FEMS
Microbiol. Ecol. 55:167–177.
Hernández, L. M., Blanco, J. A., Baz, J. P., Puentes, J. L., Millán, F. R., Vázquez, F. E., Fernández-
Chimeno, R. I., and Grávalos, D. G. 2000. 4′-N-methyl-5′-hydroxystaurosporine and 5′-hydroxys-
taurosporine, new indolocarbazole alkaloids from a marine Micromonospora sp. strain.
J. Antibiot. 53:895–902.
Höller, U., Wright, A. D., Matthee, G. F., Konig, G. M., Draeger, S., Aust, H. J., and Schulz, B. 2000.
Fungi from marine sponges: Diversity, biological activity and secondary metabolites. Mycol.
Res. 104:1354–1365.
Hosoya, T., Hirokawa, T., Takagi, M., and Shin-ya, K. 2012. Trichostatin analogues JBIR-109, JBIR-110,
and JBIR-111 from the marine sponge-derived Streptomyces sp. RM72. J. Nat. Prod. 75:285–289.
Huang, H., Yang, T., Ren, X., Liu, J., Song, Y., Sun, A., and Ju, J. 2012. Cytotoxic angucycline class glyco-
sides from the deep sea actinomycete Streptomyces lusitanus SCSIO LR32. J. Nat. Prod. 75:202–208.
Huang, X. L., Gao, Y., Xue, D. Q., Liu, H. L., Peng, C. S., Zhang, F. L., and Guo, Y. W. 2011.
Streptomycindole, an Indole alkaloid from a marine Streptomyces sp. DA22 associated with
South China sea sponge Craniella australiensis. Helv. Chim. Acta. 94:1838–1842.
Hugenholtz, P. and Pace, N. R. 1996. Identifying microbial diversity in the natural environment: A
molecular phylogenetic approach. Trends Biotechnol. 14:190–197.
Ingavat, N., Dobereiner, J., Wiyakrutta, S., Mahidol, C., Ruchirawat, S., and Kittakoop, P. 2009.
Aspergillusol A, an α-glucosidase inhibitor from the marine-derived fungus Aspergillus acu-
leatus. J. Nat. Prod. 72:2049–2052.
Ingavat, N., Mahidol, C., Ruchirawat, S., and Kittakoop, P. 2011. Asperaculin A, a sesquiterpenoid
from a marine-derived fungus, Aspergillus aculeatus. J. Nat. Prod. 74:1650–1652.
Izumikawa, M., Khan, S. T., and Komaki, H. 2009. JBIR-37 and -38, novel glycosyl benzenediols,
isolated from the sponge-derived fungus, Acremonium sp. SpF080624G1f01. Biosci. Biotechnol.
Biochem. 73:2138–2140.
Jadulco, R., Edrada, R. A., Ebel, R., Berg, A., Schaumann, K., Wray, V., and Proksch, P. 2004. New
communesin derivatives from the fungus Penicillium sp. derived from the Mediterranean
sponge Axinella verrucosa. J. Nat. Prod. 67:78–81.
Jensen, P. R., Dwight, R., and Fenical, W. 1991. Distribution of actinomycetes in near-shore tropical
marine sediments. Appl. Environ. Microbiol. 57:1102–1108.
Jensen, P. R., Gontang, E., Mafnas, C., Mincer, T. J., and Fenical, W. 2005. Culturable marine actino-
mycete diversity from tropical Pacific Ocean sediments. Environ. Microbiol. 7:1039–1048.
Julianti, E., Oh, H., Jang, K. H., Lee, J. K., Lee, S. K., Oh, D. C., and Shin, J. 2011. Acremostrictin, a highly
oxygenated metabolite from the marine fungus Acremonium strictum. J. Nat. Prod. 74:2592–2594.
Julianti, E., Oh, H., Lee, H.-S., Oh, D.-C., Oh, K.-B., and Shin, J. 2012. Acremolin, a new 1H-azirine
metabolite from the marine-derived fungus Acremonium strictum. Tetrahedron Lett. 53:2885–2886.
Kaeberlein, T., Lewis, K., and Epstein, S. S. 2002. Isolating “uncultivable” microorganisms in pure
culture in a simulated natural environment. Science 296:1127–1129.
Kaewkla, O. and Franco, C. M. 2013. Kribbella endophytica sp. nov., an endophytic actinobacterium
isolated from the surface-sterilized leaf of a native apricot tree. Int. J. Syst. Evol. Microbiol.
63:1249–1253.
Kanoh, K., Kamino, K., Leleo, G., Adachi, K., and Shizuri, Y. 2003. Pseudoalterobactin A and B,
new siderophores excreted by marine bacterium Pseudoalteromonas sp. KP20–4. J. Antibiot.
56:871–875.
Kawahara, T., Takagi, M., and Shin-ya, K. 2012. Three new depsipeptides, JBIR-113, JBIR-114 and
JBIR-115, isolated from a marine sponge-derived Penicillium sp. fS36. J. Antibiot. 65:147–150.
Kennedy, J., Baker, P., Piper, C., Cotter, P. D., Walsh, M., Mooij, M. J., and Dobson, A. D. 2009. Isolation
and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simu-
lans collected from Irish waters. Mar. Biotechnol. 11:384–396.
Kim, T. K., Garson, M. J., and Fuerst, J. A. 2005. Marine actinomycetes related to the ‘Salinospora’
group from the Great Barrier Reef sponge Pseudoceratina clavata. Environ. Microbiol. 7:509–518.
Kito, K., Ookura, R., Yoshida, S., Namikoshi, M., Ooi, T., and Kusumi, T. 2007. Pentaketides relating
to aspinonene and dihydroaspyrone from a marine-derived fungus, Aspergillus ostianus. J. Nat.
Prod. 70:2022–2025.
Kito, K., Ookura, R., Yoshida, S., Namikoshi, M., Ooi, T., and Kusumi, T. 2008. New cytotoxic
14-membered macrolides from marine-derived fungus Aspergillus ostianus. Org. Lett. 10:225–228.
Koehn, F. E. and Carter, G. T. 2005. The evolving role of natural products in drug discovery. Nat. Rev.
Drug Discovery. 4:206–220.
König, G. M., Kehraus, S., Seibert, S. F., Abdel-Lateff, A., and Müller, D. 2006. Natural products from
marine organisms and their associated microbes. ChemBioChem 7:229–238.
Krick, A., Kehraus, S., Eberl, L., Riedel, K., Anke, H., Kaesler, I., and König, G. M. 2007. A marine
Mesorhizobium sp. produces structurally novel long-chain N-acyl-l-homoserine lactones. Appl.
Environ. Microbiol. 73:3587–3594.
Lam, K. S. 2006. Discovery of novel metabolites from marine actinomycetes. Curr. Opin. Microbiol.
9:245–251.
Lazzarini, A., Cavaletti, L., Toppo, G., and Marinelli, F. 2001. Rare genera of actinomycetes as poten-
tial producers of new antibiotics. Antonie Van Leeuwenhoek 79:399–405.
Lee, H.-S., Shin, H. J., Jang, K. H., Kim, T. S., Oh, K.-B., and Shin, J. 2005. Cyclic peptides of the
nocardamine class from a marine-derived bacterium of the genus Streptomyces. J. Nat. Prod.
68:623–625.
Lee, Y. M., Dang, H. T., Li, J., Zhang, P., Hong, J., Lee, C. O., and Jung, J. H. 2011. A cytotoxic fellut-
amide analogue from the sponge-derived fungus Aspergillus versicolor. Bull. Korean Chem. Soc.
32:3817–3820.
Lee, Y. M., Hong, J., Lee, C.-O., Bae, K. S., Kim, D.-K., and Jung, J. H. 2010. A cytotoxic lipopeptide
from the sponge-derived fungus Aspergillus versicolor. Bull. Korean Chem. Soc. 31:205–208.
Li, C. S., An, C. Y., Li, X. M., Gao, S. S., Cui, C. M., Sun, H. F., and Wang, B. G. 2011a. Triazole and dihy-
droimidazole alkaloids from the marine sediment-derived fungus Penicillium paneum SD-44.
J. Nat. Prod. 74:1331–1334.
Li, D., Xu, Y., Shao, C. L., Yang, R. Y., Zheng, C. J., Chen, Y. Y., and Wang, C. Y. 2012. Antibacterial
Bisabolene-type sesquiterpenoids from the sponge-derived fungus Aspergillus sp. Mar. Drugs
10:234–241.
Li, F., Maskey, R. P., Qin, S., Sattler, I., Fiebig, H. H., Maier, A., and Laatsch, H. 2005. Chinikomycins
A and B: Isolation, structure elucidation, and biological activity of novel antibiotics from a
marine Streptomyces sp. isolate M045#, 1. J. Nat. Prod. 68:349–353.
Li, J. L., Zhang, P., Lee, Y. M., Hong, J., Yoo, E. S., Bae, K. S., and Jung, J. H. 2011b. Oxygenated hexyli-
taconates from a marine sponge-derived fungus Penicillium sp. Chem. Pharm. Bull. 59:120–123.
Li, Z.-Y., He, L.-M., Wu, J., and Jiang, Q. 2006. Bacterial community diversity associated with four
marine sponges from the South China Sea based on 16S rDNA-DGGE fingerprinting. J. Exp.
Mar. Biol. Ecol. 329:75–85.
Lin, W. H., Fu, H. Z., Li, J., and Proksch, P. 2001a. Novel chromone derivatives from marine fungus
Aspergillus versicolor isolated from the sponge Xestospongia exigua. Chin. Chem. Lett. 12:235–238.
Lin, W. H., Li, J., Fu, H. Z., and Proksch, P. 2001b. Four novel hydropyranoindeno-derivatives from
marine fungus Aspergillus versicolor. Chin. Chem. Lett. 12:435–438.
Liu, H. B., Edrada-Ebel, R., Ebel, R., Wang, Y., Schulz, B., Draeger, S., and Proksch, P. 2011. Ophiobolin
sesterterpenoids and pyrrolidine alkaloids from the sponge‐derived fungus Aspergillus ustus.
Helv. Chim. Acta. 94:623–631.
Magarvey, N. A., Keller, J. M., Bernan, V., Dworkin, M., and Sherman, D. H. 2004. Isolation and char-
acterization of novel marine-derived actinomycete taxa rich in bioactive metabolites. Appl.
Maldonado, L., Fragoso-Yáñez, D., Pérez-García, A., Rosellón-Druker, J., and Quintana, E. 2009.
Actinobacterial diversity from marine sediments collected in Mexico. Antonie Van Leeuwenhoek
95:111–120.
Maldonado, L. A., Fenical, W., Jensen, P. R., Kauffman, C. A., Mincer, T. J., Ward, A. C., and
Goodfellow, M. 2005a. Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov.,
obligate marine actinomycetes belonging to the family Micromonosporaceae. Int. J. Syst. Evol.
Microbiol. 55:1759–1766.
Maldonado, L. A., Stach, J. E. M., Pathom-Aree, W., Ward, A. C., Bull, A. T., and Goodfellow, M.
2005b. Diversity of cultivable actinobacteria in geographically widespread marine sediments.
Antonie Van Leeuwenhoek 87:11–18.
Malmstrøm, J., Christophersen, C., Barrero, A. F., Oltra, J. E., Justicia, J., and Rosales, A. 2002.
Bioactive metabolites from a marine-derived strain of the fungus Emericella variecolor. J. Nat.
Prod. 65:364–367.
Mehbub, M., Lei, J., Franco, C., and Zhang, W. 2014. Marine sponge derived natural products between
2001 and 2010: Trends and opportunities for discovery of bioactives. Mar. Drugs 12:4539–4577.
Mehbub, M. F. and Amin, A. K. M. R. 2012. Isolation and identification of actinobacteria from two
South Australian marine sponges Aplysilla rosea and Aplysina sp. BRP. 7:345–354.
Mincer, T. J., Fenical, W., and Jensen, P. R. 2005. Culture-dependent and culture-independent
diversity within the obligate marine actinomycete genus Salinispora. Appl. Environ. Microbiol.
71:7019–7028.
Mincer, T. J., Jensen, P. R., Kauffman, C. A., and Fenical, W. 2002. Widespread and persistent popula-
68:5005–5011.
Mitova, M., Popov, S., and De Rosa, S. 2004. Cyclic peptides from a Ruegeria strain of bacteria associ-
ated with the sponge Suberites domuncula. J. Nat. Prod. 67:1178–1181.
Mitova, M. I., Lang, G., Wiese, J., and Imhoff, J. F. 2008. Subinhibitory concentrations of antibiotics
induce phenazine production in a marine Streptomyces sp. J. Nat. Prod. 71:824–827.
Mohamed, I. E., Gross, H., Pontius, A., Kehraus, S., Krick, A., Kelter, G., and König, G. M. 2009.
Epoxyphomalin A and B, prenylated polyketides with potent cytotoxicity from the marine-
derived fungus Phoma sp. Org. Lett. 11:5014–5017.
Mohamed, I. E., Kehraus, S., Krick, A., König, G. M., Kelter, G., Maier, A., and Gross, H. 2010. Mode
of action of epoxyphomalins A and B and characterization of related metabolites from the
marine-derived fungus Paraconiothyrium sp. J. Nat. Prod. 73:2053–2056.
Montalvo, N. F., Mohamed, N. M., Enticknap, J. J., and Hill, R. T. 2005. Novel actinobacteria from
marine sponges. Antonie Van Leeuwenhoek 87:29–36.
Motohashi, K., Inaba, K., Fuse, S., Doi, T., Izumikawa, M., Khan, S. T., and Shin-ya, K. 2011. JBIR-56 and
JBIR-57, 2(1H)-pyrazinones from a marine sponge-derived Streptomyces sp. SpD081030SC-03.
J. Nat. Prod. 74:1630–1635.
Motohashi, K., Inaba, S., Takagi, M., and Shin-ya, K. 2009. JBIR-15, a new aspochracin derivative,
isolated from a sponge-derived fungus, Aspergillus sclerotiorum Huber Sp080903f04. Biosci.
Biotechnol. Biochem. 73:1898–1900.
Motohashi, K., Takagi, M., and Shin-ya, K. 2010. Tetrapeptides possessing a unique skeleton, JBIR-34
and JBIR-35, isolated from a sponge-derived actinomycete, Streptomyces sp. Sp080513GE-23.
J. Nat. Prod. 73:226–228.
Muldoon, J., Shashkov, A. S., Sof’ya, N. S., Tomshich, S. V., Komandrova, N. A., Romanenko, L.
A., and Savage, A. V. 2001. Structure of an acidic polysaccharide from a marine bacterium
Pseudoalteromonas distincta KMM 638 containing 5-acetamido-3,5,7,9-tetradeoxy-7-formamido-
l-glycero-l-manno-nonulosonic acid. Carbohydr. Res. 330:231–239.
Nagai, K., Kamigiri, K., Arao, N., Suzumura, K. I., Kawano, Y., Yamaoka, M., and Suzuki, K. 2003.
YM-266183 and YM-266184, novel thiopeptide antibiotics produced by Bacillus cereus isolated
from a marine sponge. I. Taxonomy, fermentation, isolation, physico-chemical properties and
biological properties. J. Antibiot. 56:123–128.
Newman, D. J. and Cragg, G. M. 2007. Natural products as sources of new drugs over the last
25 years. J. Nat. Prod. 70:461–477.
the period 1981–2002. J. Nat. Prod. 66:1022–1037.
Neumann, K., Abdel-Lateff, A., Wright, A. D., Kehraus, S., Krick, A., and König, G. M. 2007. Novel
Sorbicillin derivatives with an unprecedented carbon skeleton from the sponge‐derived fun-
gus Trichoderma species. Eur. J. Org. Chem. 2007:2268–2275.
Neumann, K., Kehraus, S., Gütschow, M., and König, G. 2009. Cytotoxic and HLE-inhibitory tetra-
mic acid derivatives from marine-derived fungi. Nat. Prod. Commun. 4:347–354.
Ookura, R., Kito, K., Ooi, T., Namikoshi, M., and Kusumi, T. 2008. Structure revision of circumdatins
A and B, benzodiazepine alkaloids produced by marine fungus Aspergillus ostianus, by x-ray
crystallography. J. Org. 73:4245–4247.
Otoguro, M., Hayakawa, M., Yamazaki, T., and Iimura, Y. 2001. An integrated method for the enrich-
ment and selective isolation of Actinokineospora spp. in soil and plant litter. J. Appl. Microbiol.
91:118–130.
Pathom-Aree, W., Stach, J. E., Ward, A. C., Horikoshi, K., Bull, A. T., and Goodfellow, M. 2006.
Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the
Mariana Trench. Extremophiles 10:181–189.
Paul, V. J. and Ritson-Williams, R. 2008. Marine chemical ecology. Nat. Prod. Rep. 25:662–695.
Pawlik, J. R. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar.
Biol. Annu. Rev. 30:273–335.
Paz, Z., Komon-Zelazowska, M., Druzhinina, I. S., Aveskamp, M. M., Shnaiderman, A., Aluma, Y.,
and Yarden, O. 2010. Diversity and potential antifungal properties of fungi associated with a
Mediterranean sponge. Fungal Divers. 42:17–26.
Pérez, M., Schleissner, C., Rodríguez, P., Zúñiga, P., Benedit, G., Sánchez-Sancho, F., and de la Calle, F.
2009. PM070747, a new cytotoxic angucyclinone from the marine-derived Saccharopolyspora
taberi PEM-06-F23–019B. J. Antibiot. 62:167–169.
Piel, J. 2004. Metabolites from symbiotic bacteria. Nat. Prod. Rep. 21:519–538.
Piel, J. 2006. Bacterial symbionts: Prospects for the sustainable production of invertebrate-derived
pharmaceuticals. Curr. Med. Chem. 13:39–50.
Piel, J. 2009. Deep sequencing reveals exceptional diversity and modes of transmission for bacterial
sponge symbionts. Nat. Prod. Rep. 26:338–362.
Piel, J., Hui, D., Wen, G., Butzke, D., Platzer, M., Fusetani, N., and Matsunaga, S. 2004. Antitumor
polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella
swinhoei. Proc. Natl. Acad Sci. U S A 101:16222–16227.
Pimentel-Elardo, S. M., Buback, V., Gulder, T. A., Bugni, T. S., Reppart, J., Bringmann, G., and
Hentschel, U. 2011. New tetromycin derivatives with anti-trypanosomal and protease inhibi-
tory activities. Mar. Drugs 9:1682–1697.
Pimentel-Elardo, S. M., Gulder, T. A. M., Hentschel, U., and Bringmann, G. 2008. Cebulactams A1
and A2, new macrolactams isolated from Saccharopolyspora cebuensis, the first obligate marine
strain of the genus Saccharopolyspora. Tetrahedron Lett. 49:6889–6892.
Pinheiro, Â., Dethoup, T., Bessa, J., Silva, A. M. S., and Kijjoa, A. 2012. A new bicyclic sesquiterpene
from the marine sponge associated fungus Emericellopsis minima. Phytochem. Lett. 5:68–70.
Prachyawarakorn, V., Mahidol, C., Sureram, S., Sangpetsiripan, S., Wiyakrutta, S., Ruchirawat, S.,
and Kittakoop, P. 2008. Diketopiperazines and phthalides from a marine derived fungus of the
order Pleosporales. Planta Med. 74:69–72.
Proksch, P., Ebel, R., Edrada, R., Riebe, F., Liu, H., Diesel, A., and Schulz, B. 2008. Sponge-associated
fungi and their bioactive compounds: The Suberites case. Bot. Mar. 51:209–218.
Proksch, P., Edrada, R., and Ebel, R. 2002. Drugs from the seas—Current status and microbiological
implications. Appl. Microbiol. Biotechnol. 59:125–134.
Pruksakorn, P., Arai, M., Kotoku, N., Vilchèze, C., Baughn, A. D., Moodley, P., and Kobayashi, M.
2010. Trichoderins, novel aminolipopeptides from a marine sponge-derived Trichoderma sp.,
are active against dormant mycobacteria. Bioorg. Med. Chem. Lett. 20:3658–3663.
Quévrain, E., Domart-Coulon, I., Pernice, M., and Bourguet-Kondracki, M. L. 2009. Novel natural
parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia nivea.
Rappe, M. S., Connon, S. A., Vergin, K. L., and Giovannoni, S. J. 2002. Cultivation of the ubiquitous
SAR11 marine bacterioplankton clade. Nature 418:630–633.
Rungprom, W., Siwu, E. R., Lambert, L. K., Dechsakulwatana, C., Barden, M. C., Kokpol, U., and
Garson, M. J. 2008. Cyclic tetrapeptides from marine bacteria associated with the seaweed
Diginea sp. and the sponge Halisarca ectofibrosa. Tetrahedron 64:3147–3152.
San-Martin, A., Rovirosa, J., Vaca, I., Vergara, K., Acevedo, L., Vina, D., and Chamy, M. C. 2011. New
butyrolactone from a marine-derived fungus Aspergillus sp. J. Chil. Chem. Soc. 56:625–627.
Sarethy, I. P., Pan, S., and Danquah, M. K. 2014. Modern taxonomy for microbial diversity, biodiver-
sity. In The Dynamic Balance of the Planet, Grillo, O. (Ed.), pp. 51–68.
Schmitt, S., Angermeier, H., Schiller, R., Lindquist, N., and Hentschel, U. 2008. Molecular microbial
diversity survey of sponge reproductive stages and mechanistic insights into vertical trans-
mission of microbial symbionts. Appl. Environ. Microbiol. 74:7694–7708.
Schneemann, I., Kajahn, I., Ohlendorf, B., Zinecker, H., Erhard, A., Nagel, K., and Imhoff, J. F. 2010a.
Mayamycin, a cytotoxic polyketide from a Streptomyces strain isolated from the marine sponge
Halichondria panicea. J. Nat. Prod. 73:1309–1312.
Schneemann, I., Ohlendorf, B., Zinecker, H., Nagel, K., Wiese, J., and Imhoff, J. F. 2010b. Nocapyrones
A−D, γ-pyrones from a Nocardiopsis strain isolated from the marine sponge Halichondria pani-
cea. J. Nat. Prod. 73:1444–1447.
Selvin, J., Shanmughapriya, S., Gandhimathi, R., Kiran, G. S., Ravji, T. R., Natarajaseenivasan, K.,
and Hema, T. A. 2009. Optimization and production of novel antimicrobial agents from sponge
associated marine actinomycetes Nocardiopsis dassonvillei MAD08. Appl. Microbiol. Biotechnol.
83:435–445.
Shindo, K., Asagi, E., Sano, A., Hotta, E., Minemura, N., Mikami, K., and Maoka, T. 2007.
Diapolycopenedioic acid xylosyl ester, a novel glyco-C-carotenoic acid produced by a new
marine bacterium Rubritalea squalenifaciens. Tetrahedron Lett. 48:2725–2727.
Shindo, K., Asagi, E., Sano, A., Hotta, E., Minemura, N., Mikami, K., Tamesada, E., Misawa, N., and Maoka,
T. 2008. Diapolycopenedioic acid xylosyl esters A, B, and C, novel antioxidative glyco-C30-carote-
noic acids produced by a new marine bacterium Rubritalea squalenifaciens. J. Antibiot. 61:185–191.
Simmons, L., Kaufmann, K., Garcia, R., Schwär, G., Huch, V., and Müller, R. 2011. Bendigoles D–F,
bioactive sterols from the marine sponge-derived Actinomadura sp. SBMs009. Biorg. Med. Chem.
19:6570–6575.
Smith, C. J., Abbanat, D., Bernan, V. S., Maiese, W. M., Greenstein, M., Jompa, J., and Ireland, C. M.
2000. Novel polyketide metabolites from a species of marine fungi. J. Nat. Prod. 63:142–145.
Speitling, M., Smetanina, O. F., Kuznetsova, T. A., and Laatsch, H. 2007. Bromoalterochromides A
and A′, unprecedented chromopeptides from a marine Pseudoalteromonas maricaloris strain
KMM 636T. J. Antibiot. 60:36–42.
Stewart, E. J. 2012. Growing unculturable bacteria. J. Bacteriol. 194:4151–4160.
Sugiyama, Y., Ito, Y., Suzuki, M., and Hirota, A. 2009. Indole derivatives from a marine sponge-
derived yeast as DPPH radical scavengers. J. Nat. Prod. 72:2069–2071.
Sun, L. L., Shao, C. L., Chen, J. F., Guo, Z. Y., Fu, X. M., Chen, M., and Wang, C. Y. 2012. New bisabo-
lane sesquiterpenoids from a marine-derived fungus Aspergillus sp. isolated from the sponge
Xestospongia testudinaria. Bioorg. Med. Chem. Lett. 22:1326–1329.
Supong, K., Thawai, C., Suwanborirux, K., Choowong, W., Supothina, S., and Pittayakhajonwut, P.
2012. Antimalarial and antitubercular C-glycosylated benz[α]anthraquinones from the marine-
derived Streptomyces sp. BCC45596. Phytochem. Lett. 5:651–656.
Sureram, S., Wiyakrutta, S., Ngamrojanavanich, N., Mahidol, C., Ruchirawat, S., and Kittakoop,
P. 2012. Depsidones, aromatase inhibitors and radical scavenging agents from the marine-
derived fungus Aspergillus unguis CRI282–03. Planta Med. 78:582–588.
Suryanarayanan, T. S. 2012. The diversity and importance of fungi associated with marine sponges.
Bot. Mar. 55:553–564.
Takagi, M., Motohashi, K., Khan, S. T., Hashimoto, J., and Shin-ya, K. 2010a. JBIR-65, a new diterpene,
isolated from a sponge-derived Actinomadura sp. SpB081030SC-15. J. Antibiot. 63:401–403.
Takagi, M., Motohashi, K., and Shin-ya, K. 2010b. Isolation of 2 new metabolites, JBIR-74 and JBIR-75,
from the sponge-derived Aspergillus sp. fS14. J. Antibiot. 63:393–395.
Takano, D., Nagamitsu, T., Ui, H., Shiomi, K., Yamaguchi, Y., Masuma, R., and Ōmura, S. 2001.
Absolute configuration of nafuredin, a new specific NADH-fumarate reductase inhibitor.
Tetrahedron Lett. 42:3017–3020.
Taylor, M. W., Radax, R., Steger, D., and Wagner, M. 2007. Sponge-associated microorganisms:
Evolution, ecology, and biotechnological potential. Microbiol. Mol. Biol. Rev. 71:295–347.
Taylor, M. W., Schupp, P. J., Dahllöf, I., Kjelleberg, S., and Steinberg, P. D. 2004. Host specificity in
marine sponge-associated bacteria, and potential implications for marine microbial diversity.
Thacker, R. and Starnes, S. 2003. Host specificity of the symbiotic cyanobacterium Oscillatoria sponge-
liae in marine sponges, Dysidea spp. Mar. Biol. 142:643–648.
Ueda, J.-Y., Khan, S. T., Takagi, M., and Shin-ya, K. 2010a. JBIR-58, a new salicylamide derivative, iso-
lated from a marine sponge-derived Streptomyces sp. SpD081030ME-02. J. Antibiot. 63:267–269.
Ueda, J.-Y., Takagi, M., and Shin-ya, K. 2010b. New xanthoquinodin-like compounds, JBIR-97, -98 and-
99, obtained from marine sponge-derived fungus Tritirachium sp. SpB081112MEf2. J. Antibiot.
63:615–618.
Vacelet, J. 1981. Algal-sponge symbioses in the coral reefs of New Caledonia: A morphological study.
Proceedings of the 4th International Coral Reef Symposium, Manila, May 18–22, 1981, Vol 2. pp. 713–719.
Vacelet, J. and Donadey, C. 1977. Electron microscope study of the association between some sponges
and bacteria. J. Exp. Mar. Biol. Ecol. 30:301–314.
Valliappan, K., Sun, W., and Li, Z. 2014. Marine actinobacteria associated with marine organisms
and their potentials in producing pharmaceutical natural products. Appl. Microbiol. Biotechnol.
98:7365–7377.
Van Soest, R. W., Boury-Esnault, N., Vacelet, J., Dohrmann, M., Erpenbeck, D., De Voogd, N. J., and
Hooper, J. N. 2012. Global diversity of sponges (Porifera). PLoS One 7:e35105.
Vandamme, P., Pot, B., Gillis, M., De Vos, P., Kersters, K., and Swings, J. 1996. Polyphasic taxonomy, a
consensus approach to bacterial systematics. Microbiol. Rev. 60:407–438.
Vanwonterghem, I., Jensen, P. D., Ho, D. P., Batstone, D. J., and Tyson, G. W. 2014. Linking microbial
community structure, interactions and function in anaerobic digesters using new molecular
techniques. Curr. Opin. Biotechnol. 27:55–64.
Varoglu, M. and Crews, P. 2000. Biosynthetically diverse compounds from a saltwater culture of
sponge-derived Aspergillus niger. J. Nat. Prod. 63:41–43.
Vogel, S. 1977. Current-induced flow through living sponges in nature. Proc. Natl. Acad Sci. U S A
74:2069–2071.
Wang, C.-Y., Wang, B.-G., Brauers, G., Guan, H.-S., Proksch, P., and Ebel, R. 2002. Microsphaerones
A and B, two novel γ-pyrone derivatives from the sponge-derived fungus Microsphaeropsis sp.
J. Nat. Prod. 65:772–775.
Webster, N. S. and Taylor, M. W. 2012. Marine sponges and their microbial symbionts: Love and
other relationships. Environ. Microbiol. 14:335–346.
Webster, N. S., Wilson, K. J., Blackall, L. L., and Hill, R. T. 2001. Phylogenetic diversity of bacteria
associated with the marine sponge Rhopaloeides odorabile. Appl. Environ. Microbiol. 67:434–444.
Wei, R. B., Xi, T., Li, J., Wang, P., Li, F. C., Lin, Y. C., and Qin, S. 2011. Lobophorin C and D, new kija-
nimicin derivatives from a marine sponge-associated actinomycetal strain AZS17. Mar. Drugs
9:359–368.
Wicke, C., Hüners, M., Wray, V., Nimtz, M., Bilitewski, U., and Lang, S. 2000. Production and struc-
ture elucidation of glycoglycerolipids from a marine sponge-associated Microbacterium species.
J. Nat. Prod. 63:621–626.
Wiese, J., Ohlendorf, B., Blümel, M., Schmaljohann, R., and Imhoff, J. F. 2011. Phylogenetic identifi-
cation of fungi isolated from the marine sponge Tethya aurantium and identification of their
secondary metabolites. Mar. Drugs 9:561–585.
Wyche, T. P., Hou, Y., Braun, D., Cohen, H. C., Xiong, M. P., and Bugni, T. S. 2011. First natural ana-
logs of the cytotoxic thiodepsipeptide thiocoraline A from a marine Verrucosispora sp. J. Org.
76:6542–6547.
Xin, Z.-H., Zhu, W.-M., Gu, Q.-Q., Fang, Y.-C., Duan, L., and Cui, C.-B. 2005. A new cytotoxic com-
pound from Penicillium aurantiogriseum, symbiotic or epiphytic fungus of sponge Mycale plu-
mose. Chin. Chem. Lett. 16:1227–1229.
Xin, Z. H., Fang, Y., Du, L., Zhu, T., Duan, L., Chen, J., and Zhu, W. M. 2007. Aurantiomides A−C,
quinazoline alkaloids from the sponge-derived fungus Penicillium aurantiogriseum SP0–19.
J. Nat. Prod. 70:853–855.
Xu, J., Takasaki, A., Kobayashi, H., Oda, T., Yamada, J., Mangindaan, R. E., and Namikoshi, M. 2006.
Four new macrocyclic trichothecenes from two strains of marine-derived fungi of the genus
Myrothecium. J. Antibiot. 59:451–455.
Yamazaki, H., Rotinsulu, H., Kaneko, T., Murakami, K., Fujiwara, H., Ukai, K., and Namikoshi, M.
2012. A new dibenz [b,e] oxepine derivative, 1-hydroxy-10-methoxy-dibenz[b,e]oxepin-6,11-di-
one, from a marine-derived fungus, Beauveria bassiana TPU942. Mar. Drugs 10:2691–2697.
Yarden, O. 2014. Fungal association with sessile marine invertebrates. Front. Microbiol. 5:228.
You, M.-X., Zhang, H.-P., and Hu, C.-Q. 2008. Isolation and characterization of three siderophores
from marine bacteria. Chin. J. Chem. 26:1332–1334.
Yu, L.-L., Li, Z.-Y., Peng, C.-S., Li, Z.-Y., and Guo, Y.-W. 2009. Neobacillamide A, a novel thiazole-
containing alkaloid from the marine bacterium Bacillus vallismortis C89, associated with South
China Sea sponge Dysidea avara. Helv. Chim. Acta. 92:607–612.
Yu, Z., Lang, G., Kajahn, I., Schmaljohann, R., and Imhoff, J. F. 2008. Scopularides A and B, cyclodep-
sipeptides from a marine sponge-derived fungus, Scopulariopsis brevicaulis. J. Nat. Prod.
71:1052–1054.
Zengler, K., Walcher, M., Clark, G., Haller, I., Toledo, G., Holland, T., and Keller, M. 2005. High‐
throughput cultivation of microorganisms using microcapsules. Methods Enzymol. 397:124–130.
Zhang, D., Yang, X., Kang, J. S., Choi, H. D., and Son, B. W. 2008a. Chlorohydroaspyrones A and B,
antibacterial aspyrone derivatives from the marine-derived fungus Exophiala sp. J. Nat. Prod.
71:1458–1460.
Zhang, P., Bao, B., Dang, H. T., Hong, J., Lee, H. J., Yoo, E. S., and Jung, J. H. 2009. Anti-inflammatory
sesquiterpenoids from a sponge-derived fungus Acremonium sp. J. Nat. Prod. 72:270–275.
Zhang, H., Lee, Y. K., Zhang, W., and Lee, H. K. 2006. Culturable actinobacteria from the marine
sponge Hymeniacidon perleve: Isolation and phylogenetic diversity by 16S rRNA gene-RFLP
analysis. Antonie Van Leeuwenhoek 90:159–169.
Zhang, H., Zhang, W., Jin, Y., Jin, M., and Yu, X. 2008b. A comparative study on the phylogenetic
diversity of culturable actinobacteria isolated from five marine sponge species. Antonie Van
Leeuwenhoek 93:241–248.
Zhang, L., An, R., Wang, J., Sun, N., Zhang, S., Hu, J., and Kuai, J. 2005. Exploring novel bioactive
compounds from marine microbes. Curr. Opin. Microbiol. 8:276–281.
Zhou, Y., Mándi, A., Debbab, A., Wray, V., Schulz, B., Müller, W. E., and Aly, A. H. 2011. New aus-
talides from the sponge‐associated fungus Aspergillus sp. Eur. J. Org. Chem. 2011:6009–6019.
section three

compounds from plants and
rhizosphere microorganisms
chapter sixteen
Antimicrobial compounds and their

chemical entities on therapeutic herbals
for agricultural and medical applications
P. Rajesh, K. Natarajan, and V. Rajesh Kannan
Contents
16.1 Introduction....................................................................................................................... 319
16.2 Mode of action of synthetic antimicrobial agents........................................................ 321
16.3 Natural plant products as antimicrobial agents........................................................... 327
16.4 Antimicrobial compounds............................................................................................... 327
16.5 History of antimicrobial compounds............................................................................. 328
16.6 Significance and importance of secondary metabolites.............................................. 329
16.7 Role of secondary metabolism in plants........................................................................ 330
16.8 Biosynthetic pathway of secondary metabolites.......................................................... 331
16.9 Classes of secondary metabolites................................................................................... 332
16.10 Genetics of secondary metabolites................................................................................. 335
16.11 Exploitation of secondary metabolites for human welfare......................................... 336
16.11.1 Agricultural benefits of secondary metabolites from medicinal plants..... 336
16.11.2 Agricultural benefits of bactericidal compounds from medicinal plants... 337
16.11.3 Agricultural benefits of fungicide compounds from medicinal plants...... 337
16.11.4 Agricultural benefits of insecticidal compounds from medicinal plants... 337
16.11.5 Medical benefits of secondary metabolites from medicinal plants............. 339
16.11.6 Antibacterial agents from synthesized secondary metabolites of plants... 339
16.11.7 Antifungal agents from synthesized secondary metabolites of plants.......340
16.11.8 Antiviral agents from synthesized secondary metabolites of plants..........340
16.12 Summary............................................................................................................................340
16.13 Conclusion.......................................................................................................................... 341
References...................................................................................................................................... 355
16.1 Introduction
The major challenge facing us today is how to control diseases and their consequent effects
on humans, animals, and plants in our society as well as on modern medicines. Following
the inevitable inadequate use of modern medicines or antibiotics, microorganisms have
developed resistance, and the world has turned to traditional antibiotic therapy for thera-
peutical application to control diseases in humans and plants.
319
However, most antibiotics kill the microorganisms by cell wall damage as well as
inhibition of cellular processes essential for survival, which leads to a strong selective
pressure to develop resistance against antibiotics. In other words, the long-term use of
large quantities of different antibiotics has contributed to antibiotic resistance in both
agriculturally and medically important bacteria, including in Pseudomonas aeruginosa
and Staphylococcus aureus. Therefore, society urgently needs an alternative approach to
develop effective therapies against microbial pathogens without affecting their growth in
order to avoid creating antibiotic-resistant bacteria in any species that belongs to animals
or plants (Keyser et al., 2008).
While dealing with antimicrobial compounds, the plant kingdom’s ability to inter-
convert and transform organic compounds in evidence-based biological activities v aries
widely. Plants are effective in synthesizing organic compounds through the process of
photosynthesis. Other than plants, microorganisms and animals must obtain the raw
materials containing organic compounds for their diet. In this way, metabolic pathways
interact using the catabolic degradation of food, while anabolic pathways biosynthesize
the specialized molecules. These two processes in combination define metabolism, and it
can be further divided into processes called primary and secondary metabolisms. Primary
metabolism involves processes that are basically the same in most organisms, such as
pathways for modifying and synthesizing carbohydrates, proteins, fats, and nucleic acids.
However, there are several organisms that also posses the ability to synthesize special-
ized compounds that are often unique to that species, and they are defined as secondary
metabolism and the processes are not essential for life.
Secondary metabolites are not produced under all conditions and provisions; in
many cases, their actual function is unknown and cannot be defined in a simple manner.
However, due to their higher investment in energy and carbon, the secondary metabolite
compounds probably have important ecological roles as protection against either biotic
factors such as herbivory, predation, and competition or abiotic factors such as UV light.
Other potential roles are included in a special character of plants that may be used as
volatile attractants. Whatever their specific functions, they probably play some role that
benefits the producing organism. It is within this area of secondary metabolites that most
biologically active compounds are found, although the actual mechanism in the boundary
of the primary and secondary metabolites is not known.
Secondary metabolites are produced in plants besides in primary biosynthetic and
metabolic routes of compounds aimed at plant growth and development as discussed
earlier. They can be regarded as products of biochemical side tracks in plant cells and
not needed for daily functioning of the plant. Phylogenetically, the secondary bioactive
compounds in plants appear to be randomly synthesized, but they are not useless junk.
Several of them are found to hold important roles and functions in living plants. For
example, the flavonoids can protect against free radicals generated during photosyn-
thesis and reduce aging as well as cell and tissue damage. The terpenoids may attract
pollinators or seed dispersers and inhibit competing plants. Alkaloids usually ward
off herbivore animals or insect attacks (phytoalexins), and other secondary metabolites
function as cellular signaling molecules or have other functions in the plants with their
specific actions. The plants producing bioactive secondary metabolites seem to be the
rule rather than the exception. Thus, most plants, even common food and feed plants,
are capable of producing such compounds. However, the typical poisonous or medici-
nal plants contain higher concentrations of more potent bioactive compounds than food
and feed plants.
Chapter sixteen: Antimicrobial compounds and their chemical entities 321
This chapter is on the significance, role, biosynthetic pathway, and classification of the
secondary metabolites, perpetual usage of antimicrobial compounds from various plant-
derived products to control agriculture and medically important pathogens, and also
deals with the resistance power of modern antibiotics.
16.2 Mode of action of synthetic antimicrobial agents

Antimicrobials and specifically antibiotics are defined as low-molecular-weight organic
natural products made by plants that are active against microorganisms at lower
concentration.
This activity develops through a limited number of mechanisms; antimicrobials inter-
fere with cell wall synthesis, cell membrane integrity, protein synthesis, DNA replication
and repair, transcription, and intermediate metabolism (Figure 16.1).
The compounds blocking the cell wall biosynthesis inhibit enzymes involved in the
synthesis of different components of the cell wall, while the secondary metabolite com-
pounds interfering with cell membrane integrity disorganize the structure or inhibit
the function of bacterial membranes. Antimicrobials affecting protein synthesis act in
impairing the ribosomal subunits, binding to prevent the translation and binding to
cause wrong translation, and producing toxic and altered proteins. Compounds having
an effect on DNA replication and repair inhibit enzymes such as gyrase, topoisom-
erase, and N-methyltransferase. Similarly, compounds affecting transcription inhibit
the subunits of the bacterial RNA polymerase blocking the entry of the first nucleo-
tide necessary to activate the polymerase. Finally, some antimicrobials interfere with
intermediate metabolism by inhibiting enzymes involved in the biosynthesis of dif-
ferent substances (Demain, 1999) such that the genetic modification with antimicrobial
resistance was intended to discover new antimicrobial agents from the plant products
(Figure 16.2).
Sulfonamides Cell wall

Trimethoprim
Cell membrane
β-Lactams
Purine synthesis Glycopeptides
Bacitracin
New proteins
Polymyxins
DNA
Polyenes
mRNA Imidazoles
Nitrofurans 50s
Nitroimidazoles
30s
Macrolides
Quinolones Lincosamides
Novobiocin Chloranphenicol
Aminoglycosides
Rifamycins Tetracyclines
Figure 16.1 Mode of action in synthetic antimicrobial agents.

OH H
N
HO
N
O H
O N
Lycroine (1) Sparteine (2)
O
O
Citronellal (3) Cineole (4)

OMe NH2 O
O
H 2N N OH
NH2
CH2
Essential oils (5) Canavanine (6)
O– O
H OH
O
S NH2
NH2+ H2C O
Azetidine-2-carboxylic acid (7) Alliin (8)
O OH O–
O
HO
O O S O
S N O
HO OH
R
N+
O
CH3 O
CH3
Berberine (9) Glucosinolates (10)

O OH OMe
OMe
MeO O
OMe
OH MeO
OH OMe O
Protocatechuic acid (11) Nobiletin (12)
Figure 16.2 Secondary metabolic chemical compounds from plant metabolisms (drawn using ISIS/
ChemDraw).(Continued)
HO CO2H OH
O HO
HO O O OH
HO
OH
OH O O
OH OH
O OH
HO O
OH OH
O OH O
HO
OH
HO
Chlorogenic acid (13) Tannins (14)
H
OH OH
H N
HO HO
O H
H H
HO O O
OH O O
HO OH
OH
Solanine (15) Phellandrene (16)
O OH OH
HO
OH
HO OH
O
Gossypol (17) Limonene (18)

N
OH
N
F
Geraniol (19) Citrol (20)
H OH
H
OH OH OH
H H N
HO HO O
O O H HO
H H O
HO O O HO OH
OH O O
HO OH
OH
Saponins (21) Arbutine (22)
Figure 16.2 (Continued) Secondary metabolic chemical compounds from plant metabolisms
(drawn using ISIS/ChemDraw). (Continued)
CH2OH H2 O
H O O C H
H OH H H O O C
OH H H OH H CN
H OH OH H
H OH OH O
Cyanogenic glycosides (23) Juglone (24)

MeO O H
OH N
O
H
O O H
O
Pisatin (25) Lupanine (26)

H
O
O O O
H
O
Furanocoumarin (27) Rishitin (28)
Polyacetylenes (29) Stilbenes (30)

HO OH HO OH OH
OH
O
HO
O O
HO OH
Simple phenols (31) Phloridzin (32)
HSe
OH O O
H 2N
O
Nonprotein amino acid (33) Coumarins (34)
OH CH2
OH
HO O CH2
OH H3C CH3
OH O
Quercetin (35) Monoterpenes (36)
CH3 HO OH
H
O HO O
H3C O
O
H H OH
O
H OH O
CH3
O
Sesquiterpene lactones (37) Morin (38)
OH O
MeO
HO O OH
OH
HO
O Rutinose
OH O
Rutin (39) Ferulic acid (40)
OH H
H H
HO
OH
Caffeic acid (41) Cucurbitacin (42)
OH H
N
OH N
OH
HO
H
Phytoecdysone (43) Nicotine (44)
O O
OH O O
O
O
O N O
O
OH
O
O O
HO O N
O
O
Aconitine (45) Colchicines (46)
N N OH
H H O
N H
H O
O
Strychnine (47) Atropine (48)
HO
N
O H
O O
H N
CH3 O
HO
Morphine (49) Piperine (50)
OH
O O
R O
H O O O
O OMe
H OH
HO
O
H
O O
O
MeO
OO
O
Azadirachtin (51) Pyrethrins (52)
H3C O
H CH2
H
O O O
O
H
H3C O
O
O
CH3
Rotenone (53) Isoflavones (54)
OH O OH O
HO H
HO
H H
H
OH O OH
Hypericin (55) Cardenolides (56)
(drawn using ISIS/ChemDraw).
16.3 Natural plant products as antimicrobial agents

The multidrug-resistant strains of many microorganisms have revealed exploration of
alternative antimicrobial agents. Medicinal plants have become the focus of intense study
in terms of validation of their traditional uses through the determination of their actual
antimicrobial effects. Synthetic drugs are not only expensive and inadequate for the treat-
ment of diseases but also often have adulterations and side effects; other than the afore-
mentioned panic condition, various microorganisms have made them resistant following
continuous usage of the antibiotics. Therefore, a search for new infection fighting strate-
gies to control microbial infections is imperative (Sieradzki et al., 1999).
Plants have a long history of antibiotic usage for curing disease used as antimicro-
bial, including antibacterial, antiviral, and antifungal agents in plants as well as for
medical purposes. Attention was refocused on plant origin, antimicrobial and antider-
matophytic agents after the discovery of penicillin, which is considered to be one of the
most important lifesaving phytodrugs and virtually known as the secondary metabo-
lites. Since then, many extracts from different plants have been tested for their antimi-
crobial activities, but there are so many unopened pages of plant life that require more
careful investigation to reveal their hidden characteristics of secondary metabolites.
Natural products are generally harmless or have minimum side effects when compared
with synthetic drugs. In addition to the antimycotic drugs, the effect of certain indig-
enous plant extracts on the spore germination of dermatophytes in vitro has also been
studied, but the work is fragmentary. Antifungal activities of some plants have been
reported by various researchers throughout the world (Sharma, 1988; Tewari et al., 1990;
Caceres et al., 1993; Rajendheran et al., 1998; Farombi, 2003; Nair et al., 2005; Mahesh and
Satish, 2008; Prusti et al., 2008).
In India, references to the curative properties of some herbs (as available in the Rigveda)
seem to be the earliest record of the use of secondary metabolites for medicinal purposes.
Singh et al. (1988) reported the exhibiting activity of the essential oil of Eucalyptus rostrata
leaves against four human pathogens, namely, Trichophyton mentagrophytes, Epidermophyton
floccosum, and Microsporum gypseum and M. canis. Iyer and Williamson (1991) investigated
the efficacy of some plant extracts and their secondary metabolites against Trichophyton
species. Rhododendron arboreum, Rhussemialata, Terminalia bellerica, and Woodfordia fruticosa
showed antibacterial activity preferably to gram-negative organisms. Generally, gram-
negative bacteria are more resistant than gram-positive bacteria (Kelmanson et al., 2000;
Parekh et al., 2005).
16.4 Antimicrobial compounds

Plants produce and accumulate an enormous variety of secondary metabolites (often
referred to as natural products). Many of these products have well-defined ecological
roles in plant defense and in mediating interactions of plants with other organisms or
microorganisms, while others have yet unknown biological roles. More than 45,000
different chemical structures of natural products have been identified. The largest
group of natural products is the terpenes with more than 25,000 structure elucida-
tions. Additionally, more than 2000 alkaloids (57) and about 8000 phenolic derivatives
are known (Croteau et al., 2000). Despite the large number of different proven chemi-
cal structures, there are an amazingly low number of biochemical pathways by which
Table 16.1 Biological activities of some secondary metabolites as plant-derived phytochemicals

Biological activity Secondary metabolic compounds
Antiviral Lycroine (A) (1) and sparteine (A) (2)
Antibacterial Citronellal (T) (3), cineole (T) (4), many essential oils (T) (5), canavanine (NP)
(6), azetidine-2-carboxylic acid (NP) (7), alliin (NP) (8), berberine (A) (9),
and glucosinolates (10)
Antifungal Protocatechuic acid (P) (11), nobiletin (F) (12), chlorogenic acid (P) (13),
tannin (P) (14), solanine (A) (15), phellandrene (T) (16), gossypol (T) (17),
limonene (T) (18), geraniol (T) (19), citrol (T) (20), saponins (S) (21),
canavanine (NP) (6), arbutine (Q) (22), cyanogenic glycosides (CG) (23),
glucosinolates (10), juglone (Q) (24), pisatin (F) (25), lupanine (A) (26),
furanocoumarin (27), rishitin (T) (28), polyacetylenes (29), and stilbenes (30)
Allelopathy Simple phenols (31); juglone (Q) (24); phloridzin (P) (32); nonprotein amino
acids (33); coumarins (34); quercetin (F) (35); polyacetylenes (29);
monoterpenes (T) (36) and sesquiterpene lactones (37)
Toxicity/repellency Quercetin (Q) (35); morin (F) (38); rutin (F) (39); ferulic acid (P) (40); caffeic
against acid (P) (41); juglone (Q) (24); tannins (P) (14); limonene (T) (18); gossypol
insects (T) (17); cucurbitacin (T) (42); saponins (T) (21); phytoecdysone (T) (43);
glucosinolates (10); monoterpenes (36); steroidal and quinolizidine
alkaloids, nicotine (A) (44); aconitine (A) (45); colchicines (A) (46);
strychnine (A) (47); atropine (A) (48); morphine (A) (49); berberine (A) (9);
piperine (A) (50); nonprotein amino acids (33); cyanogenic glycosides (23);
sesquiterpene lactones (T) (37); azadirachtin (T) (51); protease inhibitors,
lectins; pyrethrins (52); rotenone (53); and coumarins (34)
Vertebrates Isoflavones (F) (54), coumarins (34), juglone (Q) (24), tannins (14), hypericin
(F) (55), gossypol (T) (17), essential oils (T) (5), saponins (T) (21),
sesquiterpene lactones (T) (37), cardenolides (T) (56), glucosinolates (10),
alkaloids (57), nonprotein amino acids (33), and cyanogenic glycosides (23)
Attractants Flavonoids (58), anthocyanins (59), monoterpenes (36), sesquiterpenes (60),
carotenoids (T) (61), betalains (A) (62), quinines (63), phenols (31), amino
sugars (64), amino acids (65), and lipids (66)
Sources: Schlee, D., Okologische Biochemie, Springer, Berlin, Germany, 1986; Swain, T., Annu. Rev. Plant Physiol., 28,
479, 1977; Wink, M., Chemical ecology of quinolizidine alkaloids, in: Waller, G.R., ed., Allelochemicals: Role
in Agriculture, Forestry and Ecology, Am. Chem. Soc., 330, 524, 1987.
A, alkaloid; F, flavonoid; P, simple phenol; S, saponins; T, terpene; NP, nonprotein amino acids; Q, quinone.
these compounds are biosynthesized. Some of the biological activities in secondary

metabolites of plants have been derived in Table 16.1.
16.5 History of antimicrobial compounds

The use of secondary metabolites can be traced back to 2600 BC, and the first records were
written on hundreds of clay tablets in cuneiform from Mesopotamia (Newman et al., 2000).
Then more than 4000 years ago, plant secondary metabolites were used as mixtures
or plant extracts mainly in food, medicine, and to make poison. Although, plant second-
ary metabolites have been benefiting humans for thousands of years, their mysteries were
disclosed in late two centuries. The isolation of the first pure natural product, morphine
(49) from opium poppy (Papaver somniferum), by German pharmacist Friedrich Wilhelm
Serturner in 1806 opened the door to a new era of scientific plant secondary metabolite
research (Croteau et al., 2000).
He demonstrated that the active principle of plant extract could be attributed to a single
organic compound, which can be isolated. This finding initiated natural product chemis-
try and speeded up developments in synthetic, analytical, and pharmaceutical chemistry.
Even today, plant secondary metabolites are closely involved in human life; more than
30% of drugs are derived directly or indirectly from natural products and sources.
Plant secondary metabolites were thought to be simply waste products. In the 1970s,
however, new research showed that secondary plant metabolites play an indispensable
role in the survival of a plant in its environment. One of the most ground-breaking ideas
of this time argued that environmental conditions affected the evolution of plant second-
ary metabolites, and this indicated the high gene plasticity of secondary metabolites, but
this theory was ignored for about half a century before gaining acceptance. Recently, the
research around secondary plant metabolites is focused on the gene level and the genetic
diversity of plant metabolites. Biologists are trying to trace back genes to their origin and
reconstruct evolutionary pathways (Hartmann, 2007).
Plant secondary metabolism primarily has been delivered and discussed in the lat-
ter half of the nineteenth century; however, there was still much confusion over what
the exact function and use these secondary metabolites had. The well-known secondary
plant metabolites were by-products of the primary metabolism and were not crucial to
the plant’s survival. Early research only succeeded as far as categorizing the secondary
plant metabolites but did not give real insight into the actual function of the secondary
plant metabolites. In the early half of the 1900s, the main research around plant second-
ary metabolism was dedicated to the formation of secondary metabolites in plants, and
this research was compounded by the use of tracer techniques that made deducing meta-
bolic pathways much easier. However, there was still not much research being conducted
into the functions of secondary plant metabolites until around the twenty-first century
(Hartmann, 2007).
The secondary metabolites with antimicrobial compounds have been used in the
past decades in a wide manner. It was not until 1870s that Tyndall, Pasteur, and Roberts
reported on the antagonistic effects of various virulent organisms, and the antibiotics era
began in 1929 with the discovery of penicillin by Fleming. The products of penicillin effec-
tively started and augmented in the 1940s. In spite of this, there were only two classes
of naturally occurring β-lactam antibiotics known as penicillin and cephalosporin until
1970. About three decades, there was a consequence on the antibiotic products from plants
and microorganisms, but there were still revisions. The plant- and microorganism-based
natural products mentioned before are widely divergent in their chemical structures and
their antimicrobial properties. Unique peptides are having antimicrobial properties over
400 numbers that have been proven from diverse sources including plants, bacteria, fungi,
and vertebrates (Lehrer and Ganz, 1996; Steinstraesser et al., 2002).
16.6 Significance and importance of secondary metabolites

Crudely, there were huge peptides inducing the epithelial surfaces and concurrent
responses to the invading microorganisms. In addition, some of the peptides from the
plant-derived secondary metabolites are involved in the chemotaxis and promote wound
healing and contribute to the adaptive immunity by mobilizing memory T cells and
immature dendritic cells. In parallel to the screening of new antibiotics, they have been
the spotlight in finding low-molecular-weight secondary metabolites with other biolog-
ical activities. The secondary metabolites have been found to play a significant role in
activities such as enzyme inhibition and plant growth stimulation and been also used
in the production of herbicides, insecticides, and anthelmintics. Examples of two major

roles the secondary metabolites have contributed were, first, the screening of secondary
metabolites, which has been found useful for antibiotics and, second, the development of
unknown compounds using new technologies for detecting inhibition of enzymes and
biological activities and other targets. In addition, they play a significant role in agriculture
as well as in clinical medicines due to their function as antimicrobials used in the treat-
ment of microbial infections in plants and humans, respectively (Kieslich, 1986).
From the past research, there were extensive studies reported about their origin and
functionality. Being ineffective secondary metabolites have been accepted as waste prod-
ucts, but under the pressure of natural selection, they have evolved as messenger mol-
ecules that must have endured long enough to shuttle between the various components of
the microbial community.
This fact would explain why secondary metabolites have the tendency to become
small molecules as a natural consequence of their functions (Jarvis, 1995).
16.7 Role of secondary metabolism in plants

Secondary metabolites play a major role in the adaptation of plants to the changing environ-
ment and in overcoming stress constraints. This flows from the large complexity of chemical
types and interactions underlying various functions: structure stabilization, determined by
polymerization, condensation of phenols (31) and quinones, or electrostatic interactions of
polyamines with negatively charged loci in cell components (Edreva et al., 2008).
Primary metabolism in plants constitutes all metabolic pathways that are essential
to the plant’s survivability. Primary metabolites are compounds directly involved in the
growth and development of a plant, whereas secondary metabolites are compounds pro-
duced in other metabolic pathways, however important, and they are not essential to
the functioning of the plant. However, the secondary plant metabolites are useful in the
long term, often for defense purposes, and give plants characteristics such as color that is
known as pigments like chlorophyll and carotenoids. The plant secondary metabolites are
also used in signaling and regulation of primary metabolic pathways. The plant hormones
are secondary metabolites, often used to regulate the metabolic activity within cells and
overall development of the plant. As mentioned before, the plant’s secondary metabolites
help it maintain an intricate balance with the environment, often adapting to match the
environmental needs. Plant metabolites that color the plant are a good example of this as
the coloring of a plant can attract pollinators and also defend against attack by animals.
The hypothesis of the secondary metabolites went over a year, and they are as follows:
• Secondary metabolites act as an alternative defense mechanism, because only the

organisms lacking an immune system are prolific producers of these compounds.
• They have sophisticated structures, mechanisms of action, and complex and ener-
getically expensive pathways.
• Secondary metabolites act in the competition between microorganisms, plants, and
animals.
• They are produced by biosynthetic genes clusters, which would only be selected if the
product conferred a selective advantage. Some particularities of these gene clusters are
the absence of nonfunctional genes and the presence of resistance and regulatory genes.
• The production of secondary metabolites with antibiotic activities is temporarily
related with sporulation when the cells are particularly sensitive to competitors and
require special protection when a nutrient runs out.
Furthermore, the wide diversity of secondary metabolites suggests a broad range of func-
tions. Nevertheless, these functions could depend on the conditions, optimal or not, sur-
rounding the producer microorganism. Finally, due to their crucial importance, the study
and exploitation of secondary metabolites continue to progress despite the lack of agree-
ment regarding why microbes produce such chemical diversity of antimicrobial com-
pounds (Demain and Fang, 2000).
16.8 Biosynthetic pathway of secondary metabolites

The biosynthesis of secondary metabolites involves a series of reactions that are enzy-
matically catalyzed using several common mechanisms such as electrophilic additions
in alkylation, aldol, transamination, and decarboxylation, glycosylation, and redox reac-
tions (Figure 16.3). These secondary metabolites are synthesized at higher cost, requiring
a steady flow of precursors from primary metabolism together with energy-rich cofac-
tors like ATP and NAD(P)H. But the plants can compensate for the production of second-
ary metabolites higher in carbon through photosynthesis. However, for those metabolites
(P) (P) O
O (P) (P)
Dimethylallyl dishosphate Isopentenyl dishosphate
Secondary metabolites Primary metabolites
Indole alkaloids (P) (P)
O
Dimethylallyl diphosphate
O
(P) (P)
Monoterpenes (P) (P)

O
Geranyl diphosphate (GPP)
O
(P) (P)
Sesquiterpenes (P) (P) Steroids

O
Farnesyl disphosphate (FPP)
O (P) (P)
Diterpenes (P) (P) Carotenoids

O
Geranylgeranyl diphosphate (GGPP)
C50
Coenzyme Q
Figure 16.3 Biosynthetic pathway (mevalonate pathway). (From Keller, N.P., Nat. Rev., 3, 937, 2005.)
higher in limiting compounds, like nitrogen in alkaloids (57), secondary metabolism can
compete with primary pathways, such as in protein biosynthesis.
The ability to synthesize secondary metabolites has been selected through the course
of evolution in different plant lineages when such compounds address specific needs such
as the following:
Florescent volatiles and pigments have evolved to attract insect pollinators and thus
enhance fertilization.
Toxic chemicals have evolved to ward off pathogens and herbivores or to suppress the
growth of neighboring plants through synthesis.
Chemicals found in fruits prevent spoilage and act as signals (in the form of color,
aroma, and flavor) in the presence of sugars, vitamins, and flavors for animals that eat the
fruit and therefore help to disperse the seeds.
Other chemicals serve cellular functions that are unique to the particular plant in
which they occur (e.g., resistance to salt or drought).
Terpenes are biosynthesized by the isopentenoid pathway that includes two main met-
abolic branches, the mevalonic acid pathway (Figure 16.3), by which sesqui- and triterpenes
are biosynthesized, and the deoxyxylulose diphosphate pathway, by which mono-, di-, and
tetraterpenes are formed (Croteau et al., 2000). Most phenolic derivatives are biosynthesized
either by the shikimic acid pathway or through the malonate–acetate pathway, and most
alkaloids (57) are biosynthesized from amino acids. The monoterpenoids such as linalool,
menthol, thymol, and limonene (18) are derived from geranyl diphosphate. The different
conversions from GPP are catalyzed by a group of enzymes termed monoterpene synthases.
These enzymes share many properties such as cofactor requirements, molecular size, and
protein sequence similarity, but still they are different enough to allow for the catalysis of
the different products according to their specificity (McGarvey and Croteau, 1995). Sesqui-
and triterpenes, as well as many triterpene-derived metabolites such as the saponins (21)
and sterols, are synthesized from the 15-carbon intermediate farnesyl diphosphate (FPP).
Similar to monoterpene synthases, minor changes in the sesquiterpene synthase enzymes
(and their genes) are responsible for catalyzing the conversion of FPP to the different sesquiter-
penes, respectively. Still, at least in angiosperms, sesquiterpene synthase genes are significantly
similar to each other and also similar to angiosperm monoterpene synthases (Bohlmann et al.,
2000). In an analogous way, it seems that the ubiquitous pathway to lignin has been specifically
adapted in many plants for the production of unique natural products and modifications of
existing genes and enzymes to allow for the specific conversions. Thus, phenylpropanoids are
mostly derived from l-phenylalanine, not only a precursor of lignin, anthocyanins, and other
flavonoids (58) but also a precursor of t-anethole in anise (Pimpinella anisum) (Manitto et al.,
1974a) and infennel (Foeniculum vulgare) (Kaneko, 1960). The l-phenylalanine is also a precursor
of cinnamaldehyde in cinnamon in Cinnamomum zeylanicum (Senanayake et al., 1977) and of
estragole in sweet basil in Ocimum basilicum (Manitto et al., 1974b).
16.9 Classes of secondary metabolites

There is no rigid scheme for classifying natural products and their immense diversity in
structure, function, and biosynthesis that allows them to fit neatly into a few simple cate-
gories. The plants produce thousands of organic compounds that are traditionally divided
into two large classes, that is, primary and secondary metabolites. The term natural prod-
uct is generally taken to mean a secondary metabolite—a small molecule that is not essen-
tial to the growth and development of the producing organism and is not classified by
structure. Well, over 3,000,000 secondary metabolites probably exist, which are generally
classified into five categories: terpenoids (73), steroids, fatty acid–derived substances and
polyketides, alkaloids (57), and nonribosomal polypeptides and enzyme cofactors.
The plant has been derived secondary metabolites more than 1,000,000 chemical
compounds in various agricultural and medicinal purposes (Hadacek, 2002). The most
characteristic feature of secondary metabolites is the largest diversity of chemical types,
embracing representatives of all main classes of organic compounds: aliphatic, including
polyamines (67), ethylene (68), and isoprene (69); aromatic, such as phenolic alcohols (70),
phenolic acids (71), and unsaturated aromatic carbonic acids (72); hydroaromatic terpe-
noids (73) and jasmonic acid (74); and heterocyclic compounds, such as flavonoids (58) and
indole derivatives (75). Unique carbon skeletons occur along with multiplicity of func-
tional groups that were tabulated in Table 16.2 (Edreva et al., 2008).
Table 16.2 Diverse chemical types of secondary metabolites

Chemical types Chemical formula Representative
Aliphatic H2N C C C C NH2 Polyamines (67)
H2 H2 H2 H2
H2C CH2 Ethylene (68)

H2C C CH2 Isoprene (69)
H
CH3
Aromatic CH2OH Phenolic alcohols (70)
HO
COOH Phenolic acids (71)
OH
COOH Unsaturated aromatic carbonic acids (72)
HO
OMe
Hydroaromatic O CH3 Terpenoids (73)
H
O O CH3
COOH Jasmonic acid (74)
O
Heterocyclic OH O Flavonoids (58)
OH
OH
HO O
OH
Indole derivatives (75)
N
H
The majority of secondary metabolites belong to one of the families, each of which
has particular structural characteristics arising from the way in which they are built up
in nature (biosynthesis). The following are some other vast classifications of secondary
metabolites:
• Terpenoids (73) are defined by about 29,000 compounds, and terpenes constitute the
largest class of secondary metabolites and are united by their common biosynthetic
origin from acetyl-coA or glycolytic intermediates. A vast majority of the different
terpene structures produced by plants as secondary metabolites are presumed to
be involved in defense as toxins and feeding deterrents to a large number of plant-
feeding insects and mammals.
• Monoterpenes are expanded by about 1000 compounds, and their numerous deriva-
tives are important agents of insect toxicity. They show strong insecticidal responses
(neurotoxin) to insects like beetles, wasps, moths, and bees and are a popular ingre-
dient in commercial insecticides because of their low persistence in the environment
and low mammalian toxicity.
• Sesquiterpenes were defined by around 3000 compounds with their role in
plant defense characterized by a five-membered lactone ring as a cyclic ester
and their strong feeding repellence to many herbivorous insects and mammals.
Sesquiterpenes also play primarily regulatory roles in the initiation and mainte-
nance of seed and bud dormancy and plants’ response to water stress by modify-
ing the membrane properties and acting as transcriptional activators. In addition,
this increases the cytosolic calcium concentration and causes alkalinization of the
cytosol.
• Diterpenes are established by around 1000 compounds and are used as skin irritants
and internal toxins to mammals. It plays various detrimental roles in numerous plant
developmental processes such as seed germination, leaf expansion, flower and fruit
set, dry mass and biomass production, stomatal conductance, and CO2 fixation and
also the exertion of their numerous physiological effects via specific enzymes, the
synthesis of which they induce by influencing the basic process of translocation and
transcription.
• Triterpenes, steroids, and saponins (21) were expanded around 4000 compounds, and
several steroid alcohols (sterols) are important component of plant cell membranes,
especially in the plasma membrane as regulatory channels, and maintain permeabil-
ity to small molecules by decreasing the motion of fatty acid chains.
• Phenolics are in a wide range of about 8000 discovered compounds, and the plants
produce a large variety of secondary products that contain a phenol group, a
hydroxyl functional group in an aromatic ring called phenol, and a chemically het-
erogeneous group.
• They could play an important role in the plant defense system against pests and dis-
eases including root parasitic nematodes.
• Flavonoids (58) are explained in account of around 2000 compounds. This type of
secondary metabolites is one of the largest classes of plant phenolics, which perform
very different functions in plant system including pigmentation and defense. Two
other major groups of flavonoids (58) found in flowers are flavones and flavonols that
function to protect cells from UV radiation because they accumulate in epidermal
layers of leaves as well as stems and absorb light strongly in the UV-B region while
letting visible wavelengths throughout uninterrupted.
• Polyacetylenes are detected at around 1000 compounds, and they are elaborated.
• Polyketides were described to about 750 chemical compounds, and they are not
defined in a detailed manner.
• Phenylpropanoids and its derived compounds were found about 500 numbers with-
out definitions.
• Nitrogen-containing compounds are biosynthesized from common amino acids, and
all are of considerable interest because of their role in the antiherbivore defense and
toxicity to humans.
• Alkaloids (57) were coined around the numbers 12,000, and generally, most of them
are toxic to some degree and appear to serve primarily in defense against micro-
bial infection and herbivore attack. They are usually synthesized from one of the
few common amino acids, particularly from the aspartic acid, lysine, tyrosine, and
tryptophan.
• Nonprotein amino acids are defined and found to be present at around 600 com-
pounds, and many plants also contain unusual amino acids called nonprotein amino
acids (33) that incorporated into proteins but are present as free forms and act as
protective defensive substances. They exert their toxicity in various ways, and some
of them block the synthesis of or uptake of protein amino acid, while others can be
mistakenly incorporated into proteins.
• Cyanogenic glycoside compounds are found to be about 100, and they constitute a
group of N-containing protective compounds other than alkaloids (57) and release
the poisonous hydrogen cyanide (HCN). They are not in themselves toxic but are
readily broken down to give off volatile poisonous substances like HCN and hydro-
gen sulfide (H2S) when the plant is crushed.
• Glucosinolates are found to be of 100 compounds in nature.
• Amines are detected in and around 100 compounds.
16.10 Genetics of secondary metabolites

The genes regulating and ensuring synthesis of secondary metabolites and their expres-
sion can be grouped into five classes:
1. Structural genes that code for enzymes involved in the biosynthesis

2. Regulatory genes that determine the induction or repression of the structural genes
3. Genes that determine the resistance of the producing organism
4. Genes that control the permeability of the compound
5. Genes that control primary pathways
The genetic regulation of all aforementioned genes is highly complicated because many
environmental and microbial factors affect the production of these compounds (Romero-
Tabarez, 2004). The genes coding for biosynthesis of secondary metabolites has some fea-
tures that can be summarized as follows:
a. They are arranged in clusters on the chromosome.

b. These genes are organized in several transcription units, not in a single operon.
c. Within each cluster, at least some transcription units are controlled by the products
of pathway-specific regulatory genes.
d. Specific regulatory genes are situated in the closest proximity of biosynthetic genes.
e. Gene expression is primarily controlled at the level of transcription but also at the
level of translation.
f. Genes coding for resistance to a given metabolite are closely linked with the clus-
ter of biosynthetic genes. Usually, genes coding for at least two different types
of resistance to a produced secondary metabolite can be found within the gene
cluster.
g. Expression of genes coding for a secondary metabolite, including genes coding for
resistance to it, is controlled by central overruling regulatory circuits often exhibiting
pleiotropic regulatory effects (Romero-Tabarez, 2004).
16.11 Exploitation of secondary metabolites for human welfare

16.11.1 Agricultural benefits of secondary metabolites from medicinal plants
In the field of agriculture, the plant breeders select the varieties that provide maximal
yields in combination with optimal quality and resistance against pathogens, herbivores,
and other environmental stressors. Behind these panic conditions, feeding on one part
of the plant can induce systemic production of these chemicals in undamaged plant tis-
sues, and once released, these chemicals can act as signals to neighboring plants to begin
producing similar compounds. Production of these chemicals exacts a high metabolic cost
on the host plant, so many of these compounds are not produced in large quantities until
insects have begun to feed. The primary metabolites are substances produced by all plant
cells that are directly involved in the growth, development, or reproduction processes;
examples include sugars, proteins, amino acids, and nucleic acids. The secondary metabo-
lites are not directly involved in the growth or reproduction, but they are often involved
in the plant defense mechanism. These compounds usually belong to one of these three
large chemical classes: terpenoids (73), phenolics, and alkaloids (57) as discussed earlier
(Hassan Adeyemi, 2011).
The chemical protection plays a decisive role in the resistance of plants against patho-
gens and herbivores. The so-called secondary metabolites, which are a characteristic fea-
ture of plants, are especially important and can protect plants against a wide variety of
microorganisms (bacteria, viruses, and fungi) and herbivores (arthropods, vertebrates).
With the condition that all pathogens evolved in the plant systems, the chemical barrier
or bioactive secondary metabolites will be used to treat the disease or simply used as
the defense mechanism to decrease the pathogenic involvement. For example in chemi-
cal defense compounds, lupins that are of alkaloid-free varieties (sweet lupins) have been
selected by plant breeders due to their high susceptibility to a wide range of herbivores to
which the alkaloid-rich wild types were resistant (Wink, 1988).
Plant surfaces are usually covered by a hydrophobic layer consisting of antibiotic and
repellent cuticular waxes, which contains other secondary metabolites such as alkaloids
(57), flavonoids (58), terpenoids (73), and steroids. The presence of carbohydrates and lig-
nins imparts in their constitution in the cell wall as well as proliferation in the infec-
tion and wound sight of the plant. The synthesis of inhibitory proteins or enzymes could
degrade the microbial cell walls and other microbial constituents, such as peroxidase and
phenoloxidase that could help to inactivate phytotoxins of microbial origin. The synthe-
sis of low-molecular-weight compounds produces secondary metabolites that possessed
repellent or toxic properties against microorganisms and herbivores (Levin, 1976; Swain,
1977; Harborne, 1982). Color and scent performance by secondary metabolites such as fla-
vonoids (58), terpenoids (73), and other volatiles can underlie attraction or repelling of
insects and herbivores, while toxins can be involved in plant–plant allelopathic interac-
tions (Hadacek, 2002).
16.11.2 Agricultural benefits of bactericidal compounds from medicinal plants

The Panax ginseng root extracts have been used for antibacterial compounds in bacteri-
cidal activities and have been reported to have ginsenosides as their corresponding sec-
ondary metabolites (76) (Osbourn, 1996; Sparg et al., 2004). Sixteen coumarins (34), that
is, 7,8-dihydroxycoumarin (77), umbelliferone (78), scoparone (6,7-dimethoxycoumarin)
(79), esculetin (80), 6-hydroxy-7-methoxycoumarin (81), herniarin (7-methoxycouma-
rin) (82), scopoletin (83), 6,7-diacetoxy coumarin (84), 6-methoxy-7-acetylcoumarin (85)
and 6 -acetoxy-7-methoxycoumarin (86), 8-methoxypsoralen (87), 8-acetyl-7-hydroxycou-
marin (88), 7,8-dihydroxy-6-methoxycoumarin (89), 6,7-dimethoxy-4-methylcoumarin
(90), 5,7- dihydroxy-4-methylcoumarin (91), 4-hydroxycoumarin (92), and 4-hydroxy-6,7-
dimethylcoumarin (93), were tested against a panel of bacteria. Their antibacterial activ-
ity was determined depending on the microorganism such as Bacillus subtilis, Escherichia
coli, Proteus mirabilis, Klebsiella pneumoniae, Salmonella typhi, Salmonella sp., Shigella boy-
dii, Shigella sp., Enterobacter aerogenes, Enterobacter agglomerans, Sarcinalutea, Staphylococcus
epidermidis, Staphylococcus aureus, Yersinia enterocolitica, and Vibrio cholerae. In addition,
7,8-dihydroxy-6-methoxycoumarin (89), 8-acetyl-7-hydroxycoumarin (90), 7,8-dihydroxycou-
marin (77), 6,7-dimethoxy-4-methylcoumarin (90), scoparone (79), and esculetin (80) have
been found and reported an activity against V. cholera, and 8-acetyl-7-hydroxycoumarin
(88), 7,8-dihydroxy-6-methoxycoumarin (89), and 7,8-dihydroxycoumarin (77) were the most
active phytoconstituents. The dimethoxy compounds 6,7-dimethoxy-4-methylcoumarin
(90) and scoparone (79) showed and reported a significant activity against fungal strains,
especially T. mentagrophytes and Rhizoctonia solani (Cespedes et al., 2006), which are effective
repellants against viruses.
16.11.3 Agricultural benefits of fungicide compounds from medicinal plants

The Panax ginseng root extracts have also been used for antifungal compounds in fungi-
cide activities, and ginsenosides as their secondary metabolites (76) have been reported
by Osbourn (1996) and Sparg et al. (2004). The wyerone acid (94) is produced by the
broad bean (Vicia faba) when it is attacked by microorganisms and thus used as fun-
gicides. The evaluated fungi were Aspergillus niger, Penicillium notatum, Fusarium monili-
forme, Fusarium sporotrichum, R. solani, and T. mentagrophytes. The most active compounds
against gram-positive and gram-negative bacteria were the dihydroxylated coumarins
(34): 7,8-dihydroxycoumarin (77) and 7,8-dihydroxy-6-methoxycoumarin (89) (Cespedes
et al., 2006).
16.11.4 Agricultural benefits of insecticidal compounds from medicinal plants

The toxicity of secondary metabolites to insects and numerous feeding experiments has
been carried out, in which secondary compounds accumulated in plants are shown and
reported as toxic to insect herbivores that do not normally encounter them, and thus some
of these compounds have been used as insecticides. The three secondary compounds used
as insecticides are as follows: (1) pyrethroid insecticides, which are used widely in agri-
culture and horticulture and as active constituents of derris powder; (2) popular domes-
tic insecticide; and (3) nicotine (44) (tobacco, Nicotiana tabacum) used in smoke bombs for
greenhouses.
Eight numbers of isolated compounds were tested for insecticidal activity against
female adults of Aedes aegypti: β-sitosterol-3-O-β-d-glucoside (95), palmitic acid (96), linaro-
side (97), homoplantagenin (98), 5,7,3′-trihydroxy-6,4′-dimethoxy flavone-7-O-glucoside
and shikimic acid (99), protochatecuic acid (100), blepharin (101), and acetoside (102) for
adulticidal activity, respectively (Amin et al., 2012). The larvicidal activity of palmitic
acid against Culex quinquefasciatus, Anopheles stephensi, and A. aegypti was previously
reported by Abdul-Rahman et al. (2000).
Narbon bean (Vicia narbonensis), which is a grain legume, emits a foul smell to deter
animals from feeding on its seeds. The grass pea (Lathyrus sativus), which is also a grain
legume, has an enormous potential to be a food source since not only it has a nutritious
seed but it also has a high drought resistance. Unfortunately, the seed contains a number
of toxic nonprotein amino acids (33) including β-oxalyl diaminopropionic acid (103), which
was reported to cause slow progressive paralysis.
Dioclea megacarpa seeds, from a tree in the Costa Rica rain forests, are quite remark-
able in that they contain up to 13% dry weight of the toxic amino acid canavanine (6).
Canavanine (6) is similar in structure to arginine, and insects mistakenly incorporate
canavanine (6) during protein synthesis instead of arginine. The results are that proteins
fail to have the desired properties just like nonfunctioning enzymes and the insects die.
However, one species of beetle (Caryedes brasiliensis) has adapted to this toxin and can dis-
criminate between arginine and canavanine (6). This beetle can provide rich food supply
for itself without competition from other insects.
Lupanine (26), nicotine (44), tomatine (104), solanine (105), ergotamine (106), and lyser-
gic acids (107) were also natural secondary metabolites under the category of alkaloids
(57), which are susceptible to insect attacks at any use. In terpenes, isoprene (108), citronel-
lal (3) (oil of lemon), menthol (109) (peppermint oil), zingiberene (110) (ginger oil), azadi-
rachtin (51), and pyrethrins (52) were also reported for insecticidal activities.
Populus trees are deploying various types of defenses, one of which is the production of
a myriad of secondary compounds that are produced from the shikimate-phenylpropanoid
pathway. The plant secondary metabolites are used in activities against herbivores as insec-
ticides such as salicin (111), salicortin (112), tremuoloidin (113), t remulacin (114), populo-
side (115), trichocarposide (116), p-coumarate (117), caffeate (118), ferulate (119), sinapate
(120), coumaroyl quinate (121), caffeoyl quinate (122), feruloyl quinate (123), pinocembrin
chalcone (124), naringenin chalcone (125), eriodictyol chalcone (126), homoeriodictyol
chalcone (127), pinobanksin (128), aromadendrin (129), taxifolin (130), dihydromyricetin
(131), galangin (132), kaempferol (133), quercetin (35), myricetin (134), pinocembrin (135),
naringenin (136), eriodictyol (137), homoeriodictyol (138), chrysin (139), apigenin (140),
luteolin (141), condensed tannins (142), isoprene (143), (E)-β-ocimene (144), linalool (145),
(E,E)-α-farnesene (146), germacrene D (147), (Z)-3-hexenal (148), (Z)-3-hexen-1-ol (149), and
(Z)-3-hexenyl acetate (150) (Chen et al., 2009).
The Ginkgo biloba leaf extracts have been used for insecticidal and antifeedant acti
vities, and their secondary metabolites are reported to be ginkgolides A (151), B (152),
C (153), and J (154) (Ahn et al., 1997), and the Panax ginseng root extracts and their sec-
ondary metabolites ginsenosides (76) are also reported useful in insecticidal activities
(Osbourn, 1996; Sparg et al., 2004).
The use of pyrethrum in agriculture was recommended in controlling the attacks of
tulip bulb mites, iris root aphids, black citrus aphids, pea aphids, potato aphids, grape leaf-
hoppers, cotton leafhoppers, grape trips, bean trips, cabbage worms, tomato fruit worms,
parsley caterpillars, red-humped caterpillars, and many other insects. Pyrethrin (52) alone
is a naturally occurring insecticide, and industries and researchers found that synthetic
insecticides are derived from them. However, the already prepared and marketed syn-
thetic pyrethroid-derived compounds were proven to be very harmful for human beings.
They are known as DDT, permethrin, fenvalerate, deltamethrin, etofenprox, acrinathrin,
and bifenthrin. Resin acids include pimaric acid (155) (from Pinus species) and podocarpic
acid from Podocarpus cupressinum; manoyl oxides (156) were also used as insecticides.
16.11.5 Medical benefits of secondary metabolites from medicinal plants

The discovery of new bioactive natural products is still a fascinating field in organic chem-
istry as demonstrated by the recent paradigms of the anticancer drug epothilone, the
immunosuppressant rapamycin, or the proteasome inhibitor salinosporamide, to name a
few, but there are hundreds of possible examples. Finding new secondary metabolites is a
prerequisite for the development of novel pharmaceuticals, and this is an especially urgent
task in the case of antibiotics due to the rapid spreading of bacterial resistances and the
emergence of multiresistant pathogenic strains, which poses severe clinical problems in the
treatment of infectious diseases. This Thematic Series on the biosynthesis and function of
secondary metabolites deals with the discovery of new biologically active compounds from
all kinds of sources, including plants, bacteria, and fungi, and also with their biogenesis.
Biosynthetic aspects are closely related to functional investigations, because a deep under-
standing of metabolic pathways to natural products not only on a chemical but also on a
genetic and enzymatic level allows for the expression of whole biosynthetic gene clusters in
heterologous hosts. This technique can make interesting, new secondary metabolites avail-
able from noncultivable microorganisms or may be used to optimize their availability by
fermentation, for further research and also for production in the pharmaceutical industry.
Especially fascinating is the intrinsic logic of the polyketide and nonribosomal peptide
biosynthetic machineries, which is strongly correlated with the logic of fatty acid biosynthe-
sis as part of the primary metabolism. Insights into the mechanisms of modular polyketide
and nonribosomal peptide assembly lines open up the possibility for direct modifications,
for example, the oxidation states of the natural product’s carbon backbone by simple domain
knockouts within the responsible megasynthases or the introduction of a variety of alter-
native biosynthetic starters by mutasynthesis approaches, thus leading to new variants of
known metabolites, which may have improved the properties for therapeutic use. Another
interesting aspect is the usage of enzymes in chemical transformations, which can provide
synthetic organic chemistry with efficient access route to typically chiral building blocks that
may otherwise be difficult to obtain (Jeroen, 2011). Many of these secondary metabolic com-
pounds are valued for their pharmacological activities and industrial or agricultural proper-
ties that increase the commercial value of crops (Bingham et al., 1998; Paganga et al., 1999).
16.11.6 Antibacterial agents from synthesized secondary metabolites of plants

The secondary metabolites of many plants, such as essential oils (5), bisabolol (157), ngaione
(158), hymenoxin (159), and santonin (160), were reported to possess antimicrobial proper-
ties in antibacterial and antifungal activities (Bruneton, 1999; Heinrich et al., 2004) used
for medicinal purposes. Phaseolin (161) (bean) and isopimpinellin (162) (parsley) were
reported as the most significant plant secondary metabolites for the antibacterial activity
against virulence of medical pathogens.
The common herbs tarragon and thyme both contain caffeic acid (41), which are effec-
tive agents against bacteria. The catechol (163) and pyrogallol (164) both are hydroxylated
phenols, shown to be toxic to microorganisms. For that reason, the potential range of qui-
none and coumarin (34) antimicrobial effect is great. There are reports of antimicrobial
properties associated with polyamines (in particular, spermidine (165)), isothiocyanates
(166), thiosulfinates (167), and glucosides (168) (Ciocan and Bara, 2007).
16.11.7 Antifungal agents from synthesized secondary metabolites of plants

A study has shown that the antifungal agents derived from the plant heartwood of Pinus
species have used secondary metabolites such as resveratrol (169), pinosylvin (170), and
stilbenoids (171) (Harborne, 1998; Heinrich et al., 2004). The major components of this
mixture are a group of structurally related compounds, that is, apigeninidin (172) and
luteolinidin (173) (Brinker and Seigler, 1991); pisatin (25), maackiain (174), daidzein (175),
and medicarpin (176) (Graham, 1995); glyceollin (177) and keivitone (178) (Shanker et al.,
2003); phaseolin (161) (Durango et al., 2002); emodin-a (179) (Izhaki, 2002), malvone A (180)
(Veshkurova et al., 2006), umbelliferone (78), and scopoletin (83) (Uritani and Hoshiya, 1953;
Minamikawa et al., 1964; Tanaka et al., 1983); scoparone (79) (Afek et al., 1986; Kim et al.,
1992), ayapin (181), and resveratrol (169) (Langcake and Pryce, 1976); pterostilbene (182)
(Bala et al., 2000; Jeandet et al., 2002; Commun et al., 2003) and ε-viniferin (183) (Dercks
and Creasy, 1989); pinosylvins (170), pinosylvinmonomethyl ether (184), and pinosylvin
dimethyl ether (185); carvacrol (186) and thymol (187) (Muller-Riebau et al., 1995); rishi-
tin (28), lubimin (188), and solavetivone (189) (Kuc 1995); niveusin-ß (190) and costunolide
(191) (Grayer and Harborne, 1994); cichoralexin (192), lactucin (193), momilactones A (194)
(Kabuto et al., 1973) and B (195), sanguinarine (196), brassinin (197), ciclobrassinin (198),
brassilexin (199), wasalexin A (200), and arvelexin (201) (Pedras et al., 2003); caulexins A
(202), B (203), and C (204) (Pedras and Jha, 2006); and camalexins (205) and wyerone acid
(94) (Buzi et al., 2003), which were reported to have antifungal properties derived from
various plant genus and species.
The aforementioned common herbs, tarragon and thyme, are also effective
agents against fungi. Lawsone (2-hydroxy-1,4-naphthoquinone) (206) and Me-lawsone
(2-methoxy-1,4-naphthoquinone) (207) are the two main antifungal naphtha quinones
found naturally in Impatiens balsamina (Wanchai, 1998). The metabolites of many plants,
such as essential oils (5), bisabolol (157), ngaione (158), hymenoxin (159), and santo-
nin (160), were reported to possess antimicrobial properties in antifungal activities
(Bruneton, 1999; Heinrich et al., 2004) used for medicinal purposes.
16.11.8 Antiviral agents from synthesized secondary metabolites of plants

The phytoalexin resveratrol (3,4,5-trihydroxystilbene) is produced within a range of edible
plants in response to tissue damage and environmental stressors such as fungal and viral
attacks and moreover the antiviral effects against the herpes simplex virus (Docherty et al.,
1999; Faith et al., 2006).
16.12 Summary
Plants produce thousands of secondary metabolites of diverse chemical nature.
These compounds and chemical entities play major and important roles in protect-
ing plants under adverse conditions. In such way, there was a huge preparation devel-
oped under agricultural and medical purposes, which was known as pharmaceutical
preparation. It created a revolution in the world day by day due to the panic condi-
tions caused by the plant and animal diseases. Furthermore, nowadays, the beneficiary
effects have been elevated by the application secondary metabolites, which leads to
the protection of plant and animal health. Moreover, the secondary metabolites from
several plants are used in the production of medicines, dyes, insecticides, flavors, and
fragrances.
Unraveling plant secondary metabolism is the way to successful applications in

molecular farming, health food, functional food, and plant resistance. Various pathways
have been altered using genes encoding biosynthetic enzymes or regulatory proteins and
show enormous potential for the genetic engineering of plant secondary metabolism.
Recent achievements have been made in the metabolic engineering of plant secondary
metabolism.
In another way, biosynthetic aspects are closely related to functional investigations,
because of the deep understanding of metabolic pathways to natural products, not only
on a chemical but also on a genetic and enzymatic level, that allows for the expression
of whole biosynthetic gene clusters in heterologous hosts. Secondary metabolites play a
major role in the adaptation of plants to the changing environment and in overcoming
stress constraints. This results in a duly large complexity of chemical entities and interac-
tions underlying various functions: structure stabilization, determined by polymerization,
condensation of phenols and quinones, and electrostatic interactions of polyamines with
negatively charged loci in cell components; photoprotection, which is related to absor-
bance of visible light and UV radiation due to the presence of double bonds; antioxidant
and antiradical activities, governed by the availability of OH, NH2, and SH groupings, as
well as aromatic nuclei and unsaturated aliphatic chains; and signal transduction.
In discussions about secondary metabolites, there have been reviews made that
contribute to the various classifications and methods used for these metabolites in the
past 10 decades. In such way, the biosynthetic pathway of secondary metabolites, their
role in the plant’s metabolism, their significance and importance in genetics, the history of
antimicrobial compounds, the natural plant products as antimicrobial agents, the mode of
action of synthetic antimicrobial agents, and the classifications of secondary metabolites in
the field of agricultural and medical applications are discussed in this chapter in a critical
manner.
Their major classifications were defined and found as aliphatic, aromatic, hydroaro-
matic, and heterocyclic in their chemical aspects. Other subclassifications and chemical
entities were often based on their major chemical components. About 207 chemical units
were reviewed here and used for the purpose of agricultural and medical applications. In
a more detailed discussion, the chemical entities of compounds were used in the treatment
of plant diseases such as red rot disease and tikka disease and all insecticidal attacks and
used as antibacterial, antifungal, and insecticidal agents in the field of agriculture. On the
other hand, the chemical units were used for the purpose of treating animal and human
diseases like arthritis, cancers, diabetes, inflammations, ulcers, and wounds.
16.13 Conclusion
This chapter is concluded with the major classes of phytochemical compounds that are
found in medicinal plants and perpetually used for various antibacterial, antifungal, and
insecticidal activities in the field of agriculture, which employs secondary metabolites in
the treatment of plant disorders such as sheath blight, leaf blast, red rot disease, tikka
disease, and insect attacks. In addition, these secondary metabolites are used in the com-
position of antibacterial, antifungal, and antiviral compounds in the medical field to treat
various debilitating human diseases including arthritis, cancer, diabetes, and immunode-
ficiency diseases. About 207 compounds from plants with secondary metabolites, which
are most useful in agricultural and medical applications, were reviewed here. This review
about secondary metabolites as bioactive constituents will be helpful for future research
and development (Figures 16.4 and 16.5).
OH
O OH
OH H O O OH
H
O OH
OH
OH
H H
HO
H
O O
OH
HO OH
OH
Ginsenoside (76) 7,8-dihydroxycoumarin (77)
O O O
HO O O O
Umbelliferone (78) Scoparone (6,7-dimethoxycoumarin) (79)
HO O O O
H3C
HO O O HO
Esculetin (80) 6-hydroxy-7-methoxycoumarin (81)
O O OH
O O O O
CH3
Herniarin (7-methoxycoumarin) (82) Scopoletin (83)
O CH3
O
O CH3
O
O O O O
O O O
6,7-diacetoxy coumarin (84) 6-methoxy-7-acetylcoumarin (85)
O O O
CH3 O O O
O O O
CH3
6-acetoxy-7-methoxycoumarin (86) 8-methoxypsoralen (87)
OH
O O OH
HO O O
O O
8-acetyl-7-hydroxycoumarin (88) 7,8-dihydroxy-6-methoxycoumarin (89)

O O OH
O O O
O
OH
Figure 16.4 Secondary metabolic chemical compounds from plants for antibacterial, antifungal,
and insecticidal activities (drawn using ISIS/ChemDraw). (Continued)
6,7-dimethoxy-4-methylcoumarin (90) 5,7-dihydroxy-4-methylcoumarin (91)

OH OH
H3C
O O H3C O O
4-hydroxycoumarin (92) 4-hydroxy-6,7-dimethylcoumarin (93)

O Me
H 3C COOCH3
Me Me
O Me
Me
OH Me H
HO
O H H
O
HO HO
Wyerone acid (94) β-Sitosterol-3-O-β-d-glucoside (95)

O OH
HO OH
OH CH3
O O
HO
O O
O
CH3 OH O
Palmitic acid (96) Linaroside (97)

OH COOH
OH
HO
O
HO O O
OH HO OH
MeO OH
OH O
Homoplantagenin (98) Shikimic acid (99)

O H
N O
OH
HO O O
OH HO
O
HO
OH OH
Protochatecuic acid (100) Blepharin (101)
Figure 16.4 (Continued) Secondary metabolic chemical compounds from plants for antibacterial,
antifungal, and insecticidal activities (drawn using ISIS/ChemDraw). (Continued)
H O O
H O
HO
H O O N OH
O H
O H NH2
O
H H O O
O
H
H O
O O
O
H
O
H O
H
Acetoside (102) β-Oxalyl diaminopropionic acid (103)
OH OH
HO
N
O O
H HO OH
OH H3C
HO O
O OH
HO O
O
HO HO O
NH2OH
OH O
HC
H3
H3C HCH3
H HH
CH3
Tomatine (104) Solanine (105)
H
O
O N
O
H
N
N
O
HO
O
H N H N
H
O N
H
N
H
Ergometrine (106) Lysergic acid (107)
OH
Isoprene (108) Menthol (109)

CH2OH
O O
OH
OH CH2OH
OH
Zingiberene (110) Salicin (111)

CH2OH CH2OH
O O O
O
OH OH
OH CH2 OH CH2OH
OH O O
O O
OH
O
Salicortin (112) Tremuoloidin (113)

CH2OH CH2OH
O O
O O
OH OH
OH CH2 CH2
OH
O O OH O
O O
O
OH
O CH
CH
CH
Tremulacin (114) Populoside (115)

H2 HO O
HO C C O C
H H O
O O
OH
OH CH2OH
OH
H H
OH
Trichocarposide (116) p-Coumarate (117)
HO O HO O
H OH H OMe
OH OH
Caffeate (118) Ferulate (119)
HO O O O - Quinate
MeO OMe H H
OH OH
Sinapate (120) Coumaroyl quinate (121)
O O - Quinate O O - Quinate
H OH H OMe
OH OH
Caffeoyl quinate (122) Feruloyl quinate (123)

H H
H OH
HO OH HO OH
OH O OH O
Pinocembrin chalcone (124) Naringenin chalcone (125)

OH OMe
OH OH
HO OH HO OH
OH O OH O
Eriodictyol chalcone (126) Homoeriodictyol

chalcone (127)
H H
H OH
HO O HO O
H H
OH OH
OH O OH O
Pinobanksin (128) Aromadendrin (129)
OH OH
OH OH
HO O HO O
H OH
OH OH
OH O OH O
Taxifolin (130) Dihydromyricetin (131)
H H
H OH
HO O HO O
H H
OH OH
OH O OH O
Galangin (132) Kaempferol (133)
OH H
OH H
HO O HO O
OH
OH
OH O OH O
Myricetin (134) Pinocembrin (135)
H OH
OH OH
HO O HO O
OH O OH O
Naringenin (136) Eriodictyol (137)
OMe H
OH H
HO O HO O
OH O OH O
Homoeriodictyol (138) Chrysin (139)

H OH
OH OH
HO O HO O
OH O OH O
Apigenin (140) Luteolin (141)

H
OH
H
HO O
H
OH
OH
OH O
H
HO
OH
OH
Condensed tannins (142) Isoprene (143)

OH
(E)-β-ocimene (144) Linalool (145)

CH3
H2C
H3C
CH2
(E,E)-α-farnesene (146) Germacrene D (147)
O OH
(Z)-3-hexenal (148) (Z)-3-hexen-1-ol (149)
OAc O
H HO
O
O O
O T
Me Bu
O O
H
(Z)-3-hexenyl acetate (150) Ginkgolides A (151)

O O
HO HO HO HO
O O
O O O O
O T O T
Me Me Bu
Bu
O O O O
H OH
Ginkgolides B (152) Ginkgolides C (153)

O
H HO
O
O O
O T H
Me Bu
H
O O OH
Ginkgolides J (154) Pimaric acid (155)
Manoyl oxide (156)

OH
O
N
N O
H
Alkaloids (57) Flavonoids (58)
R
R
+
R O
R
R R
R
Anthocyanins (59) Sesquiterpenes (60)
HO
O
OH
OH O O
OH
+
HO N O
OH
N
OH H O
Carotenoids (61) Betalains (62)
OH OH
N
HO O
N HO
NH2
OH
O
Quinine (63) Amino sugars (64)
O H
H
N H
O
H
H
H H
HO
Amino acids (65) Lipids (66)
antifungal, and insecticidal activities (drawn using ISIS/ChemDraw).
H O
HO H
O
O
Bisabolol (157) Ngaione (158)
O
O H
O O O
H O
O
O O
O O
H
Hymenoxin (159) Santonin (160)
HO O OMe
O O O O
OMe
O
Phaseolin (161) Isopimpinellin (162)

OH OH
OH HO OH
Catechol (163) Pyrogallol (164)

H N
R
N NH2 S
H2N
Spermidine (165) Isothiocyanates (166)
O OH
S HO
R΄ O
R S O
HO OH
Thiosulfinate (167) Glucosides (168)
OH
OH
OH
HO OH
Resveratrol (169) Pinosylvins (170)
Figure 16.5 Secondary metabolic chemical compounds from plants for antibacterial, antifungal,
and antiviral activities (drawn using ISIS/ChemDraw). (Continued)
OH H
OH
HO
+
HO O
OH
OH
Stilbenoids (171) Apigeninidin (172)
OH HO O
OH
O
+
HO O
O
O
OH
Luteolinidin (173) Maackiain (174)
HO O HO O
O O
OH OMe
Daidzein (175) Medicarpin (176)

CH3 H3C CH3
H3C
O O
OH HO O
OH
O
OMe
OH O
OH
Glyceollin (177) Keivitone (178)
OH O OH OH O
HO CH3
HO OH CH3
O O
Emodin-A (179) Malvone A (180)
O OMe
O O O
MeO
Ayapin (181) Pterostilbene (182)
antifungal, and antiviral activities (drawn using ISIS/ChemDraw). (Continued)
HO HO
OH
OH
O
OH
OMe
OH
ε-Viniferin (183) Pinosylvinmonomethyl ether (184)
CH3
HO
OMe H3C CH3

OMe
Pinosylvin dimethyl ether (185) Carvacrol (186)
CH3 HO
O
OH CH3
CH2
H 3C CH3 H3C
Thymol (187) Lubimin (188)
O
H3C
CH3
HO O CH3
O H3C
OH
Solavetivone (189) Niveusin-ß (190)
H3C
H3C H H OH C
3
CH2
CH3 O
O
H CH3
H3C
Costunolide (191) Cichoralexin (192)

H CH2
H CH3
CH3
CH2
O
H
HO
O HO H
HO H OH O
O
O
Lactucin (193) Momilactone A (194)
antifungal, and antiviral activities (drawn using ISIS/ChemDraw). (Continued)
CH3 O
CH2
CH3 O O
+
H O N
CH3
H
H3C CH3
Momilactone B (195) Sanguinarine (196)
H N
N
SCH3 N S SCH3
N S
H H
Brassinin (197) Ciclobrassinin (198)
N SCH3
S SCH3
N
H
O
NOCH3
Brassilexin (199) Wasalexin A (200)
OMe CN CHO
SCH3
S
N N
H H
Arvelexin (201) Caulexin A (202)
O H CN
NH
N
OMe
N
MeO
Caulexin B (203) Caulexin C (204)
O
N
S OH
N O
H
Camalexins (205) Lawsone (206)
O
O
CH3
O
Me-lawsone (207)
antifungal, and antiviral activities (drawn using ISIS/ChemDraw).
References
Abdul-Rahman, A., Gopalakrishnan, G., Ghouse, B.S., Arumugam, S., and Himalayan, B. 2000.
Effect of Feronia limonia on mosquito larvae. Fitoterapia 71: 553.
Afek, U., Sztejnberg, A., and Carmely, S. 1986. 6,7–Dimethoxycoumarin, a citrus phytoalexin confer-
ring resistance against Phytophthora gummosis. Phytochemistry 25: 1855–1856.
Ahn, Y., Kwon, M., Park, H., and Han, C. 1997. Potent insecticidal activity of Ginkgo biloba derived
trilactone terpenes against Nilaparvata lugens. Phytochem. Pest Control 658: 90–105.
Amin, E., Radwan, M.M., El-Hawary, S.S., Fathy, M.M., Mohammed, R., Becne, J.J., and Khan, I. 2012.
Potent insecticidal secondary metabolites from the medicinal plant Acanthus montanus. Rec.
Nat. Prod. 6(3): 301–305.
Bala, A., Kollmann, A., Ducrot, P., Majira, A., Kerhoas, L., Leroux, P., Delorme, P., and Einhorn, J.
2000. Cis-ε-viniferin, a new antifungal resveratrol dehydrodimer from Cyphostemma crotalari-
oides roots. J. Phytopathol. 148: 29–32.
Bingham, S.A., Atkinson, C., Liggins, J., Bluck, L., and Coward, A. 1998. Phytoestrogens: Where are
we now. J. Nutr. 79: 393–406.
Bohlmann, J., Gershenzon, J., and S. Aubourg. 2000. Biochemical, molecular, genetic and evolu-
tionary aspects of defense-related terpenoid metabolism in conifers. In: Romeo, J.T., Ibrahim,
R., Varin, L. eds., Evolution of Metabolic Pathways. Recent Advances in Phytochemistry, vol. 34.
Elsevier Science, Oxford, U.K., pp. 109–150.
Brinker, A.M. and Seigler, D.S. 1991. Isolation and identification of piceatannol as a phytoalexin from
sugarcane. Phytochemistry 30: 3229–3232.
Bruneton, J. 1999. Pharmacognosy, Phytochemistry and Medicinal Plants. Intercept Ltd., Paris, France.
Buzi, A., Chilosi1, G., Timperio, A., Zolla, L., Rossall, S., and Magro1, P. 2003. Polygalacturonase
produced by Botrytis fabae as elicitor of two furanoacetylenic phytoalexins in Vicia faba pods.
J. Plant Pathol. 85(2): 111–116.
Caceres, A., Lopez, B., Juarez, X., Aguila, J., Garcia, S., and Del Aguila, J. 1993. Plants used in
Guatemala for the treatment of dermatophytic infections. 2. Evaluation of antifungal activity
of seven American plants. J. Ethanopharm. 40: 207–213.
Cespedes, C.L., Avila, J.G., Martinez, A., Serrato, B., Calderon-Mugica, J.C., and Salgado Garciglia, R.
2006. Antifungal and antibacterial activities of Mexican tarragon (Tagetes lucida). J. Agric. Food
Chem. 54: 3521–3527.
Chen, F., Liu, C.-J., Tschaplinski, T.J., and Zhao, N. 2009. Genomics of secondary metabolism in popu-
lus: Interactions with biotic and abiotic environments. Crit. Rev. Plant Sci. 28: 375–392.
Ciocan, I.D. and Bara, I.I. 2007. Plant products as antimicrobial agents. Analele Stiintifice ale
Universitatii, Alexandru Ioan Cuza, Sectiunea Genetica Si Biologie Moleculara, TOM VIII,
North Carolina.
Commun, C., Mauro, M., Chupeau, Y., Boulay, M., Burrus, M., and Jeandet, P. 2003. Phytoalexin
production in grapevine protoplasts during isolation and culture. Plant Physiol. Biochem. 41:
317–323.
Croteau, R., Kutchan, T.M., and Lewis, N.G. 2000. Natural products (secondary metabolites). In:
Buchanan, B., Gruissem, W., and Jones, R. (eds.), Biochemistry and Molecular Biology of Plants.
American Society of Plant Physiologists, Rockville, MD, pp. 1250–1318.
Demain, A.L. 1999. Pharmaceutically active secondary metabolites of microorganisms. Appl.
Demain, A.L. and Fang, A. 2000. The natural functions of secondary metabolites. Adv. Biochem. Eng.
Biotechnol. 69: 1–39.
Dercks, W. and Creasy, L. 1989. The significance of stilbene phytoalexins in the Plasmopara viticola
grape vine interaction. Physiol. Mol. Plant Pathol. 34: 189–202.
Docherty, J., Fu, M., Stiffler, B., Limperos, R., Pokabla, C., and DeLucia, A. 1999. Resveratrol inhibi-
tion of herpes simplex virus replication. Antiviral Res. 43: 135–145.
Durango, D., Quiñones, W., Torres, F., Rosero, Y., Gil, J., and Echeverri, F. 2002. Phytoalexin accumu-
lation in Colombian bean varieties and aminosugars as elicitors. Molecules 7: 817–832.
Edreva, A., Velikova, V., Tsonev, T., Dagnon, S., Gurel, A., Aktas, L., and Gesheva, E. 2008. Stress-
protective role of secondary metabolites: Diversity of functions and mechanisms. Gen. Appl.
Plant Physiol. 34(1–2): 67–78.
Faith, S.A., Sweet, T.J., Bailey, E., Booth, T., and Docherty, J.J. 2006. Resveratrol suppresses nuclear
factor-[kappa] B in herpes simplex virus infected cells. Antiviral Res. 72: 242–251.
Farombi, E.O. 2003. African indigenous plants with chemotherapeutic potential and biotechnologi-
cal approach to the production of bioactive prophylactic agents. Afr. J. Biotechnol. 2: 662–671.
Graham, T.L. 1995. Cellular biochemistry of phenyl propanoids responses of soybean to infec-
tion by Phytophthora sojae. In: Daniel, M. and Purkayastha, R.P. (eds.), Handbook of Phytoalexin
Metabolism and Action. Marcel Dekker, New York, pp. 85–116.
Grayer, R.J. and Harborne, J.J. 1994. A survey of antifungal compounds from higher plants.
Hadacek, F. 2002. Secondary metabolites as plant traits: Current assessment and future perspec-
tives. Crit. Rev. Plant Sci. 21: 273–322.
Harborne, J.B. 1982. Recent advances in chemical ecology. Nat. Prod. Rep. 3: 323–344.
Hartmann, T. 2007. From waste products to ecochemicals: Fifty years research of plant secondary
metabolism. Phytochemistry 68(22–24): 2831–2846.
Hassan Adeyemi, M.M. 2011. A review of secondary metabolites from plant materials for post har-
vest storage. Int. J. Pure Appl. Sci. Technol. 6(2): 94–102.
Heinrich, M., Barnes, J., Gibbons, S., and Williamson, E.M. 2004. Fundamentals of Pharmacognosy and
Phytotherapy. Churchill Livingstone, Elsevier Science Ltd., London, U.K.
Iyer, S.R. and Williamson, D. 1991. Studies on the occurrence of keratinophilic fungi in the soils of
Rajasthan- II. Indian J. Phytopathol. 44: 487–490.
Izhaki, I. 2002. Emodin-α secondary metabolites with multiple ecological functions in higher
plants—Research review. New Physiol. 155: 205–217.
Jarvis, B.B. 1995. Secondary metabolites and their role in evolution. An. Acad. Bras. Cienc. 67(3):
329–345.
Jeandet, P., Douillet, A., Bessis, R., Debord, S., Sbaghi, M., and Adrian, M. 2002. Phytoalexins from
the Vitaceae, biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activ-
ity and metabolism. J. Agric. Food Chem. 50: 2731–2741.
Jeroen, S.D. 2011. Biosynthesis and function of secondary metabolites. Beilstein J. Org. Chem. 7:
1620–1621.
Kabuto, K.C., Sasaki, N., Tsunagawa, M., Aizawa, H., Fujita, K., Kato, Y., Kitahara, Y., and Takahashi,
N. 1973. Momilactones, growth inhibitors from rice, Oryza sativa L. Tetrahedron Lett. 14:
3861–3864.
Kaneko, K. 1960. Biogenetic studies of natural products. V. Biosynthesis of anethole by Foeniculum
vulgare. Chem. Pharm. Bull. (Japan) 8: 875–879.
Keller, N.P., Turner, G., and Bennett, J.W. 2005. Fungal secondary metabolism from biochemistry to
genomics. Nat. Rev. 3: 937–947.
Kelmanson, J.E., Jager, A.K., and Van, S.J. 2000. Zulu medicinal plants with antibacterial activity.
J. Ethnopharmacol. 69: 241–246.
Keyser, P., Elofsson, M., Rosell, S., and Wolf Watz, H. 2008. Virulence blockers as alternatives to anti-
biotics: Type III secretion inhibitors against gram-negative bacteria. J. Intern. Med. 264: 17–29.
Kieslich, K. 1986. Production of drugs by microbial biosynthesis and biotransformation. Possibilities,
limits and future developments (1st communication). Arzneimittelforschung 36(4): 774–778.
Kim, S.H., Kang, S.S., and Kim, C.M. 1992. Coumarin glycosides from the roots of Angelica dahurica.
Arch. Pharm. Res. 15: 73–77.
Kuc, J. 1995. Phytoalexins stress metabolism and disease resistance in plants. Annu. Rev. Phytopathol.
33: 275–297.
Langcake, P. and Pryce, R.J. 1976. The production of resveratrol by Vitis vinifera and other members
of the Vitaceae as a response to infection or injury. Physiol. Plant Pathol. 9: 77–86.
Lehrer, R.I. and Ganz, T. 1996. Endogenous vertebrate antibiotics. Defensins, protegrins and other
cysteine-rich antimicrobial peptides. Ann. N. Y. Acad. Sci. 797: 228–239.
Levin, D.A. 1976. The chemical defenses of plants to pathogens and herbivores. Ann. Rev. Ecol. Syst.
7: 121–159.
Mahesh, B. and Satish, S. 2008. Antimicrobial activity of some medicinal plants against plant and
human pathogen. World J. Agric. Sci. 4: 839–843.
Manitto, P., Monti, D., and Gramatica, P. 1974a. Biosynthesis of anethole in Pimpinella anisum L.
Tetrahedron Lett. 17: 1467–1568.
Manitto, P., Monti, D., and Gramatica, P. 1974b. Biosynthesis of phenylpropanoid compounds. Part I.
Biosynthesis of eugenol in Ocimum basilicum. JCS Perkin I: 1727–1731.
McGarvey, D.J. and Croteau, R. 1995. Terpenoid metabolism. Plant Cell. 7: 1015–1026.
Minamikawa, T., Akazawa, T., and Uritani, I. 1964. Two glucosides of coumarin derivatives in sweet
potato roots infected by Ceratocystis fimbriata. Agric. Biol. Chem. 28: 230–233.
Muller-Riebau, F., Beger, B., and Yegen, O. 1995. Chemical composition and fungitoxic properties
to phytopathogenic fungi of essential oils selected aromatic plants growing wild in Turkey.
J. Agric. Food Chem. 43: 2262–2266.
Nair, R., Kalariya, T., and Chanda, S. 2005. Antibacterial activity of some selected Indian medicinal
flora. Turk J. Biol. 29: 41–47.
Newman, D.J., Cragg, G.M., and Snader, K.M. 2000. The influence of natural products upon drug
discovery. Nat. Prod. Rep. 17: 215–234.
Osbourn, A. 1996. Saponins and plant defence: A soap story. Trends Plant Sci. 1: 4–9.
Paganga, G., Miller, N., and Rice-Evans, C.A. 1999. The polyphenolic content of fruit and vegetables
and their antioxidant activities. What does a serving constitute? Free Radic. Res. 30: 153–162.
Parekh, P., McDaniel, M.C., Ashen, M.D., Miller, J.I., Sorrentino, M., Chan, V., Roger, S., Blumenthal,
R.S., and Sperling, L.S. 2005. Diets and cardiovascular disease: An evidence-based assessment.
J. Am. Coll Cardiol. 45: 1379–1387.
Pedras, M.S. and Jha, M. 2006. Toward the control of Leptosphaeria maculans: Design, syntheses,
biological activity, and metabolism of potential detoxification inhibitors of the crucifer

phytoalexin brassinin. Bioorg. Med. Chem. 14: 4958–4979.
Pedras, M.S., Chumala, P.B., and Suchy, M. 2003. Phytoalexins from Thlaspiarvense, a wild cruci-
fer resistant to virulent Leptosphaeria maculans, structures, synthesis and antifungal activity.
Prusti, A., Mishra, S.R., Sahoo, S., and Mishra, S.K. 2008. Antibacterial activity of some Indian
medicinal plants. Ethnobot. Leaflets 12: 227–230.
Rajendheran, J., Mani, A.M., and Navaneethakannan, K. 1998. Antibacterial activity of some selected
medicinal plants. Geobios 25: 280–282.
Romero-Tabarez, M. 2004. Discovery of the new antimicrobial compound 7-O-malonyl macrolactin
A. Aus Puerto Berrio, Kolumbien. PhD dissertation, Puerto Berrio, Colombia.
Schlee, D. 1986. Okologische Biochemie. Springer, Berlin, Germany.
Senanayake, U.M., Wills, R.B.H., and Lee, T.H. 1977. Biosynthesis of eugenol and cinnamic aldehyde
in Cinnamomum zeylanicum. Phytochemistry 16: 2032–2033.
Shanker, V., Bhalla, R., and Luthura, R. 2003. An overview of the non-mevalonic pathway for terpe-
noid biosynthesis in plants. J. Biosci. 28: 637–646.
Sharma, A. 1988. Secondary metabolites from tissue cultures of some medicinally important plants.
PhD thesis, University of Rajasthan, Jaipur, India.
Sieradzki, K., Wu, S.W., and Tomasz, A. 1999. Inactivation of the methicillin resistance gene mecA in
vancomycin-resistant Staphylococcus aureus. Micro. Drug Resist. 5(4): 253–257.
Singh, S., Singh, S.K., and Tripathi, S.C. 1988. Fungitoxic properties of essential oil of Eucalyptus
rostrata. Indian Perfum. 32: 190–193.
Sparg, S.G., Light, M.E., and Van Staden, J. 2004. Biological activities and distribution of plant sapo-
nins. J. Ethnopharmacol. 94: 219–43.
Steinstraesser, L., Tack, B.F., Waring, A.J., Hong, T., Boo, L.M., Fan, M.H., Remick, D.I., Su, G.L.,
Lehrer, R.I., and Wang, S.C. 2002. Activity of novispirin G10 against Pseudomonas aeruginosa
in vitro and in infected burns. Antimicrob. Agents Chemother. 46(6): 1837–1844.
Swain, T. 1977. Secondary compounds as protective agents. Annu. Rev. Plant Physiol. 28: 479–501.
Tanaka, Y., Data, E.S., Hirose, S., Taniguchi, T., and Uritani, I. 1983. Biochemical changes in second-
ary metabolites in wounded and deteriorated cassava roots. Agric. Bio. Chem. 47: 693–700.
Tewari, L.C., Agarwal, R.G., Pandey, M.J., Uniyal, M.R., and Pandey, G. 1990. Some traditional folk
medicine from the Himalayas (U. P. Region). Aryavaidyan 4: 49–57.
Uritani, I. and Hoshiya, I. 1953. Phytopathological chemistry of the black-rotted sweet potato. Part 6.
Isolation of coumarin substances from potato and their physiology. J. Agric. Chem. Soc. 27:
161–164.
Veshkurova, O., Golubenko, Z., Pshenichnov, E., Arzanova, I., Uzbekov, V., Sultanova, E., Salikhov, S.,
Williams, H.J., Reibenspies, J.H., and Puckhaber, L.S. 2006. Malvone A phytoalexin found in
Malva sylvestris (family Malvaceae). Phytochemistry 67(21): 2376–2379.
Wanchai, D.E. 1998. Chasing the key enzymes of secondary metabolite-biosynthesis from thai
medicinal plants. Invited lecture presented at the International Conference on Biodiversity and
Bioresources: Conservation and Utilization, Phuket, Thailand. Other presentations are published
in Pure Appl. Chem. 70(11): 1998.
Wink, M. 1987. Chemical ecology of quinolizidine alkaloids. In: Waller, G.R. (ed.), Allelochemicals:
Role in Agriculture, Forestry and Ecology. Am. Chem. Soc. 330: 524–533.
Wink, M. 1988. Plant breeding: Importance of plant secondary metabolites for protection against
pathogens and herbivores. Theor. Appl. Genet. 75: 225–233.
chapter seventeen
Role of antimicrobial compounds

from Trichoderma spp. in
plant disease management
Someshwar Bhagat, O.P. Sharma, Ajanta Birah, Natarajan
Amaresan, Israr Ahmad, Nasim Ahmad, and C. Chattopadhyay
Contents
17.1 Introduction......................................................................................................................... 359
17.2 Antimicrobial compounds in plant disease management........................................... 360
17.3 Inhibition of bioactive molecules produced by other fungi......................................... 363
17.4 Compounds favor competition for nutrients..................................................................364
17.5 Compounds favor systemic-induced resistance............................................................. 365
17.6 Compounds that enhance plant growth, development, and yield.............................. 365
17.7 Conclusion........................................................................................................................... 366
References...................................................................................................................................... 366
17.1 Introduction
Antimicrobial compounds such as secondary metabolites and cell wall–degrading
enzymes produced by Trichoderma spp. play a pivotal role in plant disease management.
Trichoderma spp. are well-known biocontrol agents that are successfully used as biopes-
ticides worldwide, and many among them are producers of secondary metabolites with
antimicrobial properties. Antimicrobial compounds produced by Trichoderma spp. are
often coupled with other mechanisms of biocontrol such as mycoparasitism and the pro-
duction of cell wall–degrading enzymes, competition for nutrients/space, and induced
resistance in the plant. The production of antimicrobial compounds by Trichoderma spp.
is strain specific, which includes volatile and nonvolatile antimicrobial compounds, such
as 6-pentyl-α-pyran-2-one, gliotoxin, viridin, harzianopyridone, harziandione, and peptai-
bols. Synergistic effects between cell wall–degrading enzymes and plethora of secondary
metabolites/antibiotics of Trichoderma spp. on fungal pathogen growth have been stud-
ied and well documented (Vinale et al., 2008). Ghisalberti and Sivasithamparam (1991)
have classified antimicrobial compounds into three categories: (1) volatile antibiotics, that
is, 6-pentyl-α-pyrone and most of the isocyanide derivates; (2) water-soluble compounds,
that is, heptelidic acid or koningic acid; and (3) peptaibols, which are linear oligopeptides
of amino acids. The involvement of toxic metabolites in plant disease development and
the interactions between beneficial and pathogenic fungi have been conclusively demon-
strated (Howell, 1998).
359
Trichoderma spp. are the most frequently isolated soil fungi and present in plant root
systems. These fungi are opportunistic, avirulent plant symbionts (Harman et al., 2004)
and function as parasites and antagonists of many plant pathogenic fungi. Trichoderma
spp. are among the most-studied fungal biocontrol agents and commercially marketed
as biopesticides, biofertilizers, and soil amendments (Harman et al., 2004; Lorito et al.,
2004). Trichoderma, a filamentous soil-inhabiting mycoparasite, has been used in commer-
cial preparation for biological control of many fungal-induced plant diseases (Bhagat and
Pan, 2007, 2010; Bhagat et al., 2013). Trichoderma has multifaceted function in agriculture,
and it can work in several ways: (1) colonization by establishing themselves within diverse
microbial communities in the rhizosphere, (2) control of pathogenic and competitive/
deleterious microflora by using a variety of mechanisms (antimicrobial compounds), (3)
improvement of the crop health, and (4) stimulation of root growth (Harman et al., 2004;
Vinale et al., 2008; Bhagat and Pan, 2010; Bhagat et al., 2013).
The first record of antibiotics exudation in the growth medium of Trichoderma lignorum
was made by Weindling (1934) followed by isolation of crystalline compound from there
(Weindling and Emerson, 1936) and isolation of the same compound from Gliocladium fim-
briatum also with high fungicidal activity by Weindling (1937), which received much atten-
tion among the researchers and opened new area of research for plant disease management.
Several cell wall–degrading enzymes from different strains of Trichoderma have been
purified and characterized. Interestingly, when tested alone or in combinations, the purified
proteins showed inhibitory activity toward a broad spectrum of fungal pathogens (Rhizoctonia
solani, Fusarium sp., Alternaria sp., Ustilago sp., Venturia sp., and Colletotrichum sp., as well as the
Oomycetes Pythium sp. and Phytophthora sp., which lack chitin in their cell walls).
Direct application of antimicrobial compounds produced by Trichoderma spp. and sev-
eral other biocontrol agents, instead of the whole live organisms, has numerous advantages
in plant disease management and may be more acceptable for the major stakeholders. They
have very short shelf life and are difficult to store even for a shorter period. The selective
production of active compounds may be performed by modifying the growth conditions
of microbes (including Trichoderma), that is, type and composition of culture medium, tem-
perature for incubation, and pH.
Trichoderma spp. produce various kinds of secondary metabolites that are chemically
different natural compounds of relatively low molecular weight, which are mainly pro-
duced by microorganisms and typically associated to individual genera, species, or strains.
Secondary metabolites are biosynthesized from primary metabolites in specialized path-
ways (i.e., polyketides or mevalonate pathways derived from acetyl coenzyme A or amino
acids). These compounds show several biological activities possibly related to survival
functions of the organism, such as competition against other micro- and macroorganisms,
antibiosis, symbiosis, and metal transport, and play an important role in plant disease man-
agement. However, biological activities are not necessarily confined to one specific group or
any particular single metabolite (Hanson, 2003, 2005). Some of fungal antimicrobial com-
pounds can modify the growth and the metabolism of plants, whereas others seem to target
specific fungal activities such as sporulation and hyphal elongation (Keller et al., 2005). Some
of the secondary metabolites produced by Trichoderma and Gliocladium species are as follows.
17.2 Antimicrobial compounds in plant disease management

The secondary metabolites are the major constituent of antimicrobial compounds of
Trichoderma, which are a heterogeneous group of natural compounds that are considered
to help the producing organism in survival and basic functions, namely, competition,
Chapter seventeen: Role of antimicrobial compounds from Trichoderma spp. 361
symbiosis, metal transport, and differentiation. Trichoderma spp. are well-known produc-
ers of antimicrobial compounds that are toxic for phytopathogenic fungi (Ghisalberti,
2002). Some of antimicrobial compounds that directly affect plant disease management
are as follows.
Pyrones are one of the first volatile antifungal compounds isolated from Trichoderma
species. This compound was identified by Collins and Halim (1972) in Trichoderma viride.
Later on, it has been isolated from several Trichoderma species and strains. The pyrone
6-pentyl-2H-pyran-2-one (6-pentyl-α-pyrone) is a metabolite commonly purified in the cul-
ture filtrate of different Trichoderma species (T. viride, T. atroviride, T. harzianum, T. koningii)
and is directly linked with the coconut aroma released by axenically developed colonies.
6-Pentyl-α-pyrone has shown both in vivo and in vitro antifungal activities toward several
plant pathogenic fungi, and a strong relationship has been found between the biosynthesis
of this metabolite and the biocontrol ability of the producing microbe (Bhagat et al., 2013;
Vinale et al., 2014). Takahiro et al. (2013) have isolated another pyrone named cytosporone
S from a Trichoderma sp., which has been reported to have in vitro antimicrobial activity
against several bacteria and fungi (Table 17.1).
Koninginins are complex pyrones isolated from T. koningii, T. harzianum, and
T. aureoviride. Koninginins A, B, D, E, and G showed in vitro antibiotic activity toward the
take-all pathogen Gaeumannomyces graminis var. tritici. Koninginin D can also inhibit the
growth of other important soilborne plant pathogens such as Pythium middletonii, R. solani,
Phytophthora cinnamomi, Fusarium oxysporum, and Bipolaris sorokiniana.
Another antifungal compound viridin, isolated from T. koningii, T. viride, and
T. virens (Singh et al., 2005; Vinale et al., 2014) prevented spore germination of Botrytis
allii, Colletotrichum lini, Fusarium caeruleum, Penicillium expansum, Aspergillus niger, and
Stachybotrys atra (Vinale et al., 2014). T. viride, T. hamatum, and certain Gliocladium species
produce viridiol, another member of viridins, with antifungal and phytotoxic metabolite.
T. viride, T. hamatum, and certain Gliocladium species produce viridiol, a similar antifungal
and phytotoxic metabolite for which the in vivo activity has been demonstrated (Howel
and Stipanovic, 1994; Vinale et al., 2014).
Harzianopyridone, a nitrogen heterocyclic compound, obtained from T. harzianum
metabolite is a potent antibiotic effective against B. cinerea, R. solani (Vinale et al., 2014),
G. graminis var. tritici, and P. ultimum (Vinale et al., 2006). A new compound named harzianic
acid, characterized by the presence of a pyrrolidindione ring system, has been isolated
from T. harzianum. This tetramic acid derivative exhibited in vitro antibiotic activity against
Pythium irregulare, Sclerotinia sclerotiorum, and R. solani (Vinale et al., 2009). Recently, Vinale
et al. (2013) have also reported harzianic acid from Trichoderma with antimicrobial and
plant growth promotion activities.
The azaphilones isolated from T. harzianum T22 are natural products containing a
highly oxygenated bicyclic core and a chiral quaternary center. A new azaphilone, named
T22 azaphilone, showed a marked growth inhibition of several plant pathogens (R. solani,
P. ultimum, and G. graminis var. tritici) in vitro (Vinale et al., 2006).
Harzianolide and its derivatives, butenolide and deydroharzianolide, were isolated
from different strains of T. harzianum (Vinale et al., 2006, 2014). The in vitro antifungal activ-
ities of these antimicrobial compounds were established against several phytopathogenic
agents (Vinale et al., 2006, 2014). A novel hydroxy-lactone derivative, named cerinolactone,
has been isolated from culture filtrates of Trichoderma cerinum. The isolated compound
showed in vitro antifungal activity against R. solani, P. ultimum, and B. cinerea.
Trichoderma spp. also produce isocyano metabolites. But isolation and separation of
these compounds are very difficult due to their instability (Reino et al., 2008). Dermadin
Table 17.1 Antimicrobial activities of secondary metabolites isolated from

Trichoderma sp.
Metabolites derived from Trichoderma spp. Antimicrobial activity
Acetate derivative
Ferulic acid Gliocladium virens Antiviral, bactericide,
and fungicide
Tricarboxylic acid derivatives
Viridiofungins A, B, C T. viride Antifungal
Polyketides
Benzoquinones and quinhydrones G. roseum Antibiotic
Oxygen heterocyclic compounds
Nectariapyron G. vermoesenii Antibiotic
Harzianopyridone T. harzianum Antifungal
Butenolide harzianolide T. harzianum Antifungal
Harzianic acid T. harzianum Antimicrobial
Pyrones
6-pentyl-a-pyrone Trichoderma species Antifungal
Antimicrobial
Dehydroderivative of 6-pentyl-a-pyrone T. viride and Antifungal
T. koningii
Massoilactone and d-decanolactone Trichoderma species Antifungal
T. koningii Antifungal
and T. harzianum
Octaketides G. virens and T. Antifungal
Sesquiterpenes viride
Daucane sesquiterpenes
Trichothecenes Trichoderma species Antifungal
Triterpenes and sterol
Helvolic acid Gliocladium Antibiotic
species
Ergokonin A and B T. koningii Antifungal
Viridin Gliocladium virens Antibiotic
Isocyano derivatives
Dermadine Trichoderma spp. Antibiotic and
antifungal
Diketopiperazines
Gliotoxin Gliocladium virens Antibiotic, antiviral
Epitrisulfide Gliocladium virens Antibiotic
Diketopiperazines
Gliovirin Gliocladium virens Antibiotic, antiviral
Polypeptides
Trichopolyns T. polysporum Antibiotic
Source: Sivasithamparam, K. and Ghisalberti, E.L., Secondary metabolism in Trichoderma and
Gliocladium, In: Trichoderma and Gliocladium, Vol. 1, Harman, G.E., Kubicek, C.P., Eds.,
London, U.K.,Taylor & Francis Ltd., pp. 139–191, 1998.
from T. viride, T. koningii, and T. hamatum is an antibiotic metabolite. The isonitrile tricho-
viridin isolated from T. koningii and T. viride showed in vitro antimicrobial properties.
Several dermadin and trichoviridin analogues have also been isolated.
Gliotoxin and gliovirin are the two most important Trichoderma antimicrobial com-
pounds belonging to diketopiperazines. P group strains of Trichoderma (Gliocladium)
virens produced the gliovirin, which was very active against P. ultimum, but inactive
against R. solani. Similarly, strains of the Q group produced gliotoxin, which was very
active against R. solani, but less active against P. ultimum (Howell, 1999; Vinale et al., 2014).
Seedling bioassay tests revealed that the strains of the P group were able to effectively
control Pythium damping off on cotton, while Q group strains gave better results toward
the same disease caused by R. solani (Howell, 1991; Howell et al., 1993; Vinale et al., 2014).
These studies clearly indicated the potential role of antibiotic production in the biocontrol
mechanism of the gliotoxin/gliovirin producers. The T. virens veA ortholog vel1 (VELVET
protein Vel1) is involved in regulation of gliotoxin biosynthesis, biocontrol activity, and
many other secondary metabolism-related genes (Mukherjee et al., 2010, 2013).
Peptaibols are linear peptides rich in nonproteinogenic amino acids (α-aminoisobutyric
acid and isovaline); acetylated at the N-terminal group and the C-terminus is an amino
alcohol (phenylalaninol, valinol, leucinol, isoleucinol, or tryptophanol). Lorito et al. (1996)
demonstrated that peptaibols inhibited β-glucan synthase activity in the host fungus, but
acting synergistically with T. harzianum β-glucanases. The inhibition of glucan synthase
prevented the reconstruction of the pathogen cell walls, thus facilitating the disruptive
action of β-glucanases. The most important peptaibol is the T. viride alamethicin. The terms
peptaibiome and peptaibiomics (peptide antibiotics or peptaibiotics) have been suggested
to describe the analysis and study of all peptaibols expressed in an organism or tissue using
spectrometric methods, like LC/ESI-MSn or intact-cell MALDI-TOF (Neuhof et al., 2007).
Mukherjee et al. (2012b) reviewed the genes that determine the range of secondary metabo-
lites produced by Trichoderma, including both useful and toxic compounds. Polyketide syn-
thases and nonribosomal peptide synthases (NRPSs) define two major classes of secondary
metabolites (Mukherjee et al., 2012a; Baker et al., 2012).
17.3 Inhibition of bioactive molecules produced by other fungi

Some of antimicrobial compounds (secondary metabolites) produced by Trichoderma spp.
inhibit certain bioactive molecules formed by other fungi. They may be produced by cer-
tain antagonistic and endophytic fungi also in pure culture. Detoxification of fungal toxins
by beneficial microorganisms also represents interesting possibilities for disease manage-
ment. Pure cultures of fungi able to detoxify mycotoxins have been obtained from complex
microbial populations by using enrichment culture techniques (Karlovsky, 1999). T. viride
and other fungal species were able to degrade aflatoxin B1. Moreover, T. harzianum hydro-
lases were able to degrade aflatoxin B1 (AFB1) and ochratoxin A (OTA) in vitro.
In substrates with high carbon/nitrogen ratios, T. virens produced a metabolite similar
to the antibiotic viridin, called viridiol that acts as a plant growth inhibitor. This com-
pound, isolated also from T. hamatum, inhibited the 5′-hydroxyaverantin dehydrogenase,
involved in aflatoxin biosynthesis, thus reducing or completely blocking the production
of this mycotoxin during the fungal interactions (Sakuno et al., 2000; Wipf and Kerekes,
2003). Elad (1996) found that mycoparasitism and antibiosis were not the main biocon-
trol mechanisms of T. harzianum strain T39 against B. cinerea. These authors indicated that
the antagonist interferes with the infection process by affecting the pathogen conidia in
the early stages of the interaction. They suggested that T. harzianum (T39) acts directly by
inhibiting B. cinerea hydrolytic enzymes or indirectly by blocking plant responses that

induce enzymatic activity in B. cinerea. Subsequently, Elad and Kapat (1999) demonstrated
that a Trichoderma protease is involved in the biocontrol of B. cinerea by degrading the
hydrolytic enzymes required for infection by the pathogen.
17.4 Compounds favor competition for nutrients

Competition for carbon, nitrogen, and iron plays an important role during the interactions
between beneficial and pathogenic fungi and is often associated with the biocontrol mech-
anisms of nonpathogenic Fusarium and Trichoderma species (Vinale et al., 2008). Trichoderma
has a strong ability to mobilize and take up soil nutrients, which makes it more efficient
and competitive than many other soil microbes. This process could be related also to the
production of organic acids, namely, fumaric, gluconic, and citric acids, which decrease
soil pH and allow the solubilization of micronutrients, phosphates, and mineral cations
such as iron, manganese, and magnesium (Vinale et al., 2008). Iron is a mineral essential
nutrient for numerous microorganisms, both bacteria and fungi. In the aerobic environ-
ment (with oxygen and neutral pH), iron exists mainly as Fe3+ in immobilized form rather
than hydroxides and oxyhydroxides forms, making it unavailable for microbial growth
(Miethke, 2013). Microorganisms that excrete siderophores are able to grow in natural
environments poor in iron using residual immobilized iron. Most fungi produce vari-
ous siderophores, which help the microbes to overcome adverse conditions (Winkelmann,
2007). The production of microbial siderophores can be beneficial to plants for two reasons:
(1) siderophores can solubilize iron unavailable for the plant, and (2) siderophore produc-
tion by nonpathogenic microorganisms can also suppress the growth of plant pathogens
by depriving them of iron sources. The majority of the fungal siderophores isolated so far
belong to hydroxamate class and can be divided into three structural families: fusarinines,
coprogens, and ferrichromes. Fungi typically produce more than one siderophore, even if
restricted to a particular family.
Segarra et al. (2010) analyzed the effect of iron during the interaction of Trichoderma
(T34) with F. oxysporum f.sp. lycopersici on tomato plants, to establish the importance of iron
concentration for the activity of a Trichoderma asperellum strain (T34). These results clearly
indicated a role of siderophores during the interaction with the pathogen and the plants,
although no siderophores have been isolated and characterized thus far from the culture
filtrate of this beneficial microbe (De Santiago et al., 2009). But Vinale et al. (2013) demon-
strated the ability of tetramic acid from Trichoderma sp. to bind with a good affinity to Fe3+,
which explains the mechanism of iron solubilization that significantly changes nutrient
availability in the soil environment for other microorganisms and the host plant. In addi-
tion to iron transport, siderophores produced by microorganisms (including Trichoderma
sp.) have other functions and effects, including enhancement of virulence of pathogens,
storage of intracellular iron, and suppression of microbial growth during the competition
with other microbes (Miethke, 2013).
Anke et al. (1991) isolated siderophores from all different structural families simultane-
ously from a culture filtrate of Trichoderma spp. The culture filtrate of this fungus obtained
in iron-deficient condition contained coprogen, coprogen B, fusarinine C, and ferricrocin.
T. harzianum produced the highest number of siderophores and did not have any unique
compound, while Trichoderma reesei biosynthesized one cisfusarinine as the major sidero-
phore and three others that were present only in T. harzianum. The variation of siderophores
produced by Trichoderma spp. is expected due to further modifications of the NRPS products
rather than diverse NRPS-encoding genes (Lehner et al., 2013).
17.5 Compounds favor systemic-induced resistance

Several antimicrobial compounds (metabolites) produced by Trichoderma are involved
in the induction of plant resistance, such as (1) proteins with enzymatic activity, that is,
xylanase; (2) avirulence-like gene products able to induce defense reactions in plants; and
(3) low-molecular-weight compounds released from either fungal or plant cell walls, by spe-
cific enzyme activities. Some of the low-molecular-weight degradation products released
from fungal cell walls have been purified and characterized that consist of short oligosac-
charides with two types of monomers, with and without an amino acid residue (Woo et al.,
2006; Woo and Lorito, 2007). These compounds elicited a reaction in the plant when applied
to leaves or when injected into root or leaf tissues. They also stimulated the biocontrol
ability of fungi such as Trichoderma by activating the mycoparasitic gene expression cas-
cade. Some Trichoderma secondary metabolites may act as elicitors of plant defense mecha-
nisms against pathogens. A reduction of disease symptoms on tomato and canola seedlings
treated with 6PP and inoculated with the pathogens B. cinerea or Leptosphaeria maculans has
been reported. Moreover, soil drench applications of 6PP 4 days before inoculation with
Fusarium moniliforme showed considerable suppression of seedling blight and significant
plant growth promotion, as compared to untreated control (El-Hasan and Buchenauer,
2009). Application of 6PP on maize seedlings distinctly enhanced the activities of β-1,3-
glucanase, peroxidase, and polyphenoloxidase in both shoot and root tissues indicating an
induction of defense responses in maize plants (El-Hasan and Buchenauer, 2009).
Peptaibols are another class of plant defense elicitors produced by Trichoderma.
Application of alamethicin, a long sequence peptaibol with 20 residues produced by
T. viride, induced defense responses in Phaseolus lunatus (lima bean) (Engelberth et al.,
2000) and Arabidopsis thaliana (Chen et al., 2003). Mukherjee et al (2012b) demonstrated
that mutation in one of the polyketide synthase/nonribosomal peptide synthases (PKS/
NRPS) hybrid genes caused reduction in induction of the defense response gene phenyl-
alanine ammonia lyase of T. virens, suggesting a putative role for the associated metabolite
(Mukherjee et al., 2012b). These results provide evidence that a PKS/NRPS hybrid enzyme
responsible for the metabolite production is involved in Trichoderma–plant interactions
resulting in induction of defense response in maize.
17.6 Compounds that enhance plant growth,

development, and yield
Trichoderma species can improve the plant growth, development, and yield (Harman et al.,
2004; Bhagat et al., 2013; Mukherjee et al., 2013). Several strains belonging to the Trichoderma
genus have been found to be able to stimulate plant development (Kumar et al., 2011),
especially at the root level (i.e., formation of more lateral roots), by activating an auxin-
dependent mechanism (Contreras-Cornejo et al., 2009; Vinale et al., 2014) and/or produc-
ing indole-3-acetic acid (IAA) or auxin analogues (Hoyos-Carvajal et al., 2009; Vinale
et al., 2014). Growth promotion of plant by antimicrobial compounds of Trichoderma has
been demonstrated (Vinale et al., 2012b). Koninginins, 6PP, trichocaranes A–D, harziano-
pyridone, cyclonerodiol, harzianolide, and harzianic acid are examples of isolated com-
pounds that promote plant growth in a concentration-dependent manner (Vinale et al.,
2014). A novel metabolite, named cerinolactone, has been isolated and characterized from
T. cerinum and was able to positively alter the growth of tomato seedlings 3 days after
treatment (Vinale et al., 2012a, 2014). The dose–effect response of plant growth to second-
ary metabolites produced by Trichoderma spp. clearly indicated need for further systematic
research. These metabolites seem to act as auxin-like molecules, which have a positive
effect at low concentrations with inhibitory effect at higher doses. A hormone activity was
detected on etiolated pea stems treated with harzianolide and 6PP. These compounds also
affected the growth of tomato and canola seedlings in a manner depending on the concen-
tration and/or the application method used (Vinale et al., 2008, 2014).
17.7 Conclusion
A plethora of antimicrobial compounds produced by Trichoderma spp. have been isolated
and characterized. The fungal secondary metabolites with a direct antimicrobial activity
against plant pathogens have been mainly isolated from biocontrol strains of the genus
Trichoderma (Sharma et al., 2004). Even though many secondary metabolites are known,
elite strains usually produce only a few main secondary metabolites (Bhagat, 2008;
Mukherjee et al., 2012a; Bhagat et al., 2013). The quality and quantity of secondary metabo-
lites synthesized depend on (1) the compound considered, (2) the species and the strain,
(3) the occurrence of other microorganisms, (4) the equilibrium among elicited biosynthe-
sis and biotransformation rate, and (5) the growth conditions. Furthermore, in some cases,
the biocontrol agent was able to modulate the production of toxic secondary metabolites
according to the presence or the absence of the target pathogen (Vinale et al., 2009; Bhagat
and Pan, 2010).
Interestingly, antimicrobial compounds produced by Trichoderma spp. may also be
involved in biocontrol mechanism through the inhibition of bioactive products. It is well
recognized that biocontrol fungi, such as selected agents of Trichoderma spp., are able to
produce compounds with multiple activities, including direct/indirect toxic effects against
plant pathogens, plant defense induction, or growth promotion (Vinale et al., 2008; Sharma
et al., 2004). A hormone-like effect has been proposed for some Trichoderma secondary
metabolites, and specific antimicrobial compounds having this characteristic have been
detected in plant–fungus cultures. In fact, treatment with Trichoderma metabolites pro-
duces extensive changes of the plant expressome, proteome, and metabolome, by acting
on specific pathways involved in the synthesis of major growth hormones and induction
of resistance to biotic/abiotic stresses and nutrient uptake (Vinale et al., 2012; Mukherjee
et al., 2012a; Bhagat et al., 2013). These recent findings have suggested new strategies for the
development of novel bioformulations based on antimicrobial compounds alone or in com-
bination with live organisms, in order to maximize the desired beneficial effects and reduce
the risks associated with the release of microorganisms into the diverse crop ecosystem.
References
Almassi, F., Ghisalberti, E.L., Narbey, M.J., and Sivasithamparam, K. 1991. New antibiotics from
strains of Trichoderma harzianum. J Nat Prod 54: 396–402.
Anke, H., Kinn, J., Bergquist, K.E., and Sterner, O. 1991. Production of siderophores by strains of the
genus Trichoderma—Isolation and characterization of the new lipophilic coprogen derivative,
palmitoylcoprogen. Biometals 4: 176–80.
Baker, S.E., Perrone, G., Richardson, N.M., Gallo, A., and Kubicek, C.P. 2012. Phylogenetic analysis
and evolution of polyketide synthase-encoding genes in Trichoderma. Microbiology 158: 147–154.
Bhagat, S., Bambawale, O.M., Tripathi, A.K., Ahmad, I., and Srivastava, R.C. 2013. Biological manage-
ment of fusarial wilt of tomato by Trichoderma spp. in Andamans. Ind J Hort 70: 397–403.
Bhagat, S. and Pan, S. 2007. Mass multiplication of T. harzianum on agricultural byproducts and their
evaluation in management of seedling blight (R. solani) of mung bean and collar rot (S. rolfsii)
of groundnut. Ind J Agric Sci 77: 583–588.
Bhagat, S. and Pan, S. 2010. Biological management of root and collar rot of French bean. Ind J Agric
Sci 42: 42–50.
Bhagat, S. 2008. Biocontrol potential of some isolates of Trichoderma spp. from Andaman and Nicobar
Islands. A Ph.D. thesis submitted to Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, West
Bengal, India.
Brian, P.W. and McGowan, J.C. 1945. Viridin: A highly fungistatic substance produced by Trichoderma
viride. Nature 156: 144–145.
Chen, F., D’Auria, J.C., and Tholl, D. 2003. An Arabidopsis thaliana gene for methylsalicylate biosyn-
thesis, identified by a biochemical genomics approach, has a role in defence. Plant J 36: 577–588.
Claydon, N., Hanson, J.R., Truneh, A., and Avent, A.G. 1991. Harzianolide, a butenolide metabolite
from cultures of Trichoderma harzianum. Phytochemistry 30: 3802–3803.
Collins, R.P. and Halim, A.F. 1972. Characterisation of the major aroma constituent of the fungus
Trichoderma viride (Pers.). J Agric Food Chem 20: 437–438.
Contreras-Cornejo, H.A., Macìas-Rodrìguez, L., Cortés-Penagos, C., and López-Bucio, J. 2009.
Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral
root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiol 149: 1579–1592.
De Santiago, A., Quintero, J.M., Avilés, M., and Delgado, A. 2009. Effect of Trichoderma asperellum
strain T34 on iron nutrition in white lupin. Soil Biol Biochem 41: 2453–2459.
Dickinson, J.M., Hanson, J.R., Hitchcock, P.B., and Claydon, N. 1989. Structure and biosynthesis of
harzianopyridone, an antifungal metabolite of Trichoderma harzianum. J Chem Soc Perkin Trans
1: 1885–1887.
Elad, Y. 1996. Mechanisms involved in the biological control of Botrytis cinerea incited diseases. Eur
J Plant Pathol 102: 719–732.
Elad, Y. and Kapat, A. 1999. The role of Trichoderma harzianum protease in the biocontrol of Botrytis
cinerea. Eur J Plant Pathol 105: 177–189.
El-Hasan, A. and Buchenauer, H. 2009. Actions of 6-pentyl-alpha-pyrone in controlling seedling
blight incited by Fusarium moniliforme and inducing defence responses in maize. J Phytopathol
157: 697–707.
Engelberth, J., Koch, T., Schuler, G., Bachmann, N., Rechtenbach, J., and Boland, W. 2000. Ion
channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling.
Cross talk between jasmonate and salicylate signaling in lima bean. Plant Physiol 125: 369–377.
Ghisalberti, E.L. 2002. Anti-infective agents produced by the hyphomycetes genera Trichoderma and
Glioclaudium. Curr Med Chem 1: 343–374.
Ghisalberti, E.L. and Sivasithamparam, K. 1991. Antifungal antibiotics produced by Trichoderma
spp. Soil Biol Biochem 23: 1011–1020.
Golder, W.S. and Watson, T.R. 1980. Lanosterol derivatives as precursors in the biosynthesis of viri-
din. Part 1. J Chem Soc Perkin Trans 1: 422–425.
Hanson, J.R. 2003. Natural Products: The Secondary Metabolites. Vol. 17 Ed. Cambridge, U.K.: Royal
Society of Chemistry.
Hanson, J.R. 2008. The Chemistry of Fungi. Ed. Cambridge, U.K.: Royal Society of Chemistry.
Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., and Lorito, M. 2004. Trichoderma species opportu-
nistic, avirulent plant symbionts. Nat Rev Microbiol 2: 43–56.
Hoyos-Carvajal, L., Orduz, S., and Bissett, J. 2009. Growth stimulation in bean (Phaseolus vulgaris L.)
by Trichoderma. Biol Control 51: 409–416.
Howell, C.R. 1991. Biological control of Pythium damping off of cotton with seed coating prepara-
tions of Gliocladium virens. Phytopathology 81: 738–741.
Howell, C.R., Stipanovic, R.D., and Lumsden, R.D. 1993. Antibiotic production by strains of
Gliocladium virens and its relation to the biocontrol of cotton seedling diseases. Biocontrol Sci
Technol 3: 435–441.
Howell, C.R. 1998. The role of antibiosis in biocontrol. In: Kubiecek, C.P., Harman, G.E., editors.
Trichoderma and Gliocladium, Vol 1. London, U.K.: Taylor & Francis; 173–184.
Howell, C.R. 1999. Selective isolation from soil and separation in vitro of P and Q strains of Trichoderma
virens with differential media. Mycologia 91: 930–934.
Howell, C.R. 2003. Mechanisms employed by Trichoderma species in the biological control of plant
diseases: The history and evolution of current concepts. Plant Dis 87: 4–10.
Karlovsky, P. 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and
food production. Nat Toxins 7: 1–23.
Keller, N.P., Turner, G., and Bennett, J.W. 2005. Fungal secondary metabolism—From biochemistry
to genomics. Nat Rev Microbiol 3: 937–947.
Lehner, S.M., Atanasova, L., and Neumann, N.K. 2013. Isotope-assisted screening for iron-containing
metabolites reveals high diversity among known and unknown siderophores produced by
Trichoderma spp. Appl Environ Microbiol 79: 18–31.
Leong, J. 1986. Siderophores: Their biochemistry and possible role in the biocontrol of plant patho-
gens. Ann Rev Phytopathol 24: 187–209.
Lorito, M. 2008. A novel role for Trichoderma secondary metabolites in the interactions with plants.
Physiol Mol Pl Pathol 72: 80–86.
Lorito, M., Farkas, V., Rebuffat, S., Bodo, B., and Kubicek, C.P. 1996. Cell wall synthesis is a major
target of mycoparasitic antagonism by Trichoderma harzianum. J Bacteriol 178: 6382–6385.
Miethke, M. 2013. Molecular strategies of microbial iron assimilation: From high-affinity complexes
to cofactor assembly systems. Metallomics 5: 15–28.
Moffatt, J.S., Bu’Lock, J.D., and Yuen, T.H. 1969. Viridiol, a steroid-like product from Trichoderma
viride. J Chem Soc Chem Commun 14: 849.
Mukherjee, P.K. and Kenerley, C.M. 2010. Regulation of morphogenesis and biocontrol properties in
Trichoderma virens by a VELVET protein, Vel1. Appl Environ Microbiol 76: 2345–2352.
Mukherjee, P.K., Horwitz, B.A., and Kenerley, C.M. 2012a. Secondary metabolism in Trichoderma—A
genomic perspective. Microbiology 158: 35–45.
Mukherjee, P.K., Buensanteai, N., Moran-Diez, M.E., Druzhinina, I.S., and Kenerley, C.M. 2012b.
Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens
reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic
resistance response in maize. Microbiology 158: 155–165.
Mukherjee, P.K., Horwitz, B.A., Herrera-Estrella, A., Schmoll, M., and Kenerley, C.M. 2013. Trichoderma
research in the genome era. Ann Rev Phytopathol 51:105–129.
Neuhof, T., Dieckmann, R., Druzhinina, I.S., Kubicek, C.P., and von Dohren, H. 2007. Intact-cell MALDI-
TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: Can
molecular phylogeny of species predict peptaibol structures? Microbiology 153: 3417–3437.
Ordentlich, A., Wiesman, Z., Gottlieb, H.E., Cojocaru, M., and Chet, I. 1992. Inhibitory furanone pro-
duced by the biocontrol agent Trichoderma harzianum. Phytochemistry 31: 485–486.
Reino, J.L., Guerrero, R.F., Hernández-Galán, R., and Collado, I.G. 2008. Secondary metabolites from
species of the biocontrol agent Trichoderma. Phytochem Rev 7: 89–123.
Sakuno, E., Yabe, K., Hamasaki, T., and Nakajima, H.A. 2000. New inhibitor of 5’-hydroxyaveran-
tin dehydrogenase, an enzyme involved in aflatoxin biosynthesis, from Trichoderma hamatum.
J Nat Prod 63: 1677–1678.
Scarselletti, R. and Faull, J.L. 1994. In Vitro activity of 6-pentyl-a-pyrone, a metabolite of Trichoderma
harzianum, in the inhibition of Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici. Mycol
Res 98:1207–1209.
Segarra, G., Casanova, E., Avilès, M., and Trillas, I. 2010. Trichoderma asperellum strain T34 controls
Fusarium wilt disease in tomato plants in soilless culture through competition for iron. Microb
Ecol 59: 141–149.
Sharma, O.P., Jeswani, M.D., Bambawale, O.M., and Murthy, K.S. 2004. Trichoderma as a potential
bio-agent for the management of diseases: Taxonomy, biology and mode of action. In: Microbial
Biotechnology, Trivedi, P.C., Eds. Jaipur, India: Avishankar Publishers & Distributors.
Sivasithamparam, K. and Ghisalberti, E.L. 1998. Secondary metabolism in Trichoderma and
Gliocladium. In: Trichoderma and Gliocladium, Vol. 1, Harman, G.E., Kubicek, C.P., Eds. London,
U.K.: Taylor & Francis Ltd. pp. 139–191.
Singh, S., Dureja, P., Tanwar, R.S., & Singh, A. 2005. Production and antifungal activity of secondary
metabolites of Trichoderma virens. Pestic Res J 17: 26–29.
Takahiro, I., Nonaka, K., Suga, T., Masuma, R., Omura, S., and Shiomi, K. 2013. Cytosporone S with
antimicrobial activity, isolated from the fungus Trichoderma sp. FKI-6626. Bioorg Med Chem Lett
23: 679–681.
Vinale, F., Marra, R., Scala, F., Ghisalberti, E.L., Lorito, M., and Sivasithamparam, K. 2006. Major sec-
ondary metabolites produced by two commercial Trichoderma strains active against different
phytopathogens. Lett App Microbiol 43: 143–148.
Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Marra, R., Woo, S.L., and Lorito, M. 2008.
Trichoderma-plant-pathogen interactions. Soil Biol Biochem 40: 1–10.
Vinale, F., Ghisalberti, E.L., and Sivasithamparam, K. 2009. Factors affecting the production of
Trichoderma harzianum secondary metabolites during the interaction with different plant
pathogens. Lett Appl Microbiol 48: 705–711.
Vinale, F., Arjona, G.I., and Nigro, M. 2012a. Cerinolactone, a hydroxylactone derivative from
Trichoderma cerinum. J Nat Prod 75: 103–106.
Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Ruocco, M., Woo, S., and Lorito, M. 2012b.
Trichoderma secondary metabolites that affect plant metabolism. Nat Prod Commun 7: 1545–1550.
Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Woo, S.L., Nigro, M., Marra, R., Lombardi, N.,
Pascale, A., Ruocco, M., Lanzuise, S., Manganiello, G., and Lorito, M. 2014. Trichoderma second-
ary metabolites active on plants and fungal pathogens. The Open Mycol J 8: 127–139.
Vinale, F., Nigro, N., Sivasithamparam, K., Flematti, G., Ghisalberti, E.L., Ruocco, M., Varlese, R.
et al. 2013. Harzianic acid: A novel siderophore from Trichoderma harzianum. FEMS Microbiol
Lett 347: 123–129.
Weindling, R. 1934. Studies on a lethal principle effective in the parasitic action of Trichoderma ligno-
rum on Rhizoctonia solani and other soil fungi. Phytopathology 24: 1153–1179.
Weindling, R. 1937. The isolation of toxin substances from the culture filtrates of Trichoderma and
Gliocladium. Phytopathology 27: 1175–1177.
Weindling, R. and Emersion, O.H. 1936. The isolation of a toxic substance from the culture filtrate of
a Trichoderma. Phytopathology 26: 1068–1070.
Winkelmann, G. 2007. Ecology of siderophores with special reference to the fungi. Biometals 20:
379–392.
Wipf, P. and Kerekes, A.D. 2003. Structure reassignment of the fungal metabolite TAEMC161 as the
phytotoxin viridiol. J Nat Prod 66: 716–871.
Woo, S.L. and Lorito, M. 2007. Exploiting the interactions between fungal antagonists, patho-
gens and the plant for biocontrol. In: Vurro, M. and Gressel, J., Eds., Novel Biotechnologies for
Biocontrol Agent Enhancement and Management. Amsterdam, the Netherlands: IOS, Springer
Press, pp. 107–130.
Woo, S.L., Scala, F., Ruocco, M., and Lorito, M. 2006. The molecular biology of the interactions
between Trichoderma spp., phytopathogenic fungi, and plants. Phytopathology 96: 181–185.
Worasatit, N., Sivasithamparam, K., Ghisalberti, E.L., and Rowland, C. 1994. Variation in pyrone
production, pectic enzymes and control of rhizoctonia root rot of wheat among single-spore
isolates of Trichoderma koningii. Mycol Res 98: 1357–1363.
chapter eighteen
Antimicrobial compounds from

rhizosphere bacteria and their role
in plant disease management
Natarajan Amaresan, Nallanchakravarthula Srivathsa, Velusamy
Jayakumar, Someshwar Bhagat, and Nooruddin Thajuddin
Contents
18.1 Introduction......................................................................................................................... 371
18.2 Plant–microbe interaction in rhizosphere....................................................................... 372
18.3 Rhizosphere bacteria in plant disease management..................................................... 373
18.4 Antimicrobial compounds of rhizosphere bacteria....................................................... 374
18.4.1 2,4-Diacetylphloroglucinol.................................................................................... 374
18.4.2 Phenazines............................................................................................................... 375
18.4.3 Pyrrolnitrin.............................................................................................................. 375
18.4.4 Bacteriocins.............................................................................................................. 375
18.5 Mechanism of action.......................................................................................................... 376
18.5.1 Antibiosis................................................................................................................. 376
18.5.2 Lytic enzyme production....................................................................................... 376
18.5.3 Degradation of pathogen virulence..................................................................... 377
18.5.4 Competition............................................................................................................. 377
18.5.5 Induced resistance.................................................................................................. 377
18.6 Formulations and field application.................................................................................. 378
18.7 Future perspectives............................................................................................................ 379
References...................................................................................................................................... 379
18.1 Introduction
Plants are in intimate relationship with microorganisms, and many of those associations
are permanent. Microbial association starts when a seed falls onto the soil and continues
with its germination, and its growth ceases at one point and it dies. The populations of
microorganisms surrounding/on the surface of plants exceeds 106 bacteria/g of leaves and
109 bacteria/g of roots. Many of these organisms are possibly saprophytic in nature (no
deleterious or beneficial effects can be conferred by their presence), nourishing themselves
from the nutrients produced by the plants. However, the presence of some microorgan-
isms can be detrimental in nature (e.g., pathogens), while some may be beneficial in nature
(e.g., plant growth-promoting rhizobacteria (PGPR), biocontrol agents (BCA), nitrogen-
fixing bacteria, and other symbionts).
371
Plants allocate carbon below ground in the form of root exudates, thereby influ-
encing the microbial communities (Morgan et al., 2005; Drigo et al., 2010). The term
“rhizosphere” was coined by Lorenz Hiltner (Hiltner, 1904) to define the volume of soil
in close proximity to roots that are characterized by elevated microbial populations.
The rhizosphere is under the continuous influence of living roots and the rich nutrient
supply (rhizodeposition), which enables microorganisms to have direct influence on
plant growth. The root exudates consist of simple and complex sugars, growth regula-
tors, primary and secondary compounds such as amino acids, organic acids, phenolic
acids, flavonoids, fatty acids, enzymes, steroids, alkaloids, and vitamins (Uren, 2000;
Philippot et al., 2013). Some of these root exudates are known to play a role in shaping
the microbial communities in the rhizosphere (Haichar et al., 2008; Bressen et al., 2009;
Drigo et al., 2010).
PGPR are one of the most commonly studied rhizosphere components in terms of
direct plant growth promotion and biological control and are isolated from different envi-
ronments (Lugtenberg and Kamilova, 2009; Amaresan et al., 2014a, b). Bacteria that are
intimately associated with plant roots, for example, endophytes, are also known for their
plant growth promotion and biological control (Kumar et al., 2011; Amaresan et al., 2012a).
The rhizobacteria are the dominant driving forces in recycling of soil nutrients, and subse-
quently, they are crucial for soil fertility (Glick, 2012). Rhizobacteria or the bacteria in the
rhizosphere help the plant directly or indirectly through various mechanisms and associa-
tions. Nitrogen fixation, synthesis of various phytohormones, and solubilization of various
minerals are some of the direct effects (Basak and Biswas, 2010; Panhwar et al., 2012), and
production of antagonistic substances specifically against plant pathogens is known to be
an indirect effect (Hao et al., 2011).
The antagonistic potential of these rhizobacteria as well as root-associated bacteria has
been shown to control various root, foliage, and postharvest diseases of agricultural crops
(Glick and Bashan, 1997; Goel et al., 2002; Weller, 2007; Amaresan et al., 2012b). Not only
bacteria but also fungi are known to have antagonistic effects against plant pathogens by
producing an array of antagonistic compounds (Kumar et al., 2012; Philippot et al., 2013).
When these organisms are used in a controlled manner, they can enhance overall soil
fertility and plant health (Berg, 2009). Rhizobacteria are being used as biocontrol agents,
as they are in the vicinity of plant roots and can act as frontline of defense against soil-
borne plant pathogens (Dowling and O’Gara, 1994). Biocontrol can be explained as the
“harnessing of disease-suppressive microorganisms for plant protection.”
The synergistic plant growth–promoting effects of fluorescent Pseudomonas sp. and
Mesorhizobium sp. cicer strain in both sterile and “wilt sick” soil conditions of chickpea
crop were reported by Sindhu et al. (2002). Their coinoculation resulted in the enhanced
nodulation by Mesorhizobium sp. as well as increased shoot dry weight in comparison
with uninoculated controls. The disease suppression and/or the plant growth–promoting
effects could be the result of various multifarious interactions with the other members of
the rhizosphere community.
18.2 Plant–microbe interaction in rhizosphere

Most microorganisms are uncultivable in nature (Torsvik and Ovreas, 2002), and
it is important for us to unravel their interactions with plants. With the advent of
cultivation-independent methods and molecular methods, the untangling of the relation-
ships involved was made easier to study. It is of fundamental importance to understand
the various mechanisms and processes that regulate soil ecosystem functioning. A recent
Chapter eighteen: Antimicrobial compounds from rhizosphere bacteria 373
review (Philippot et al., 2013) entitled “Going back to the roots: The microbial ecology
of the rhizosphere” discusses the various microbial interactions and processes that are
occurring in the rhizosphere and their importance to sustainable agriculture, among oth-
ers. For sustainable agriculture, it is crucial to understand the importance of biofertiliz-
ers and biopesticides in enhancing nutrient acquisition and sustainable plant protection
(Huang et al., 2014).
Plants pump carbon below ground in the form of root exudates. The plant roots
select from a wide pool of soil microorganisms (Nallanchakravarthula et al., 2014); the
selected microorganisms play a role in plant protection and nutrient acquisition by releas-
ing various antimicrobial compounds or increasing nutrient uptake. The rhizosphere has
been described as both a playground and battlefield for soilborne pathogens and ben-
eficial microorganisms (Raaijmakers et al., 2008). Plant carbon is also known to attract
plant pathogens, thereby competition occurs in between the pathogens and other micro-
organisms, wherein the rhizosphere microbial community dynamics change. There are
two types of plant-originated organic compounds that are released into their surrounding
environment. One of which is exuded by the plants at the root surface, termed as root
exudates. A rough estimate by Uren (2000) on root exudates indicates about 10% of the
photosynthate is being allocated to the root. The second one originates from the decaying
plant cells as a result of cell death. As a result, the rhizosphere is considered as nutrient
rich (heterotrophic medium) when compared with bare soils, which remain nonconducive
or poor medium for plant growth. The quality and quantity of root exudates depend on
the plant species, cultivar, age, and the environmental conditions (Rovira, 1956; Haichar
et al., 2008). There is accumulating evidence that different plant species select different
microorganisms (Costa et al., 2006; Haichar et al., 2008).
18.3 Rhizosphere bacteria in plant disease management

Many rhizosphere bacteria are known to produce antagonistic compounds that play a role
in encouraging or inhibiting soilborne plant pathogens. The populations of antagonis-
tic microorganisms can be increased artificially by fertilizer application, organic amend-
ments, foliar spraying of chemicals, etc. To be a successful antagonist, rhizobacteria should
also compete for nutrients. The root exudate composition is known to influence rhizo-
sphere microbial communities (Bressen et al., 2009; Drigo et al., 2010). In a study by Wu
et al. (2008), susceptible cotton cultivar to Verticillium dahliae is known to produce more
amino acids and sugars in comparison with resistant cultivar. In another recent study
(Li et al., 2013) of peanut cultivars, the susceptible cultivar root exudate composition signif-
icantly differed in sugars, alanine, and total amino acids, whereas p-hydroxybenzoic acid,
benzoic acid, p-coumaric acid, and total phenolic acids were higher in midresistant culti-
var. It was also shown that the plant can exploit the microbial consortia from soil in order
to counteract the plant pathogen (Mendes et al., 2011). Rudrappa et al. (2008) demonstrated
the stimulation of growth of Bacillus subtilis in response to signals released by Arabidopsis
plants challenged by Pseudomonas syringae pv. tomato. In in vitro studies, Pseudomonas iso-
lates have been shown to inhibit Phytophthora capsici by the production of biosurfactant
(Ozyilmaz and Benlioglu, 2013). Soil amendments are also shown to increase the suppres-
siveness to plant pathogens and modulate bacterial members (Giotis et al., 2012; Cretoiu
et al., 2013). It has newly been reported that many pathogens are attacking the new crop
plants, implying the need to identify and formulate new compounds or biocontrol agents
or methods to counteract the pathogens (Jayakumar et al., 2009; Singh et al., 2011; Sharma
et al., 2013).
18.4 Antimicrobial compounds of rhizosphere bacteria

Common soil bacterial genera such as Pseudomonas, Burkholderia, Enterobacter, and Bacillus
are also known to be successful endophytes (Lodewyckx et al., 2002; Amaresan et al.,
2012a, b, 2014b). Many secondary metabolites are being produced by these genera such
as antibiotics, anticancer, volatile organic compounds, antifungal, antiviral, insecticidal,
and immunosuppressant compounds. In spite of various compounds isolated from these
endophytes and rhizosphere bacteria, they are still an untapped array of various second-
ary metabolites that remain to be exploited.
Antibiotics and its production are known to be a major mechanism by which rhizo-
bacteria play a key role in plant protection. Their production has also been reported from
various extreme sources. Using purified antibiotics, they were shown to suppress diseases
by mutant analysis and biochemical studies. They subdue or, in some cases, kill the plant
pathogenic fungi by inhibition of spore germination, fungistasis, and lysis of fungal myce-
lia and/or by exerting fungicidal effects. DeCoste et al. (2010) showed an increase in the
populations of Pseudomonas sp. LBUM300 associated with strawberry plants exposed to
V. dahliae. V. dahliae presence increased the expression of “hcnC” gene, but the presence of
Pseudomonas spp. did not alter the colonization by the pathogen. It was reported that anti-
biotic compounds such as 2,4-diacetylphloroglucinol (2,4-DAPG), oomycin A, phenazines,
pyrrolnitrin, pyrroles, and pyocyanin are produced by rhizobacteria, which are known to
inhibit the plant pathogen growth (Bender et al., 1999).
18.4.1 2,4-Diacetylphloroglucinol
2,4-DAPG is a natural phenolic compound found in certain strains of gram-negative
bacteria such as Pseudomonas fluorescens. It is active against organisms ranging from
viruses, bacteria, plants, to nematodes; its enhanced activity has always been found
against plant pathogens (Keel et al., 1992; Maurhofer et al., 1992; Mazzola et al., 1995;
Cronin et al., 1997; Delany et al., 2001; Dwivedi and Johri, 2003; Siddiqui and Shaukat,
2003a; Islam and Fukushi, 2010). 2,4-DAPG from Pseudomonas fluorescens CHA0 has been
shown to induce plant resistance against pathogens (Iavicoli et al., 2003; Siddiqui and
Shaukat, 2003b, 2004). It has also been shown that application of 2,4-DAPG-producing
pseudomonads increased crop soybeans yields (McSpadden Gardener et al., 2006).
Pseudomonas protegens is also known to produce 2,4-DAPG and showed similar effect
on plant pathogens such as P. fluorescens (Ramette et al., 2011). Its activity was observed
both in vitro and in vivo, when tested with both wild type and nonproducing mutants
(Vincent et al., 1991; Fenton et al., 1992; Keel et al., 1992). In a comparison study, the
2,4-DAPG-producing strains protected plants better than the nonproducing strains
(Cronin et al., 1997; Rezzonico et al., 2007). The polyketide 2,4-DAPG has shown to inhibit
the growth of plant pathogenic bacteria such as Erwinia carotovora (Cronin et al., 1997),
Pythium spp., and other pathogenic fungi such as Rhizoctonia solani, Thielaviopsis basicola,
and Gaeumannomyces graminis var. tritici (Howell and Stipanovic 1979; Keel et al., 1992;
Shanahan et al., 1992; Cronin et al., 1997), including nematodes (Cronin et al., 1997).
The best-known example of disease suppression by the 2,4-DAPG-producing strains is
take all of wheat caused by G. graminis var. tritici (Mendes et al., 2011; Raaijmakers and
Mazzola, 2012; Kwak and Weller, 2013).
The molecular mechanism of 2,4-DAPG action against a soilborne phytopathogenic
peronosporomycete Pythium ultimum var. sporangiferum has been described by de Souza
et al. (2003) wherein it alters the plasma membrane and vacuolizes, thereby disintegrating
the hyphal cell contents. The motility of zoospores and zoo sporogenesis in downy mil-
dew pathogen, Plasmopara viticola, and a damping-off pathogen, Aphanomyces cochlioides, is
known to be effected by its derivatives (Islam and von Tiedemann, 2011).
18.4.2 Phenazines
Phenazine-1-carboxylic acid is an antifungal metabolite (Haynes et al., 1956; Thomashow
and Weller, 1988; Hass and Defago, 2005) that is produced by Pseudomonas. It is a hetero-
cyclic nitrogen compound showing colored pigmentation and is produced exclusively by
bacteria belonging to genera Pseudomonas, Streptomyces, Nocardia, Sorangium, Brevibacterium,
Burkholderia (Turner and Messenger, 1986), and Bacillus (Kim, 2000). Other derivatives of
phenazines include pyocyanin (King et al., 1954), phenazine-1-carboxamide (Birkofer,
1947), idoinin (Gerber, 1969), aeruginosin A (Holliman, 1969), and aeruginosin B (Herbert
and Holliman, 1969).
The biocontrol capacity of P. fluorescens 2–79 (Thomashow and Weller, 1988),
Pseudomonas chlororaphis PCL1391, and Pseudomonas aeruginosa PNA1 (Tambong and Hofte,
2001) is known to be caused by production of phenazines. It was also suggested in control-
ling soilborne plant pathogen R. solani (Rosales et al., 1995; Huang et al., 2004). Bull et al.
(1991) showed that P. fluorescens 2–79 controls G. graminis var. tritici, which causes take all of
wheat by production of phenazine-1-carboxylic acid. They showed that the population of
phenazine-producing strains is inversely proportional to the number of lesions caused by
the pathogen and reported during primary infection of roots; phenazine-1-carboxylic acid
is a major factor in suppression of G. graminis var. tritici. These heterocyclic antibiotics are
reported to show wide-spectrum antimicrobial activity, particularly by Pseudomonas spp.
(Hu et al., 2005; Sunish Kumar et al., 2005; Ravindra Naik and Sakthivel, 2006). Attempts
are made in order to understand the scale and quantitative aspects of phenazine produc-
tion in natural settings (Mavrodi et al., 2012).
18.4.3 Pyrrolnitrin
Very few gram-negative bacteria such as Enterobacter agglomerans, Serratia spp., and
Pseudomonas spp. are known to produce this compound. It is an organohalogenic com-
pound derived from tryptophan and has an antifungal activity (Hammer et al., 1999; Haas
and Defago, 2005; Costa et al., 2009). Its production has been linked with certain bacteria
in controlling plant diseases, especially fungal pathogens (Haas and Defago, 2005; Costa
et al., 2009). Burkholderia cepacia strain 5.5B was identified with the production of pyrrolni-
trin, in suppression of stem rot of poinsettia caused by R. solani. This compound has also
shown to be an antagonistic toward Botrytis cinerea, R. solani, and Sclerotinia sclerotiorum
(Hammer and Evensen, 1993; Fernando et al., 2005).
18.4.4 Bacteriocins
Bacteriocins are another group of antibiotic compounds produced by bacteria. They are
produced by many gram-negative and gram-positive bacteria and are known to inhibit
other related strains of same species due to their high specificity. Application of such
bacteria for controlling soilborne and phyllosphere-inhabiting bacterial plant pathogens
seems to be promising. An avirulent strain of Agrobacterium is known to produce a peptide
bacteriocin trifolitoxin that enhances the biological control of Agrobacterium vitis crown
gall (Herlache and Triplett, 2002). In a field study, the bean nodulation of Rhizobium etli
CE3 increases with the expression of trifolitoxin genes from Rhizobium leguminosarum bv.
trifolii T24 (Robleto et al., 1998). Serratia plymithicum produces “serracin P,” a phage tail–like
bacteriocin, which was employed in controlling the fire blight caused by Erwinia amylovora
(Jabrane et al., 2002). Xanthomonas campestris pv. glycines shows antibacterial activity
against phytopathogenic Xanthomonas spp. by secretion of “glycinecin A” (Heu et al., 2001).
Pseudomonas syringae subsp. savastanoi, the causal agent of olive knot disease, is known to
be inhibited by bacteriocin produced from Pseudomonas syringae pv. ciccaronei.
18.5 Mechanism of action

Rhizobacteria impedes growth of various phytopathogenic microorganisms using diverse
mechanisms that include (1) antibiosis, (2) lytic enzyme productions, (3) degradation of
pathogen virulence, (4) production of siderophores, (5) reduction in ethylene production,
and (6) induction of systemic resistance.
18.5.1 Antibiosis
In general, rhizobacteria show antibiosis activity in inhibiting a wide variety of microor-
ganisms. PGPR are known to exhibit antibiosis to native microflora (Burr et al., 1978), but
it was not known whether it was a result of antagonism in soil resulted from antibiosis
or competition or both. In in vitro studies, it is unclear that the antibiosis effect by PGPR
is linked to production of inhibitory substances on root surfaces (Kloepper and Schroth,
1981). Antibiotic production by microorganisms was demonstrated in soil organic mat-
ter (Wright, 1956). In spite of their importance, determining their role in the rhizosphere
remains elusive. Rationally, the antibiotic production in the rhizosphere would equip the
producers to be better colonizers than their counterparts in competing against nutrients.
Frequently, antibiosis is used by many biocontrol agents such as fluorescent Pseudomonas
spp., Bacillus spp., Streptomyces spp., and Trichoderma spp. A wide array of chemicals have
been identified, and their roles in suppression of many plant pathogens have been docu-
mented (Fravel, 1988; Loper and Lindow, 1993; Weller and Thomashow, 1993, Raaijmakers
and Mazzola, 2012). Not only antibiotics but also bacteriocins, enzymes such as cell
wall– degrading enzymes, and volatile compounds with antifungal activity show anti-
biosis. Pseudomonas fluorescens CHAO is known to produce siderophores, phenazines,
2,4-diacetylphloroglucinol, and cyanide, and various combinations of these metabolites are
responsible for its antagonism against G. graminis var. tritici and Chalara elegans (Defago and
Haas, 1990).
18.5.2 Lytic enzyme production

Microorganisms are known to excrete hydrolytic enzymes that are lytic in nature. These
enzymes play an important role in mycoparasitism. Fungal cell walls are made up of chi-
tin and β-1,3-glucans (Lam and Gaffney, 1993). The action of chitinases or β-1,3-glucanase
(laminarinase) in the lysis of fungal cell walls, in combination as well as alone, is well
documented (Harman et al., 1993; Lam and Gaffney, 1993; Lorito et al., 1993, 1994a, b).
Pseudomonas stutzeri has been shown to produce chitinase and laminarinase, which
caused the lysis of Fusarium solani germ tube and its mycelia. Markedly, this effect is more
on mycelial growth inhibition rather than spore germination (Lim et al., 1991). The preva-
lence of diseases caused by R. solani, Sclerotium rolfsii, and Pythium ultimum was declined
due to the enzymatic action of β-1,3-glucanase produced by Pseudomonas cepacia. In a green-
house study, a chitinase (ChiA)-deficient Serratia marcescens mutant was shown to increase
the fungal pathogen germ tube elongation and Fusarium wilt of pea seedling (Lam and
Gaffney, 1993). In a separate study, a non-biocontrol agent Escherichia coli is transformed

with ChiA from S. marcescens; the resultant transgenic bacterium showed a reduced disease
incidence of southern blight of bean caused by S. rolfsii (Shapira et al., 1989).
18.5.3 Degradation of pathogen virulence

Degradation of pathogen virulence is another way of biological control. For example, albi-
cidin toxin produced by Xanthomonas albilineans is detoxified by Pantoea dispersa (Zhang
and Birch, 1996). Binding of proteins to toxins is another way of mediation of their detoxi-
fication effect. The toxins produced by Klebsiella oxytoca and Alcaligenes denitrificans are
known to be inhibited reversibly by unknown protein (Walker et al., 1988; Basnayake and
Birch, 1995). Enzymes also play a role in the detoxification. X. albilineans produces albici-
din, a polyketide–peptide compound that is known to inhibit the supercoiling activity of
DNA gyrase both in plants and bacteria; P. dispersa esterase is known to detoxify irrevers-
ibly this toxin (Zhang and Birch, 1997). Certain phytotoxins (fusaric acid) produced by
Fusarium species are known to be detoxicated by B. cepacia and Ralstonia solanacearum by
hydrolyzing fusaric acid (Toyoda et al., 1988). Pathogen toxins show broad-spectrum activ-
ity against other microorganisms, especially its competitors, or even detoxify the antibiot-
ics produced by their antagonists as a self-defense mechanism (Schouten et al., 2004).
18.5.4 Competition
Nutrient limitation occurs in poor soils. Most often, essential elements for microbial or plant
growth are not in free forms, and they bound to soil particles or organic matter or form chem-
ical complexes, which need to be mineralized for their availability. Of these nutrients, iron
is very important, and its bioavailability is limited by the solubility of Fe3+. Microorganisms
produce siderophores to chelate iron from minerals, especially under nutrient-limiting condi-
tions. The studies on siderophores started decades ago on the discovery of fungal ferrichrome
(Neilands, 1952). Siderophores transport not only iron but also other elements such as Al, Cd,
Cu, Ga, In, Pb, and Zn, as well as with radionuclides including U and Np (Kiss and Farkas,
1998; Neubauer et al., 2000a, b). Siderophores also play a role in antagonism against plant patho-
gens (Buyer and Leong, 1986; Solanki et al., 2014; Suman and Veena, 2014). Many soil bacteria
belonging to Erwinia, Pseudomonas, Nocardia, Streptomyces, Arthrobacter, and Chromobacterium
are known to produce siderophores (Meyer and Abdallah, 1980; Muller and Raymond, 1984;
Buyer and Leong, 1986; Berner et al., 1988; Berner and Winkelmann, 1990; Gunter et al., 1993;
Wei et al., 2007). The presence of these siderophores has been suggested to depend on the soil
physiochemical and biological properties (Bossier et al., 1988; Nelson et al., 1988). It was shown
that there was an interspecies utilization of the siderophores in fluorescent pseudomonad,
and it was suggested that a specific outer membrane receptor protein might play a role for this
interspecies siderophore utilization (Buyer and Leong 1986).
18.5.5 Induced resistance

Some of the biocontrol agents are known to induce certain physiological changes to the
plant, thereby increasing its tolerance/resistance toward a pathogen by induced systemic
resistance (ISR). This resistance can be localized or systemic in nature. The genes that
are responsible for ISR are also known to induce systemic acquired resistance (SAR),
but not always. When a localized infection occurs or an attenuated pathogen attacks the
plant, SAR will be expressed as a resistance to wide pathogens (Uknes et al., 1992). Jones
and Dangl (2006) describe how a plant immune system responds in general to microbes
including nonpathogens. There are two pathways by which host resistance is mediated.
First, SAR is mediated by salicylic acid that initiates the expression of pathogenesis-related
proteins including a variety of enzymes. Second pathway involves the ISR, which is medi-
ated by jasmonic acid and/or ethylene. BCA are known to induce disease resistance in
many ways such as Bacillus mycoides; a biocontrol agent is able to stimulate sugar beet to
produce peroxidase, chitinase, and β-1,3-glucanase (Bargabus et al., 2003). Bacillus subtilis
strains GB03 and IN97 and Pseudomonas putida shown to produce 2,3-butanediol and lipo-
polysaccharide in Arabidopsis (Ryu et al., 2004; Meziane et al., 2005), siderophore produc-
tion by S. marcescens in cucumber (Press et al., 2001). Root colonizers such as Pseudomonas
spp. and Trichoderma spp. are found to be potential elicitors of plant host defenses. It was
also reported that PGPR strains upon inoculation elicit SAR and ISR by salicylic acid, sid-
erophore, lipopolysaccharides, and 2,3-butanediol and other volatile substances (van Loon
et al., 1998; Ryu et al., 2004; Ongena et al., 2004). Quorum sensing is also known to play a
role in the ISR. S. marcescens 90–166 has been reported to elicit ISR in tobacco plants in a
pathogen-dependent manner (Ryu et al., 2013).
18.6 Formulations and field application

The biocontrol agents are used for the control of soilborne, seed-borne, and airborne fun-
gal, bacterial, and viral plant pathogens. Apart from controlling the plant pathogens, they
are also reported to enhance crop growth. There are a number of carrier substrates that
are available to formulate biocontrol organisms for field applications. Some of the carrier
molecules include diatomaceous earth granules, wheat bran, wheat bran–saw dust mix-
ture, wheat bran–peat mixture, vermiculite–wheat bran acid formulation, alginate pellets,
talc, granules, wettable powder, pellets, sticks, and powder.
The preparatory method of talc-based formulation of Pseudomonas is given in the suc-
ceeding text; proteose peptone, 20 g; dipotassium hydrogen phosphate, 2.5 g; glycerol,
15 mL; magnesium sulphate, 6 g; and distilled water, 1000 mL.
King’s B broth medium is prepared in conical flasks and sterilized in autoclave for
20 min. A loop of antagonistic bacteria from 24 h old culture is inoculated into the broth
medium and incubated in a rotary shaker at 150 rpm for 2 days at room temperature. The
grown-up culture will be used directly as inoculum or used as mother culture for mass
production in the fermenter.
For mass production by fermentation, method is being employed, using King’s B broth
medium. The media would be prepared and sterilized; after sterilization, the mother cul-
ture is added to the fermenter at 100 mL/10 L of medium and maintained in optimum
condition for 2–3 days. One kilogram of talc powder is taken in a metal tray, and the pH
was adjusted to neutral by adding CaCO3 at 150 g/kg. The bacterial growth from the conical
flask or fermenter is mixed with talc powder at 1:2.5 ratio (i.e., 400 mL culture in 1 kg of talc
powder). The mixture is air dried and mixed with 5 g of carboxymethyl cellulose per 1 kg
of talc powder. It is then packed in polythene bag, sealed, and can be used within 4 months.
Application to crop
1. Seed treatment
• 4 g/kg of seed
• 600 g in 65 L of water—soak 50 kg seed for 12 h
2. Seedling dip—1%
3. Soil application—2.5 kg/ha
4. Spray—0.1%
18.7 Future perspectives

Many rhizobacteria are known to exert their beneficial effects under laboratory condi-
tions; but when tested under field or greenhouse conditions, they fail to show their
beneficial effects. There could be many reasons for their failure. To be a better BCA or
PGPR, they should show successful root colonization, be able to produce secondary
metabolites, and be able to withstand both biotic and abiotic stresses. Understanding
their mechanisms that are pivotal for their successful expression would lead us to iso-
late improved bacteria. Commercially available biocontrol agents are not genetically
modified, and the employment of wild-type or isolated strains as a biocontrol agent
is likely to continue. Engaging rational screening methods that stimulate field condi-
tions are expected to generate new isolates. Employing the use of multiple organisms
for biocontrol is being increasingly used for effective plant disease management and
growth promotion. This could be attributed to the various arrays of secondary metabo-
lites produced by the different microorganisms present in the mixed culture. It might
be possible to formulate a microbial consortium that has improved biocontrol prop-
erties, especially under natural conditions such as siderophore producers, phloroglu-
cinols, and phenazines. But much is needed to be done on the impact of biocontrol
agents and PGPRs and their associations including their limitations. The advent of the
advanced molecular methods and high-throughput sequencing may reveal the intrica-
cies involved in their associations and mechanisms, thereby providing an empirical
basis for sustainable agriculture.
References
Amaresan N., Jayakumar V., Kumar K., and Thajuddin N. 2012a. Isolation and characterization of
plant growth promoting endophytic bacteria and their effect on tomato (Lycopersicon esculen-
tum) and chilli (Capsicum annuum) seedling growth. Ann Microbiol. 62:805–810.
Amaresan N., Jayakumar V., Kumar K., and Thajuddin N. 2012b. Endophytic bacteria from tomato
and chilli, their diversity and antagonistic potential against Ralstonia solanacearum. Arch
Phytopathol Pl Protect. 45:344–355.
Amaresan N., Kumar K., Sureshbabu K., and Madhuri K. 2014a. Plant growth-promoting poten-
tial of bacteria isolated from active volcano sites of Barren Island, India. Lett Appl Microbiol.
58:130–137.
Amaresan N., Jayakumar V., and Thajuddin N. 2014b. Isolation and characterisation of endophytic
bacteria associated with chilli (Capsicum annuum) grown in coastal agricultural ecosystem. Ind
J Biotechnol. 13:247–255.
Bargabus, R. L., Zidack, N. K., Sherwood, J. E., and Jacobsen, B. J. 2003. Oxidative burst elicited by
Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensi-
tive cell death in sugar beet. Mol Plant Microbe Interact. 16:1145–1153.
Basak, B. B., and Biswas, D. R. 2010. Co-inoculation of potassium solubilizing and nitrogen fixing
bacteria on solubilization of waste mica and their effect on growth promotion and nutrient
acquisition by a forage crop. Biol Fertil Soils. 46:641–648.
Basnayake, W. V. S., and Birch, R. G. 1995. A gene from Alcaligenes denitrificans that confers albicidin
resistance by reversible antibiotic binding. Microbiology. 141:3551–560.
Bender, C. L., Rangaswamy, V., and Loper, J. E. 1999. Polyketide production by plant-associated
pseudomonads. Ann Rev Phytopathol. 37:175–196.
Berg, G. 2009. Plant–microbe interactions promoting plant growth and health: Perspectives for con-
trolled use of microorganisms in agriculture. Appl Microbiol Biotechnol. 84:11–18.
Berner, I., and Winkelmann, G. 1990. Ferrioxamine transport mutants and the identification of the
ferrioxamine receptor protein FoxA in Erwinia herbicola (Enterobacter agglomerans). Biol Metals.
2:197–202.
Berner, I., Konetschny Rapp, S., Jung, G., and Winkelmann, G. 1988. Characterization of ferriox-
amine E 449 as the principal siderophore of Erwinia herbicola (Enterobacter agglomerans). Biol
Metals. 1:51–56.
Birkofer, L. 1947. Chlororaphin, ein weiteres farbiges stoffwechselproduckt des Bacillus pyocyaneus.
Chem Ber. 80:212–214.
Bossier, P., Hofte, M., and Verstraete, W. 1988. Ecological significance of siderophores in soil. Adv
Microb Ecol. 10:385–414.
Bull, C. T., Weller, D. M., and Thomashow, L. S. 1991. Relationship between root colonization and
suppression of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens strain 2–79.
Phytopathology. 81:954–959.
Burr, T. J., Schroth, M. N., and Suslow, T. 1978. Increased potato yields by treatment of seed pieces
with specific strains of Pseudomonas fluorescens and P. putida. Phytopathology. 68:1377–1383.
Buyer, S. J. and Leong, J. 1986. Iron transport-mediated antagonism between plant growth-promoting
and plant deleterious Pseudomonas strains. J Biol Chem. 261:791–794.
Chin-A-Woeng, T. F. C., Bloemberg, G. V., van der Bij, A. J., et al., 1998. Biocontrol by phenazine-
1-carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by
Fusarium oxysporum f. sp. radicis-lycopersici. Mol Plant-Microbe Interact. 11:1069–1077.
Costa, R., Götz, M., Mrotzek, N., Lottmann, J., Berg, G., and Smalla, K. 2006. Effects of site and plant
species on rhizosphere community structure as revealed by molecular analysis of microbial
guilds. FEMS Microbiol Ecol. 56:236–249.
Costa, R., van Aarle, I. M., Mendes, R., and van Elsas, J. D. 2009. Genomics of pyrrolnitrin biosyn-
thetic loci: Evidence for conservation and whole-operon mobility within gram-negative bacte-
ria. Environ Microbiol. 11:159–175.
Cretoiu, M.S., Korthals, G.W., Visser, J.H.M., and Elsas, J.D. van. 2013. Chitin amendment increases
soil suppressiveness toward plant pathogens and modulates the actinobacterial and oxa-
lobacteraceal communities in an experimental agricultural field. Appl Environ Microbiol.
79:5291–5301.
Cronin, D., Moenne-Loccoz, Y., Fenton, A., et al., 1997. Role of 2,4-diacetylphloroglucinol in the
interactions of the biocontrol Pseudomonas strain F113 with the potato cyst nematode Globodera
rostochiensis. Appl Environ Microbiol. 63:1357–1361.
DeCoste, N.J., Gadkar, V.J., and Filion, M. 2010. Verticillium dahliae alters Pseudomonas spp. popula-
tions and HCN gene expression in the rhizosphere of strawberry. Can J Microbiol. 56:906–915.
Defago, G. and Haas, D. 1990. Pseudomonas as antagonists of soil borne plant pathogens: Mode of
action and genetic analysis. In: Soil Biochemistry, ed. J. M. Bollag and G. Stotzky, 249–291. New
York: Marcel Dekker.
Delany, I. R., Walsh, U. F., Ross, I. et al., 2001. Enhancing the biocontrol efficacy of Pseudomonas
fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol. Plant
Soil. 232:195–205.
de Souza, J. T., Arnould, C., Deulvot, C., et al., 2003. Effect of 2,4-diacetylphloroglucinol on Pythium:
Cellular responses and variation in sensitivity among propagules and species. Phytopathol.
93:966–975.
Dowling, D. N. and O’Gara, F. 1994. Metabolites of Pseudomonas involved in the biocontrol of plant
disease. Trends in Biotechnol. 12:133–141.
Drigo, B., Pijl, A. S., Duyts, H., Kielak, et al., 2010. Shifting carbon flow from roots into associ-
ated microbial communities in response to elevated atmospheric CO2. Proc Natl Acad Sci.
107:10938–10942.
Dwivedi, D. and Johri, B. N. 2003. Antifungals from fluorescent pseudomonads: Biosysnthesis and
regulation. Curr Sci. 85:1693–1703.
Fenton, A. M., Stephens, P. M., Crowley, J., et al., 1992. Exploitation of gene(s) involved in
2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas
strain. Appl Environ Microbiol. 58:3873–3878.
Fernando, W. D. G., Nakkeeran, S., and Zhang, Y. 2005. Biosynthesis of antibiotics by PGPR and its
relation in biocontrol of plant diseases. In PGPR: Biocontrol and Biofertilizer, ed. Z. A. Siddiqui,
67–109. the Netherlands: Springer Science.
Fravel, D. 1988. Role of antibiosis in biocontrol of plant disease. Annu Rev Phytopathol. 26:75–91.
Gerber, N. N. 1969. New microbial phenazines. J Heterocyclic Chem. 6:297–300.

Giotis, C., Theodoropoulou, A., Cooper, J., Hodgson, R., Shotton, P., Shiel, R., et al., 2012. Effect of
variety choice, resistant rootstocks and chitin soil amendments on soil-borne diseases in soil-
based, protected tomato production systems. Eur J Plant Pathol. 134:605–617.
Glick, B. R. 2012. Plant growth-promoting bacteria: Mechanisms and applications. Scientifica.
2012:1–15.
Glick, B. R. and Bashan, Y. 1997. Genetic manipulation of plant growth-promoting bacteria to
enhance biocontrol of phytopathogens. Biotechnol Adv. 15:353–378.
Goel, A. K., Sindhu, S. S., and Dadarwal, K. R. 2002. Stimulation of nodulation and plant growth of
chickpea (Cicer arietinum L.) by Pseudomonas spp. antagonisctic to fungal pathogens. Biol Fertil
Soils. 36:391–396.
Gunter, K., Toupet, C., and Schupp, T. 1993. Characterization of an iron-regulated promoter involved
in desferrioxamine B synthesis in Streptomyces pilosus: Repressor-binding site and homology
to the diphtheria toxin gene promoter. J Bacteriol. 175:3295–3302.
Haas, D. and Keel, C. 2003. Regulation of antibiotic production in root-colonizing Pseudomonas spp.
and relevance for biological control of plant disease. Annu Rev Phytopathol. 41:117–153.
Haichar, F. el. Z., Marol, C., Berge, O., et al., 2008. Plant host habitat and root exudates shape soil
bacterial community structure. ISME J 2:1221–1230.
Hammer, P. E. and Evensen, K. B. 1993. Post harvest control of Botrytis cinerea on cut flowers with
pyrrolnitrin. Plant Dis. 77:283–286.
Hammer, P. E., Burd, W., Hill, D. S., Ligon, J. M., and van Pee, K. 1999. Conservation of the pyrrolni-
trin biosynthetic gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol Lett.
180:39–44.
Hao, D., Gao, P., Liu, P., Zhao, J. et al. 2011. AC3–33, a novel secretory protein, inhibits Elk1 transcrip-
tional activity via ERK pathway. Mol Biol Rep. 38:1375–1382.
Harman, G. E., Hayes, C. K., Lorito, M. et al., 1993. Chitinolytic enzymes of Trichoderma harzianum
purification of chitobiosidase and endochitinase. Phytopathology. 83:313–318.
Hass, D., and Defago, G. 2005. Biological control of soil borne pathogens by fluorescent pseudomo-
nads. Nat Rev Microbiol. 3:307–319.
Haynes, W. C., Stodola, F. H., Locke, J. M. et al., 1956. Pseudomonas aureofaciens Kluyver and Phenazine
α-carboxylic acid, its characteristic pigment. J Bacteriol. 72:412–417.
Herbert, R. B. and Holliman, F. G. 1969. Pigments of Pseudomonas aeruginosa. J Biol Chem. 159:725–750.
Herlache, T. C. and Triplett, E. W. 2002. Expression of a crown gall biological control phenotype in an
avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance
genes. BMC Biotechnol. 2:2.
Heu, S., Oh, J., Kang, Y., Ryu, S., Cho, S. K., Cho, Y., and Cho, M. 2001. gly gene cloning and expres-
sion and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv.
glycines 8ra. Appl Environ Microbiol. 67:4105–4110.
Hiltner, L. 1904. Uber neuere erfahrungen und probleme auf dem gebiete der bodenbakteriologie
unter besonderer berucksichtigung der grundungung und brache. Arb Dtsch Landwirtsch Ges.
98:59–78.
Holliman, F. G. 1969. Pigments of Pseudomonas sp. part I. Structure and synthesis of aeruginosin A.
J Chem Soc C. 18:2514–2516.
Howell, C. R., and Stipanovic, R. D. 1979. Control of Rhizoctonia solani on cotton seedlings with
Pseudomonas fluorescens and with an antibiotic produced by the bacterium. Phytopathol.
69:480–482.
Hu, H. B., Xu, Y. Q., Chen, F., Zhang, X. H., and Hur, B. K. 2005. Isolation and characterization
of a new fluorescent Pseudomonas strain that produces both phenazine-1-carboxylic acid and
pyoluteorin. J Microbiol Biotechnol.15:86–90.
Huang, Z. Y., Bonsall, R. F., Mavrodi, D. V., Weller, D. M., and Thomashow, L. S. 2004. Transformation
of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves
biocontrol of Rhizoctonia root rot and in situ antibiotic production. FEMS Microbiol Ecol.
49:243–251.
Huang, X. F., Chaparro, J. M., Reardon, K. F., Zhang, R., Shen, Q., and Vivanco, J. M. 2014. Rhizosphere
interactions: Root exudates, microbes, and microbial communities. Botany. 92:267–275.
Iavicoli, A., Boutet, E., Buchal, A., and Métraux, J. P. 2003. Induced systemic resistance in Arabidopsis
thaliana in response to root inoculation with Pseudomonas fluorescens CHA0. Mol Plant-Microbe
Interact. 16: 851–858.
Islam, M. T. and Fukushi, Y. 2010. Growth inhibition and excessive branching in Aphanomyces cochli-
oides induced by 2,4-diacetylphloroglucinol is linked to disruption of filamentous actin cyto-
skeleton in the hyphae. World J Microbiol Biotechnol. 26:1163–1170.
Islam, M. T. and von Tiedemann, A. 2011. 2,4-Diacetylphloroglucinol suppresses zoosporogen-
esis and impairs motility of Peronosporomycete zoospores. World J Microbiol Biotechnol.
27:2071–2079.
Jabrane, A., Sabri, A., Compère, P., et al., 2002. Characterization of serracin P, a phage-tail-like
bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen. Appl Environ
Microbiol. 68:5704–5710.
Jayakumar V., Usha Rani G.K., Amaresan N., and Rajalakshmi S. 2009. First report of anthracnose
disease of black pepper (Piper nigrum) caused by an unknown species of Colletotrichum. Plant
Disease. 93:199–199.
Jones, J. D. and Dangl, J. L. 2006. The plant immune system. Nature. 444:323–329.
Keel, C., Schnider, U., Maurhofer, M., et al., 1992. Suppression of root diseases by Pseudomonas fluore-
scens CHA0: Importance of the bacterial secondary metabolite, 2,4-diacetylphloroglucinol. Mol
Plant Microbe Interact. 5:4–13.
Kim, K. 2000. Phenazine-1-carboxylic acid resistance in phenazine-1-carboxylic producing Bacillus
sp B-6. J Biochem Mol Biol. 33:332–336.
King, E. O., Ward, M. K., and Raney, D. E. 1954. Two simple media for the demonstration of pyocya-
nin and fluorescein. J Lab Clin Med. 44:301–307.
Kiss, T. and Farkas, E. 1998. Metal-binding ability of desferrioxamine B. J Inclusion Phenomena Mol
Recog Chem. 32:385–403.
Kloepper, J. W. and Schroth, M. N. 1981. Relationship of in vitro antibiosis of plant growth-promoting
rhizobacteria to plant growth and displacement of root microflora. Phytopathology. 71:1020–1024.
Kumar, K., Amaresan, N., Bhagat, S., Madhuri, K., and Srivastava, R. C. 2011. Isolation and charac-
terization of rhizobacteria associated with coastal agricultural ecosystem of rhizosphere soils
of cultivated vegetable crops. World J Microbiol Biotechnol. 27:1625–1632.
Kumar, K., Amaresan, N., Bhagat, S., Madhuri, K., and Srivastava, R. C. 2012. Isolation and charac-
terization of Trichoderma spp. for antagonistic activity against root rot and foliar pathogens. Ind
J Microbiol. 52:137–144.
Kwak, Y. S. and Weller, D. M. 2013. Take-all of wheat and natural disease suppression: A review.
Plant Pathol. J. 29: 125–135.
Lam, S. T., and Gaffney, T. D. 1993. Biological activities of bacteria used in plant pathogen control.
In: Biotechnology in Plant Disease Control, ed. I. Chet, 291–320. New York: John Wiley & Sons.
Li, X., Zhang, T., Wang, X., Hua, K., Zhao, L., and Han, Z. 2013. The composition of root exudates
from two different resistant peanut cultivars and their effects on the growth of soil-borne
pathogen. Int J Biol Sci. 9: 164–173.
Lim, H. S., Kim, Y. S., and Kim, S. D. 1991. Pseudomonas stutzeri YPL-1 genetic transformation
and antifungal mechanism against Fusarium solani, an agent of plant root rot. Appl Environ
Microbiol. 57:510–516.
Lodewyckx, C., Vangronsveld, J., Porteous, F., et al., 2002. Endophytic bacteria and their potential
applications. Crit Rev Plant Sci. 21:583–606.
Loper, J. E. and Lindow, S. E. 1993. Roles of competition and antibiosis in suppression of plant dis-
eases by bacterial control agents. In: Pest Management: Biologically Based Technologies, ed. R. D.
Lumsden, and J. L. Vaughn, 144–155. Washington, DC: American Chemical Society.
Lorito, M., Harman, G. E., Hayes, C. K., et al., 1993. Chitinolytic enzymes produced by Trichoderma
harzianum: Antifungal activity of purified endochitinase and chitobiosidase. Phytopathology.
83:302–307.
Lorito, M., Hayes, C. K., Di Pietro, A., Woo, S. L., and Harman, G. E. 1994a. Purification, characteriza-
tion, and synergistic activity of a glucan 1,3-glucosidase and an N-acetyl-beta-glucosaminidase
from Trichoderma harzianum. Phytopathology. 84:398–405.
Lorito, M., Hayes, C. K., Zoina, A., Scala, F., Del-Sorbo, G., Woo, S. L., and Harman, G. E. 1994b.
Potential of genes and gene products from Trichoderma spp. and Gliocladium spp. for the devel-
opment of biological pesticides. Mol Biotechnol. 2:209–217.
Lugtenberg, B. and Kamilova, F. 2009. Plant growth-promoting rhizobacteria. Annu Rev Microbiol.
63:541–556.
Maurhofer, M., Keel, C., Schnider, U., Viosard, C., Haas, D., and Defago, G. 1992. Influence of
enhanced antibiotic production in Pseudomonas fluorescens strain CHA0 on its disease suppres-
sive capacity. Phytopathology. 82:190–195.
Mavrodi, D.V., Mavrodi, O.V., Parejko, J.A., Bonsall, R.F., Kwak, Y.-S., Paulitz, T.C., et al., 2012.
Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland cere-
als. Appl Environ Microbiol. 78:804–812.
Mazzola, M., Fujimoto, D. K., Thomashow, L. S., and Cook, R. J. 1995. Variation in sensitivity of
Gaeumannomyces graminis to antibiotics produced by fluorescent pseudomonads spp. and
effect on biological control of take-all disease. Appl Environ Microbiol. 61:2554–2559.
McSpadden Gardener, B., Benitez, M. S., Camp, A., and Zumpetta, C. 2006. Evaluation of a seed treat-
ment containing a phlD+ strain of Pseudomonas fluorescens on organic soybeans, 2005. Biological
and cultural tests for control of plant diseases report. 21: FC046.
Mendes, R., Kruijt, M., de Bruijn, I., Dekkers, E., van der Voort, M., Schneider, J.H.M., et al.,
(2011) Deciphering the rhizosphere microbiome for disease-suppressive bacteria. Science.
332:1097–1100.
Meyer, J. M. and Abdallah, M. A. 1980. The siderochromes of non fluorescent pseudomonads:
Production of nocardamine by Pseudomonas stutzeri. J Gen Microbiol. 118:125–129.
Meziane, H., van der Sluis, I., van Loon, L. C., Höfte, M., and Bakker, P. A. 2005. Determinants
of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants. Mol Plant
Pathol. 6:177–185.
Morgan, J. A. W., Bending, G., and White, P. 2005. Biological costs and benefits to plant-microbe
interactions in the rhizosphere. J Exp Bot. 56:1729–1739.
Muller, G. and Raymond, K. N. 1984. Specificity and mechanism of ferrioxamine-mediated iron
transport in Streptomyces pilosus. J Bacteriol. 160:304–312.
Nallanchakravarthula, S., Mahmood, S., Alström, S., and Finlay, R. D. 2014. Influence of soil type,
cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal micro-
biome of Strawberry. PLoS ONE. 9:e111455.
Neilands, J. B. 1952. A crystalline organo-iron pigment from a rust fungus (Ustilago sphaerogena).
J Am Chem Soc. 74:4846.
Nelson, M., Cooper, C. R., Crowley, D. E., Reid, C. P. P., and Szaniszlo, P. J. 1988. An Escherichia coli
bioassay of individual siderophores in soil. J Plant Nutr. 11:915–924.
Neubauer, U., Furrer, G., Kayser, A., and Schulin, R. 2000a. Siderophores, NTA, and citrate: Potential
soil amendments to enhance heavy metal mobility in phytoremediation. Int J Phytoremediation.
2:353–368.
Neubauer, U., Nowak, B., Furrer, G., and Schulin, R. 2000b. Heavy metal sorption on clay minerals
affected by the siderophore desferrixamine B. Environ Sci Technol. 34:2749–2755.
Ongena, M., Duby, F., Rossignol F., et al., 2004. Stimulation of the lipoxygenase pathway is associated
with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain. Mol Plant
Microbe Interact. 17:1009–1018.
Ozyilmaz, U. and Benlioglu, K. 2013. Enhanced biological control of Phytophthora blight of pepper
by biosurfactant-producing Pseudomonas. Plant Pathol J. 29(4):418–426.
Panhwar, Q. A., Othman, R., Rahman, Z. A., Meon, S., and Ismail, M. R. 2012. Isolation and charac-
terization of phosphate-solubilizing bacteria from aerobic rice. Afr J Biotechnol. 11:2711–2719.
Philippot, L., Raaijmakers, J. M., Lemanceau, P., and van der Putten, W. H. 2013. Going back to the
roots: The microbial ecology of the rhizosphere. Nat Rev Microbiol. 11:789–799.
Press, C. M., Loper, J. E., and Kloepper, J. W. 2001. Role of iron in rhizobacteria-mediated induced
systemic resistance of cucumber. Phytopathology. 91:593–598.
Raaijmakers, J. M., and Mazzola, M. 2012. Diversity and natural functions of antibiotics produced by
beneficial and plant pathogenic bacteria. Annu Rev Phytopathol. 50:403–424.
Raaijmakers, J. M., Paulitz, T. C., Steinberg, C., et al., 2008. The rhizosphere: A playground and battle-
field for soilborne pathogens and beneficial microorganisms. Plant Soil. 321:341–361.
Ramette, A., Frapolli, M., Saux, M. F. L., et al., 2011. Pseudomonas protegens sp. nov., widespread plant-
protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyo-
luteorin. Syst Appl Microbiol. 34:180–188.
Ravindra Naik, P. and Sakthivel, N. 2006. Functional characterization of a novel hydrocarbonoclas-
tic Pseudomonas sp. strain PUP6 with plant-growth-promoting traits and antifungal potential.
Res Microbiol. 157:538–546.
Rezzonico, F., Zala, M., Keel, C., et al., 2007. Is the ability of biocontrol fluorescent pseudomonads to
produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher
plant protection? New Phytol. 173:861–872.
Robleto, E. A., Kmiecik, K., Oplinger, E. S., Nienhuis, J., and Triplett, E. W. 1998. Trifolitoxin produc-
tion increases nodulation competitiveness of Rhizobium etli CE3 under agricultural conditions.
Appl Environ Microbiol. 64:2630–2633.
Rosales, A. M., Thomashow, L., Cook, R. J., and Mew, T. W. 1995. Isolation and identification of antifungal
metabolites produced by rice-associated antagonistic Pseudomonas spp. Phytopathology 85:1028–1032.
Rovira, A. D. 1956. Plant root excretions in relation to the rhizosphere effect. Plant Soil. 7:178–194.
Rudrappa, T., Czymmek, K. J., Paré, P. W., and Bais, H. P. 2008. Root-secreted malic acid recruits
beneficial soil bacteria. Plant Physiol. 148:1547–1556.
Ryu, C. M, Farag, M. A., Hu, C. H., et al., 2004. Bacterial volatiles induce systemic resistance in
Arabidopsis. Plant Physiol. 134:1017–1026.
Ryu, C.-M., Choi, H. K., Lee, C.-H., Murphy, J. F., Lee, J.-K., and Kloepper, J. W. 2013. Modulation of
quorum sensing in acylhomoserine lactone-producing or degrading tobacco plants leads to
alteration of induced systemic resistance elicited by the Rhizobacterium Serratia marcescens
90–166. Plant Pathol. J. 29: 182–192.
Schouten, A., van Den Berg, G., Edel-Hermann, V., et al., 2004. Defense responses of Fusarium oxy-
sporum to 2,4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas
fluorescens. Mol Plant-Microbe Interact. 17:1201–1211.
Shanahan, P., O’sullivan, D. J., Simpson, P., Glennon, J. D., and O’Gara, F. 1992. Isolation of
2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiologi-
cal parameters influencing its production. Appl Environ Microbiol. 58:353–358.
Shapira, R., Ordentlich, A., Chet, I., and Oppenheim, A. B. 1989. Control of plant diseases by chitin-
ase expressed from cloned DNA in Escherichia coli. Phytopathology. 79:1246–1249.
Sharma P., Meena P. D., and Singh Y. P. 2013. New record of twig blight on Catharanthus roseus in
India. Afr J Microbiol Res. 7(38):4680–4682.
Siddiqui, I. A. and Shaukat, S. S. 2003a. Plant species, host age and host genotype effects on
Meloidogyne incognita biocontrol by Pseudomonas fluorescens strain CHA0 and its genetically-
modified derivatives. J Phytopathol. 151:231–238.
Siddiqui, I. A. and Shaukat, S. S. 2003b. Suppression of root-knot disease by Pseudomonas fluorescens
CHA0 in tomato: Importance of bacterial secondary metabolite, 2,4-diacetylphloroglucinol.
Soil Biol Biochem. 35:1615–1623.
Siddiqui, I. A. and Shaukat, S. S. 2004. Suppression of Meloidogyne incognita by Pseudomonas fluroscens
strain CHA0 and its genetically modified derivatives: II. The influence of sodium chloride.
Nematol Mediterranea. 32:127–130.
Sindhu, S. S., Suneja, S., Goel, A. K., Parmar, N., and Dadarwal, K. R. 2002. Plant growth promoting
effects of Pseudomonas sp. on coinoculation with Mesorhizobium sp. cicer strain under sterile
and wilt sick soil conditions. Appl Soil Ecol. 19:57–64.
Singh N., Sharma P., and Verma O.P. 2011. First report of Choanophora sp. causing twig blight of
Boerhavia diffusa in India. New Dis Rep. 23:29. doi:10.5197/j.2044–0588.2011.023.029
Solanki, M. K., Singh, R. K., Srivastava, S., et al., 2014. Isolation and characterization of siderophore
producing antagonistic rhizobacteria against Rhizoctonia solani. J Basic Microbiol 54:585–597.
Suman, K. and Veena, K. 2014. Effect of antagonistic rhizobacteria coinoculated with Mesorhizobium
ciceris on control of fusarium wilt in chickpea (Cicer arietinum L.). Afr J Microbiol Re. 8:1255–1265.
Sunish Kumar, R., Ayyadurai, N., Pandiaraja, P., et al., 2005. Characterization of antifungal metabo-
lite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad spectrum
antifungal activity and biofertilizing traits. J Appl Microbiol. 98:145–154.
Tambong, J. T. and Hofte, M. 2001. Phenazines are involved inbiocontrol of Pythium myriotylum on
cocoyam by Pseudomonas aeruginosa PNA1. Eur J Plant Pathol. 107:511–521.
Thomashow, L. S. and Weller, D. M. 1988. Role of a phenazine antibiotic from Pseudomonas fluorescens
in biological control of Gaeumannomyces graminis var. tritici. J Bacteriol. 170:3499–3508.
Torsvik, V. and Ovreas, L. 2002. Microbial diversity and function in soil: From genes to ecosystems.
Curr Opin Microbiol. 5:240–245.
Toyoda, H., Hashimoto, H., Utsumi, R., Kobayashi, H., and Ouchi, S. 1988. Detoxification of fusaric
acid by a fusaric acid-resistant mutant of Pseudomonas solanacearum and its application to bio-
logical control of fusarium wilt of tomato. Phytopathology. 78:1307–1311.
Turner, J. M. and Messenger, A. J. 1986. Occurrence, biochemistry and physiology of phenazine pig-
ment production. Adves Microb Physiol. 27:211–275.
Uknes, S., Mauch-Mani, B., Moyer, M., et al., 1992. Acquired resistance in Arabidopsis. Plant Cell.
4:645–656.
Uren, N. C. 2000. Types, amounts, and possible functions of compounds released into the rhizo-
sphere by soil-grown plants. In: The Rhizosphere: Biochemistry and Organic Substances at the Soil-
Plant Interface, ed. R. Pinton, Z. Varanini, and P. Nannipiero, 19–40. New York: Marcel Dekker.
Van Loon, L. C., Bakker, P. A., and Pieterse, C. M. 1998. Systemic resistance induced by rhizosphere
bacteria. Annu Rev Phytopathol. 36:453–483.
Vincent, M. N., Harrison, L. A., Brackin, J. M., et al., 1991. Genetic analysis of the antifungal activity
of a soilborne Pseudomonas aureofaciens strain. Appl Environ Microbiol. 57:2928–2934.
Walker, M. J., Birch, R. G., and Pemberton, J. M. 1988. Cloning and characterization of an albicidin
resistance gene from Klebsiella oxytoca. Mol Microbiol. 2:443–454.
Wei, X., Sayavedra-Soto, L. A., and Arp, D. J. 2007. Characterization of the ferrioxamine uptake sys-
tem of Nitrosomonas europaea. Microbiology. 153:3963–3972.
Weller, D. M. 2007. Pseudomonas biocontrol agents of soil borne pathogens: Looking back over
30 years. Phytopathology. 97:250–256.
Weller, D. M. and Thomashow, L. S. 1993. Use of rhizobacteria for biocontrol. Curr Opin Biotechnol.
4:306–311.
Wright, J. M. 1956. The production of antibiotics in soil. IV. Production of antibiotics in coats of seeds
grown in soil. Ann Appl Biol. 44:561–566.
Wu, Y., Fang, W., Zhu, S., Jin, K., and Ji, D. 2008. The effects of cotton root exudates on the growth
and development of Verticillium dahliae. Front Agric China. 2:435–440.
Zhang, L. and Birch, R. G. 1996. Biocontrol of sugar cane leaf scald disease by an isolate of Pantoea
dispersa which detoxifies albicidin phytotoxins. Lett Appl Microbiol. 22:132–136.
Zhang, L. and Birch, R. G. 1997. The gene for albicidin detoxification from Pantoea dispersa encodes
an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane. Proc Natl
Acad Sci USA. 94:9984–9989.
section four
Synthetic chemical compounds as

broad spectrum antimicrobials
chapter nineteen
Microbe-mediated synthesis
of silver nanoparticles
A new drug of choice against
pathogenic microorganisms
Contents
19.1 Introduction......................................................................................................................... 389
19.2 Microbe-mediated synthesis of silver nanoparticles..................................................... 390
19.2.1 Synthesis of silver nanoparticles using bacteria................................................ 391
19.2.2 Synthesis of silver nanoparticles using actinomycetes..................................... 392
19.2.3 Synthesis of silver nanoparticles using fungi..................................................... 393
19.3 Antimicrobial mechanism of silver nanoparticles........................................................ 393
19.3.1 Antibacterial activity of silver nanoparticles...................................................... 394
19.3.2 Antifungal activity of silver nanoparticles......................................................... 395
19.4 Antimicrobial activity of silver nanoparticles in various applications...................... 395
19.4.1 Silver nanoparticles as a dressing material........................................................ 395
12.4.2 Silver nanoparticles as air disinfectant............................................................... 396
12.4.3 Silver nanoparticles in wound healing................................................................ 396
12.4.4 Silver nanoparticles for agriculture application................................................. 396
12.4.5 Silver nanoparticles for disinfecting drinking water........................................ 396
References...................................................................................................................................... 397
19.1 Introduction
In writing this chapter, the first question that came to our mind was “what are these
nanoparticles?” What is so interesting about them that researchers around the globe are
behind it? Well the answer lies in the unique properties possessed by these particles and,
besides, due to the fact that they can be revolutionized the way we want. The term nano is
adapted from the Greek word meaning “dwarf” and acts as a bridge between bulk materi-
als and atomic or molecular structures (Thakkar et al., 2010).
Although the concept of nanoparticles was first presented by Richard Feynman
through his famous lecture entitled “There’s a plenty of room at the bottom” at the
American Institute of Technology (Hulkoti and Taranath, 2014), nanoparticles have been
used since antiquity. For example, the Chinese used gold nanoparticles as an inorganic
dye to introduce red color into their ceramic porcelains more than a thousand years ago.
389
A Roman period glass called the Lycurgus Cup contained metal nanoparticles, which
provided beautiful colors. In medieval times, nanoparticles were used for decoration of
cathedral windows. Siddhars, the great Indian ancient scientists, practiced the use of
Rajat Bhasma (Rajat is silver and Bhasma means fine powder) in medicine (Hansen et al.,
2008; Manikprabhu and Lingappa, 2014).
Among various nanoparticles, silver nanoparticles are of great importance. The
word “silver” is of Gothic origin, meaning shiny white; the Latin name “argentines”
originates from an Aryan root, which means white and shining. Silver has been popu-
lar for domestic use since the ancient times; historically, silver was equated with the
moon due to white brightness of silver. Silver antimicrobial properties were known
from antiquity, having historical associations with humans dating back to 4000 BC.
Silver vessels were used to preserve water and wine. Hippocrates, the father of medi-
cine, promoted the use of silver for healing injuries. Alexander the Great was advised
by Aristotle to store water in silver vessels and boil prior to use. Evidence of the use of
silver nitrate as an antibacterial agent in the Roman pharmacopeia also exists. During
the late eighteenth century, Crede, a German obstetrician, popularized the use of pro-
phylactic 1% silver nitrate eye solution for the prevention of ophthalmia neonatorum.
During the mid-nineteenth century, Joseph Lister and Marion Sims promoted the use
of silver wire sutures in order to reduce the incidences of septic complications (Pradeep
and Anshup, 2009).
At present, silver nanoparticles are used as antimicrobial agents in most public places
such as elevators and railway stations in China. The mutation resistant antimicrobial
activities of silver are being used in different pharmaceutical formulations such as anti-
bacterial clothing, burn ointments, and coating for medical devices (Prabhu and Poulose,
2012; Manikprabhu and Lingappa, 2013a). Various physical and chemical methods were
reported for the synthesis of silver nanoparticles, but most of these methods cause poten-
tial environmental and biological hazards. Compared to physical and chemical methods,
biological synthesis using microbes and plants was regarded as a safe and eco-friendly pro-
cess (Manikprabhu and Lingappa, 2013b). In this chapter, we focus on microbe-mediated
synthesis of silver nanoparticles and antimicrobial activity against various pathogenic
microorganisms.
19.2 Microbe-mediated synthesis of silver nanoparticles

Microbe-mediated synthesis of silver nanoparticles (Figure 19.1) requires a special
ability, that is, “resistance of the organism to withstand silver ions.” It is noted that
those microorganisms that synthesize silver nanoparticles are vulnerable to higher
concentrations of silver ions. Even though the organism has the resistance to silver
ions, it becomes useless at the higher concentration. That is why silver can be called
a “moiety with two functions.” One is inducing the organism to synthesize nanopar-
ticles at lower concentration; another is the induction of cell death at higher concentra-
tion (Deepak et al., 2011). The synthesis of nanoparticles was preliminary confirmed
by a color change from colorless to brown. Further, synthesis was confirmed by UV–
visible absorption spectroscopy; the maximum absorption between 400 and 450 nm
due to surface plasmon resonance in the visible region indicates the synthesis of sil-
ver nanoparticles. In x-ray diffraction pattern, the intense peaks corresponding to
(111), (200), (220), and (311) correspond to crystalline nature of silver nanoparticles
(Manikprabhu and Lingappa, 2013a).
Chapter nineteen: Microbe-mediated synthesis of silver nanoparticles 391
Figure 19.1 Biological synthesis of silver nanoparticles.
19.2.1 Synthesis of silver nanoparticles using bacteria

One reason why bacteria are preferred for nanoparticle synthesis is because they are easy
to manipulate. The first evidence of bacteria-synthesizing nanoparticles was established
using Pseudomonas stutzeri AG259 strain, which was isolated from a silver mine (Slawson
et al., 1992).
The most widely accepted mechanism of silver biosynthesis is the presence of the
nitrate reductase enzyme. During the reduction, nitrate is converted into nitrite and the
electron is transferred to the silver ion; hence, the silver ion is reduced to silver (Ag+ to
Ag0) (Prabhu and Poulose, 2012). Further, in different microorganisms, various enzymes
are believed to take part in the bioreduction process involving the transport of electrons
from certain electron donors to metal electron acceptors. Some studies of nonenzymatic
reduction mechanism suggested that some organic functional groups of microbial cell
walls could be responsible for the bioreduction process (Baker et al., 2013), for example,
cells of Lactobacillus sp. A09 can reduce silver ions by the interaction of the silver ions
with the groups on the microbial cell wall (Fu et al., 2000). Apart from these, proteins and
microbial pigment were also used as reducing and stabilizing agent for the synthesis of
nanoparticles (Jain et al., 2011; Manikprabhu and Lingappa, 2013a). All the mentioned
mechanisms could result in the intracellular or extracellular complexion and the deposi-
tion of metal nanoparticles.
The mechanisms involved in the intracellular synthesis of nanoparticles are as fol-
lows. First, the cell wall of the microorganism is being negatively charged, which inter-
acts electrostatically with the positively charged metal ions. Then, the enzymes present
in the cell wall bioreduce the metal ions. Finally, aggregation of particles and synthesis
of nanoparticles take place. The advantages of intracellular nanoparticles synthesis are
that, the nanoparticles are small in size and nearly monodispersed, but in order to release
intracellular-synthesized nanoparticles, additional processing steps such as ultrasound
treatment or reaction with suitable detergents are required (Mukherjee et al., 2001; Sharma
et al., 2007). Various bacteria have been reported for intracellular synthesis of nanopar-
ticles like recently, an airborne Bacillus sp. isolated from the atmosphere was found to
reduce Ag+ ions to Ag0. This bacterium accumulated metallic silver of 5–15 nm in size in
the periplasmic space of the cell. Lactobacillus sp. in buttermilk produced silver crystals
of well-defined morphology within the cell with no disturbance in its viability (Nair and
Pradeep, 2002; Di Gregorio et al., 2005). Spherical silver nanoparticles of size 10–20 nm
Table 19.1 Synthesis of silver nanoparticles by different bacteria

Synthesized from Location References
Staphylococcus aureus Extracellular Nanda and Saravanan (2009)
Bacillus subtilis EWP-46 Extracellular Velmurugan et al. (2014)
Pseudomonas aeruginosa Extracellular Kumar and Mamidyala (2011)
Escherichia coli Extracellular Gurunathan et al. (2009)
Bacillus licheniformis Extracellular Kalishwaralal et al. (2008)
Klebsiella pneumoniae Extracellular Kalpana and Lee (2013)
Enterobacter cloacae Extracellular Shahverdi et al. (2007)
Brevibacterium casei Extracellular Kalishwaralal et al. (2010)
Salmonella typhimurium Extracellular Ghorbani (2013)
Bacillus megaterium Extracellular Karkaj et al. (2013)
were synthesized using Proteus mirabilis PTCC 1710 (Samadi et al., 2009). Synthesis at
extreme conditions was also reported, for example, Corynebacterium sp. SH09 produced
silver nanoparticles at 60°C on the cell wall in the size range of 10–15 nm (Zhang et al.,
2005). Further synthesis at high concentration of silver nitrate was also reported by the
metal-tolerant bacteria Idiomarina sp. PR58–8 (Seshadri et al., 2012). Although several bac-
teria for intracellular synthesis of silver nanoparticles were reported, most of them are
difficult to implement for industrial use due to the tedious recovery process. In this regard,
extracellular synthesis of silver nanoparticles is the current focus of research. Extracellular
synthesis using bacteria isolated from different environments was reported. Bacillus strain
CS 11 isolated from the industrial area produce spherical extracellular silver nanoparticles
of 42–92 nm size range (Das et al., 2014). Gram-negative marine bacteria Pseudomonas aeru-
ginosa (Shivakrishna et al., 2013) and Stenotrophomonas synthesized silver nanoparticles
in a range of 35–46 and 40–60 nm, respectively (Malhotra et al., 2013). Synthesis using
the gram-positive marine bacteria Bacillus sp. was also reported (Maruthamuthu, 2012).
The first thermophilic bacterium reported for silver nanoparticles was Geobacillus stearo-
thermophilus, which synthesized nanoparticles extracellularly (Fayaz et al., 2011). Similarly,
the extremophilic bacteria Ureibacillus thermosphaericus strain reported synthesis of silver
nanoparticles at 80°C with the particle size range of 10–100 nm (Juibari et al., 2011). The
endophytic bacterium Bacillus cereus (Sunkar and Nachiyar, 2012), the psychrophilic bac-
teria Pseudomonas antarctica, Pseudomonas proteolytica, Pseudomonas meridiana, Arthrobacter
kerguelensis, and Arthrobacter gangotriensis, and the mesophilic bacteria Bacillus indicus and
Bacillus cecembensis were also reported for green synthesis of silver nanoparticles (Shivaji
et al., 2011). Further, detailed information regarding extracellular synthesis of nanopar-
ticles by bacteria is mentioned in Table 19.1.
19.2.2 Synthesis of silver nanoparticles using actinomycetes

Actinomycetes are gram-positive bacteria but share some important characteristics of
fungi and at present are the center of attraction due to their ability to produce a large
number of secondary metabolites. The first actinomycete that was reported to synthesize
nanoparticles was Thermomonospora sp. mainly due to its ability to survive in a wide range
of environmental conditions (Rautaray et al., 2004). Since then, many other actinomycetes
were explored for the synthesis of nanoparticles. Streptomyces sp. 09 PBT 005 was able
to synthesize silver nanoparticles extracellularly, which had antibacterial activity (Kumar
et al., 2015). Similarly, Streptomyces aureofaciens, Streptomyces coelicolor klmp33, Streptomyces
rochei, Streptomyces sp. ERI-3, and Streptomyces hygroscopicus were also reported to synthe-
size extracellular silver nanoparticles (Prabhu et al., 2011; Manikprabhu and Lingappa,
2013a; Golinska et al., 2014; Zonooz and Salouti, 2011; Sadhasivam et al., 2010). Further
reports on marine Streptomyces parvulus SSNP11 and Streptomyces albidoflavus CNP10
reduced silver ions by reducing nitrate to nitrite and ammonium were also reported (Shetty
and Kumar, 2012; Baker et al., 2013). Streptomyces sp. BDUKAS10 isolated from mangrove
reported to synthesize nanoparticles (Sivalingam et al., 2012). Nanoparticles synthesized
by Streptomyces sp. VITPK1 showed anticandidal activity (Sanjenbam et al., 2014). Similarly,
silver nanoparticles synthesized using Streptomyces sp. JF714876 (Vidyasagar et al., 2012),
Streptomyces sp. JAR1 (Chauhan et al., 2013), and Streptomyces sp. VITSTK7 (Thenmozhi
et al., 2013) showed good antimicrobial activity. Not only the genus Streptomyces was
the center of attraction, but other genera such as Rhodococcus sp., Actinomycetes sp., and
Nocardiopsis sp. (Golinska et al., 2014) were also reported to synthesize silver nanoparticles.
19.2.3 Synthesis of silver nanoparticles using fungi

Fungi are eukaryotic, sporeforming, and filamentous branched organisms. Fungi can
accumulate metals by physicochemical and biological mechanisms, including extracellular
binding by metabolites and polymers, binding to specific polypeptides, and metabolism-
dependent accumulation (Gade et al., 2010). Many fungi such as Cladosporium cladospo-
rioides (Balaji et al., 2009), Neurospora crassa (Castro-Longoria et al., 2011), and Penicillium
brevicompactum (Shaligram et al., 2009) were exploited for the synthesis of metal nanopar-
ticles. Intracellular synthesis of silver nanoparticles by Verticillium of size range 2–25 nm
deposited to the surface of the cytoplasmic membrane was reported by Sastry et al. (2003).
“Green synthesis” of highly stabilized nanocrystalline silver particles by a nonpathogenic
and agriculturally important fungus, Trichoderma asperellum, was reported by Mukherjee
et al. (2008). The marine-derived fungus Aspergillus flavus synthesized intracellular sil-
ver nanoparticles in acidic pH, while the same fungus in alkaline pH range supported
rapid extracellular synthesis of silver nanoparticles (Vala et al., 2014). This indicates that
the synthesis on nanoparticles is pH dependent. Not only terrestrial fungi were explored
but marine fungi too. The marine fungi Penicillium fellutanum isolated from coastal man-
grove sediment synthesized nanoparticles using proteins as a reducing agent (Kathiresan
et al., 2009). The mushroom Agaricus bisporus was reported to synthesize nanoparticles,
which showed remarkable antimicrobial activity (Dhanasekaran et al., 2013). Similarly, the
edible mushroom Pleurotus florida synthesized polydispersed nanoparticles of size 20 ±
5 nm (Bhat et al., 2011). Further detailed information regarding extracellular synthesis of
nanoparticles is mentioned in Table 19.2.
The significant drawback of using these bio-entities in nanoparticles synthesis is that
the genetic manipulation of eukaryotic organisms as a means of over-expressing specific
enzymes is difficult when compared with bacteria.
19.3 Antimicrobial mechanism of silver nanoparticles

Silver nanoparticles are ideal bacterial agents due to their slow oxidation and liberation
of Ag+ ions to the surroundings. Moreover, the small size of these particles facilitates
their penetration through cell membranes to affect intracellular processes from inside.
Additionally, the excellent antibacterial properties exhibited by nanoparticles are due
to their well-developed surface that provides maximum contact with the environment
(Krutyakov et al., 2008).
Table 19.2 Synthesis of silver nanoparticles by different fungi

Size of
Synthesized from Location nanoparticles (nm) References
Aspergillus fumigatus Extracellular 5–25 Bhainsa and D’Souza (2006)
Phanerochaete chrysosporium Extracellular 50–200 Vigneshwaran et al. (2006)
Fusarium solani Extracellular 5–35 Ingle et al. (2009)
Amylomyces rouxii strain KSU-09 Extracellular 5–27 Musarrat et al. (2010)
Alternaria alternate Extracellular 20–60 Gajbhiye et al. (2009)
Aspergillus niger Extracellular 15–20 Gade et al. (2008)
Fusarium acuminatum Extracellular 4–50 Ingle et al. (2008)
The exact antimicrobial mechanism of silver nanoparticles is still not clear. However,
various theories of the action of silver nanoparticles on microbes to cause the antimicro-
bial effect were proposed. One is that silver nanoparticles have the ability to anchor
to the bacterial cell wall and subsequently penetrate inside the cell, thereby causing
structural changes in the cell membrane like the permeability of the cell membrane lead-
ing to death of the cell (Manikprabhu and Lingappa, 2013a). Another theory is that the
formation of free radicals by the silver nanoparticles may be considered to be another
mechanism by which the cells die. The electron spin resonance spectroscopy studies
suggested that there is formation of free radicals by the silver nanoparticles when in con-
tact with the bacteria, and these free radicals have the ability to damage the cell mem-
brane and make it porous, which can ultimately lead to cell death (Danilcauk et al., 2006).
Yet another theory has also been proposed that there can be a release of silver ions by
the nanoparticles, which can interact with the thiol groups of many vital enzymes and
inactivate them and finally lead to the death of the cell (Matsumura et al., 2003). Another
fact is that DNA has sulfur and phosphorus as its major components; the nanoparticles
can act on these soft bases and destroy the DNA, which would definitely lead to cell
death. The interaction of the silver nanoparticles with the sulfur and phosphorus of the
DNA can lead to problems in the DNA replication of the bacteria and thus terminate the
microbes (Manikprabhu and Lingappa, 2013a). It has also been found that the nanopar-
ticles can modulate the signal transduction in bacteria. It is a well-established fact that
phosphorylation of protein substrates in bacteria influences bacterial signal transduc-
tion. Nanoparticles dephosphorylate the peptide substrates on tyrosine residues, which
lead to signal transduction inhibition and thus the stoppage of growth (Prabhu and
Poulose, 2012).
19.3.1 Antibacterial activity of silver nanoparticles

Silver nanoparticles have been demonstrated as an effective antibacterial agent against
a broad bacterial spectrum, including both gram-negative and gram-positive bacte-
ria (Marambio-Jones and Hoek, 2010). Silver nanoparticles showed antibacterial activ-
ity against various pathogenic bacteria like Escherichia coli, Bacillus subtilis, Enterococcus
faecalis, Salmonella typhimurium, Staphylococcus epidermidis, and Staphylococcus aureus
(Schrofel et al., 2014; Gopinath et al., 2015). The investigation of antimicrobial activity of
silver nanoparticles on gram-negative and gram-positive bacteria showed that E. coli was
inhibited at a low concentration of Ag-NPs (3.3 nM), which was 10 times less than the
minimum inhibitory concentration on S. aureus (33 nM), that may be due to difference
in cell membrane composition and permeability of gram-negative and gram-positive

bacteria (Tran and Le, 2013). This conclusion was supported by Jung et al. (2008), which
suggested that the thickness of the peptidoglycan layer of gram-positive bacteria may
prevent the action of the silver ions to some extent.
The antibacterial activity of silver nanoparticles against different drug-resistant
pathogens like ampicillin-resistant E. coli and erythromycin-resistant Streptococcus pyo-
genes was evaluated, and the minimum inhibitory concentrations and minimum bacte-
ricidal concentrations of silver nanoparticles ranged between 30 and 100 mM (Lara et al.,
2010). Recently, our study of methicillin-resistant S. aureus showed encouraging results;
silver nanoparticles of average size 50 nm showed good antimicrobial activity; further, the
synergistic activity of silver nanoparticles and antibiotics increases antimicrobial activity
(Manikprabhu and Lingappa, 2013a). Similarly, antibacterial activity of silver nanoparticles
against extended-spectrum beta-lactamase E. coli was also reported (Manikprabhu and
Lingappa, 2014).
Apart from pH, the size of nanoparticles varied the antimicrobial activity. The antimi-
crobial activity of silver nanoparticles against Streptococcus mutans varied according to size;
the minimum inhibitory concentration increased with increase in size (Espinosa-Cristobal
et al., 2009). Similarly, antimicrobial activity of silver nanoparticles is dose dependent.
Further, different shapes of silver nanoparticles have different antimicrobial activity. Pal
et al. (2007) demonstrated that truncated triangular silver nanoplates displayed the stron-
gest antimicrobial action against E. coli, when compared with spherical and rod shaped
silver nanoparticles.
19.3.2 Antifungal activity of silver nanoparticles

Fungi are becoming a major concern of the world as their mycotoxins not only affect
plants and animals, but also humans, causing many diseases and economic damage
(Sanchez-Hervas et al., 2008). In this regard, silver nanoparticles were used as an anti-
fungal agent against various pathogens like Bipolaris sorokiniana, Magnaporthe grisea
(Jo et al., 2009), and Trichophyton mentagrophytes (Kim et al., 2008). The antifungal activ-
ity of silver nanoparticles on dermatophyte like Candida albicans was reported and sug-
gested that silver nanoparticles exert an antifungal activity by disrupting the structure
of the cell membrane and inhibiting the normal budding process due to the destruc-
tion of the membrane integrity (Kim et al., 2009). Fungus-mediated synthesis of silver
nanoparticles (Alternaria alternate) showed antifungal activity against Phoma glomerata,
Phoma herbarum, Fusarium semitectum, Trichoderma sp., and Candida albicans. Further, the
synergetic of silver nanoparticles with fluconazole increased the antifungal activity
(Gajbhiye et al., 2009). This indicates that silver nanoparticles are good antimicrobial
agents.
19.4 Antimicrobial activity of silver

nanoparticles in various applications
19.4.1 Silver nanoparticles as a dressing material
Wound dressing materials play a major part in wound management. Dr. Robert Burrell is
said to develop the world’s first nanosilver-based wound dressing in 1995 (Ahmad et al.,
2011). He developed Acticoat that speeds up the healing process and removes scars if any.
In recent times, the development of resistant strains of pathogens has become a major
problem; to overcome this problem, newly designed wound dressing has provided a
major breakthrough for the treatment of infection and wounds. Silver dressings make
use of delivery systems that release silver in different concentrations. But different factors
like the distribution of silver in the dressing, its chemical and physical forms, and affin-
ity of dressing to moisture also influence the killing of microorganisms (Rai et al., 2009).
Many advancements in dressing material were made; recently, one dressing material was
designed that has the potential to change color when the antibiotic is released and hence
alerting that there is an infection in the wound. Experts believe that this dressing has
great potential in treating burn victims who are susceptible to toxic shock syndrome.
With the advent of such a system, there can be a reduction in antibiotic resistance (Tian
et al., 2007).
12.4.2 Silver nanoparticles as air disinfectant

Bioaerosols are airborne particles of biological origins, including viruses, bacteria, and
fungi, which are capable of causing infectious, allergenic, or toxigenic diseases. Several
silver nanoparticle–based air disinfectant were formulated. The antimicrobial effect of
silver nanoparticles on bacterial contamination of activated carbon filters was studied.
The results showed that silver nanoparticle–activated carbon filters were effective for the
removal of bioaerosols. The antibacterial activity analysis of silver nanoparticle–activated
carbon filters indicated that two bacteria, B. subtilis and E. coli, were completely inhibited
within 10 and 60 min, respectively (Jung et al., 2008).
12.4.3 Silver nanoparticles in wound healing

Wound healing is a complex process and has been the subject of intense research for a long
time. The recent emergence of nanotechnology has provided a new therapeutic modal-
ity in silver nanoparticles for use in burn wounds. The wound-healing properties of sil-
ver nanoparticles in an animal model showed rapid healing in a dose-dependent manner
(Rickman et al., 2003).
12.4.4 Silver nanoparticles for agriculture application

Nanopesticides and nanoherbicides were being extensively used in agriculture. Several
industries were making formulations that contain 100–250 nm nanoparticles that are more
soluble in water thus increasing their activity. The water- or oil-based nanoemulsions con-
tained uniform suspensions of pesticide or herbicide nanoparticles of 200–400 nm, which
are used to control many pests (Yakub and Soboyejo, 2012).
12.4.5 Silver nanoparticles for disinfecting drinking water

Water is one of the most important substances on Earth and is essential to all living things.
Contamination of drinking water and the subsequent outbreak of waterborne dis-
eases are the leading cause of death in many developing nations. Significant interest
has arisen in the use of silver nanoparticles for water disinfection. Silver nanoparticles
decorated onto porous ceramic materials are used as an antibacterial water filter when
tested against E. coli. It was found that at a flow rate of 0.01 L min−1, the output count
of E. coli was zero (Ahmad et al., 2011). Similarly, silver nanoparticles binding with
polyurethane foams resulted due to interaction of nitrogen atom with the polyurethane
foams, which showed disinfection ability. At a flow rate of 0.5 L min−1, the output count
of E. coli was found nil when the input water had a bacterial load of 105 CFU mL−1 (Jung
et al., 2008).
Acknowledgments
This work was supported by the Key Project of International Cooperation of Ministry
of Science and Technology (MOST) (No. 2013DFA31980) and Yunnan Provincial Natural
Science Foundation (2013FA004). W.-J. Li was also supported by Guangdong Province
Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme (2014).
References
Ahmad, N., Sharma, S., Singh, V. N., Shamsi, S. F., Fatma, A., and Mehta, B. R. 2011. Biosynthesis of
silver nanoparticles from Desmodium triflorum: A novel approach towards weed utilization.
Biotechnology Research International, 2011, 1–8.
Baker, S., Harini, B. P., Rakshith, D., and Satish, S. 2013. Marine microbes: Invisible nanofactories.
Journal of Pharmaceutical Research, 6, 383–388.
Balaji, D. S., Basavaraja, S., Deshpande, R., Bedre Mahesh, D., Prabhakar, B. K., and Venkataraman, A.
2009. Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium
cladosporioides fungus. Colloids Surfaces B, 68, 88–92.
Bhainsa, K. C. and D’Souza, S. F. 2006. Extracellular biosynthesis of silver nanoparticle using the
fungus Aspergillus fumigatus. Colloids Surfaces B, 47, 160–164,
Bhat, R., Deshpande, R., Ganachari, S. V., Huh, D. S., and Venkataraman, A. 2011. Photo-irradiated
biosynthesis of silver nanoparticles using edible mushroom Pleurotus florida and their anti-
bacterial activity studies. Bioinorganic Chemistry and Applications, 2011, 1–7.
Castro-Longoria, E., Vilchis-Nestor, A. R., and Avalos-Borja, M. 2011. Biosynthesis of silver, gold and
bimetallic nanoparticles using the filamentous fungus Neurospora crassa. Colloids Surfaces B, 83,
42–48.
Chauhan, R., Kumar, A., and Abraham, J. 2013. A biological approach to the synthesis of silver
nanoparticles with Streptomyces sp JAR1 and its antimicrobial activity. Scientia Pharmaceutica,
81, 607–621.
Danilcauk, M., Lund, A., Saldo, J., Yamada, H., and Michalik, J. 2006. Conduction electron spin reso-
nance of small silver particles. Spectrochimica Acta A, 63, 189–191.
Das, V. L., Thomas, R., Varghese, R. T., Soniya, E. V., Mathew, J., and Radhakrishnan, E. K. 2014.
Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from indus-
trialized area. 3 Biotech, 4, 121–126.
Deepak, V., Kalishwaralal, K., Pandian, S. R. K., and Gurunathan, S. 2011. An insight into the bac-
terial biogenesis of silver nanoparticles, industrial production and scale-up. M. Rai and N.
Duran (eds.), in Metal Nanoparticles in Microbiology, pp. 17–35. Springer: Berlin/Heidelberg.
Dhanasekaran, D., Latha, S., Saha, S., Thajuddin, N., and Panneerselvam, A. 2013. Extracellular bio-
synthesis, characterisation and in-vitro antibacterial potential of silver nanoparticles using
Agaricus bisporus. Journal of Experimental Nanoscience, 8, 579–588.
Di Gregorio, S., Lampis, S., and Vallini, G. 2005. Selenite precipitation by a rhizospheric strain of
Stenotrophomonas sp. isolated from the root system of Astragalus bisulcatus: A biotechnological
perspective. Environment International, 31, 233–241.
Espinosa-Cristobal, L. F., Martinez-Castanon, G. A., Martinez-Martinez, R. E., Loyola-Rodriguez,
J. P., Patino-Marin, N., Reyes-Macias, J. F., and Ruiz, F. 2009. Antibacterial effect of silver
nanoparticles against Streptococcus mutans. Materials Letters, 63, 2603–2606.
Fayaz, A. M., Girilal, M., Rahman, M., Venkatesan, R., and Kalaichelvan, P. T. 2011. Biosynthesis
of silver and gold nanoparticles using thermophilic bacterium Geobacillus stearothermophilus.
Process Biochemistry, 46, 1958–1962.
Fu, J. K., Liu, Y. Y., Gu, P. Y., Tang, D. L., Lin, Z. Y., Yao, B. X., and Weng, S. Z. 2000. Spectroscopic
characterization on the biosorption and bioreduction of Ag(I) by Lactobacillus sp. A09. Acta
Physico-Chimica Sinica, 16, 770–782.
Gade, A. K., Bonde, P., Ingle, A. P., Marcato, P. D., Duran, N., and Rai, M. K. 2008. Exploitation of
Aspergillus niger for synthesis of silver nanoparticles. Journal of Biobased Materials and Bioenergy,
2, 243–247.
Gade, A., Ingle, A., Whiteley, C., and Rai, M. 2010. Mycogenic metal nanoparticles: Progress and
applications. Biotechnology Letters, 32, 593–600.
Gajbhiye, M., Kesharwani, J., Ingle, A., Gade, A., and Rai, M. 2009. Fungus-mediated synthesis of sil-
ver nanoparticles and their activity against pathogenic fungi in combination with fluconazole.
Nanomedicine: Nanotechnology, Biology and Medicine, 5, 382–386.
Ghorbani, H. R. 2013. Biosynthesis of silver nanoparticles using Salmonella typhimurium. Journal of
Nanostructure in Chemistry, 29, 1–4.
Golinska, P., Wypij, M., Ingle, A. P., Gupta, I., Dahm, H., and Rai, M. 2014. Biogenic synthesis of
metal nanoparticles from actinomycetes: Biomedical applications and cytotoxicity. Applied
Microbiology and Biotechnology, 98, 8083–8097.
Gopinath, P. M., Narchonai, G., Dhanasekaran, D., Ranjani, A., and Thajuddin, N. 2015. Mycosynthesis,
characterization and antibacterial properties of AgNPs against multidrug resistant (MDR)
bacterial pathogens of female infertility cases. Asian Journal of Pharmaceutical Sciences, 10(2),
138–145.
Gurunathan, S., Kalishwaralal, K., Vaidyanathan, R., Venkataraman, D., Pandian, S. R. K.,
Muniyandi, J., and Eom, S. H. 2009. Biosynthesis, purification and characterization of silver
nanoparticles using Escherichia coli. Colloids Surfaces B, 74, 328–335.
Hansen, S. F., Maynard, A., Baun, A., and Tickner, J. A. 2008. Late lessons from early warnings for
nanotechnology. Nature Nanotechnology, 3, 444–447.
Hulkoti, N. I. and Taranath, T. C. 2014. Biosynthesis of nanoparticles using microbes—A review.
Colloids Surfaces B, 121, 474–483.
Ingle, A., Gade, A., Pierrat, S., Sonnichsen, C., and Rai, M. 2008. Mycosynthesis of silver nanopar-
ticles using the fungus Fusarium acuminatum and its activity against some human pathogenic
bacteria. Current Nanoscience, 4, 141–144.
Ingle, A., Rai, M., Gade, A., and Bawaskar, M. 2009. Fusarium solani: A novel biological agent for the
extracellular synthesis of silver nanoparticles. Journal of Nanoparticle Research, 11, 2079–2085.
Jain, N., Bhargava, A., Majumdar, S., Tarafdar, J. C., and Panwar, J. 2011. Extracellular biosynthesis
and characterization of silver nanoparticles using Aspergillus flavus NJP08: A mechanism per-
spective. Nanoscale, 3, 635–641.
Jo, Y. K., Kim, B. H., and Jung, G. 2009. Antifungal activity of silver ions and nanoparticles on phy-
topathogenic fungi. Plant Disease, 93, 1037–1043.
Juibari, M. M., Abbasalizadeh, S., Jouzani, G. S., and Noruzi, M. 2011. Intensified biosynthesis of
silver nanoparticles using a native extremophilic Ureibacillus thermosphaericus strain. Materials
Letters, 65, 1014–1017.
Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., and Park, Y. H. 2008. Antibacterial activity
and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Applied
and Environmental Microbiology, 74, 2171–2178.
Kalishwaralal, K., Deepak V., Ramkumarpandian S., Nellaiah H., and Sangiliyandi, G. 2008.
Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheni-
formis. Materials Letters, 62, 4411–4413.
Kalishwaralal, K., Deepak, V., Pandian, S. R. K., Kottaisamy, M., BarathManiKanth, S., Kartikeyan,
B., and Gurunathan, S. 2010. Biosynthesis of silver and gold nanoparticles using Brevibacterium
casei. Colloids Surfaces B, 77, 257–262.
Kalpana, D. and Lee, Y. S. 2013. Synthesis and characterization of bactericidal silver nanoparticles
using cultural filtrate of simulated microgravity grown Klebsiella pneumoniae. Enzyme and
Microbial Technology, 52, 151–156.
Karkaj, O. S., Salouti, M., Zanjani, R. S., and Derakhshan, F. K. 2013. Extracellular deposition of silver
nanoparticles by Bacillus megaterium. Synthesis and Reactivity in Inorganic, Metal-Organic, and
Nano-Metal Chemistry, 43, 903–906.
Kathiresan, K., Manivannan, S., Nabeel, M. A., and Dhivya, B. 2009. Studies on silver nanoparticles
synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sedi-
ment. Colloids Surfaces B, 71, 133–137.
Kim, K. J., Sung, W. S., Suh, B. K., Moon, S. K., Choi, J. S., Kim, J. G., and Lee, D. G. 2008. Antifungal effect
of silver nanoparticles on dermatophytes. Journal of Microbiology and Biotechnology, 18, 1482–1484.
Kim, K. J., Sung, W. S., Suh, B. K., Moon, S. K., Choi, J. S., Kim, J. G., and Lee, D. G. 2009. Antifungal
activity and mode of action of silver nano-particles on Candida albicans. Biometals, 22, 235–242.
Krutyakov, Y. A., Kudrinskiy, A. A., Olenin, A. Y., and Lisichkin, G. V. 2008. Synthesis and properties
of silver nanoparticles: Advances and prospects. Russian Chemical Reviews, 77, 233–257.
Kumar, C. G. and Mamidyala, S. K. 2011. Extracellular synthesis of silver nanoparticles using cul-
ture supernatant of Pseudomonas aeruginosa. Colloids Surfaces B, 84, 462–466.
Kumar, P. S., Balachandran, C., Duraipandiyan, V., Ramasamy, D., Ignacimuthu, S., and Al-Dhabi, N.
A. 2015. Extracellular biosynthesis of silver nanoparticle using Streptomyces sp. 09 PBT 005 and
its antibacterial and cytotoxic properties. Applied Nanoscience, 5(2), 169–180,
Lara, H. H., Ayala-Núnez, N. V., Turrent, L. D. C. I., and Padilla, C. R. 2010. Bactericidal effect of
silver nanoparticles against multidrug-resistant bacteria. World Journal of Microbiology and
Biotechnology, 26, 615–621.
Malhotra, A., Dolma, K., Kaur, N., Rathore, Y. S., Mayilraj, S., and Choudhury, A. R. 2013. Biosynthesis
of gold and silver nanoparticles using a novel marine strain of Stenotrophomonas. Bioresource
Technology, 142, 727–731.
Manikprabhu, D. and Lingappa, K. 2013a. Antibacterial activity of silver nanoparticles against
methicillin-resistant Staphylococcus aureus synthesized using model Streptomyces sp. pigment
by photo-irradiation method. Journal of Pharmaceutical Research, 6, 255–260.
Manikprabhu, D. and Lingappa, K. 2013b. Microwave assisted rapid and green synthesis of sil-
ver nanoparticles using a pigment produced by Streptomyces coelicolor KLMP33. Bioinorganic
Chemistry and Applications, 2013, 1–5.
Manikprabhu, D. and Lingappa, K. 2014. Synthesis of silver nanoparticles using the Streptomyces
coelicolor klmp33 2 pigment: An antimicrobial agent against extended-spectrum beta-lacta-
mase (ESBL) producing Escherichia coli. Materials Science and Engineering: C, 45, 434–437.
Marambio-Jones, C. and Hoek, E. M. 2010. A review of the antibacterial effects of silver nanomateri-
als and potential implications for human health and the environment. Journal of Nanoparticle
Research, 12, 1531–1551.
Maruthamuthu, S. 2012. Extracellular synthesis of silver nanoparticles by marine thermophilic bac-
teria. International Journal of Pharmaceutical and Biological Archives, 3, 1418–1423.
Matsumura, Y., Yoshikata, K., Kunisaki, S. I., and Tsuchido, T. 2003. Mode of bacterial action of silver
zeolite and its comparison with that of silver nitrate. Applied Environmental Microbiology, 69,
4278–4281.
Mukherjee, P., Ahmad, A., Mandal, D., Senapati, S., Sainkar, S. R., Khan, M. I., Ramani, R., Parischa, R.,
Ajayakumar, P. V., Alam, M., Sastry, M., and Kumar, R. 2001. Bioreduction of AuCl4 ions by
the fungus Verticillium sp. and surface trapping of the gold nanoparticles formed. Angewandte
Chemie International Edition, 40, 3585–3588.
Mukherjee, P., Roy, M., Mandal, B. P., Dey, G. K., Mukherjee, P. K., Ghatak, J., Tyagi, A. K., and Kale, SP.
2008. Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic
and agriculturally important fungus T. asperellum. Nanotechnology, 19(7), 075103.
Musarrat, J., Dwivedi, S., Singh, B. R., Al-Khedhairy, A. A., Azam, A., and Naqvi, A. 2010. Production
of antimicrobial silver nanoparticles in water extracts of the fungus Amylomyces rouxii strain
KSU-09. Bioresource Technology, 101, 8772–8776.
Nair, B. and Pradeep, T. 2002. Coalescence of nanoclusters and formation of submicron crystallites
assisted by Lactobacillus strains. Crystal Growth & Design, 2, 293–298.
Nanda, A. and Saravanan, M. 2009. Biosynthesis of silver nanoparticles from Staphylococcus aureus
and its antimicrobial activity against MRSA and MRSE. Nanomedicine: Nanotechnology, Biology
and Medicine, 5, 452–456.
Pal, S., Tak, Y. K., and Song, J. M. 2007. Does the antibacterial activity of silver nanoparticles depend
on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli.
Applied and Environmental Microbiology, 73, 1712–1720.
Prabhu, S. and Poulose, E. K. 2012. Silver nanoparticles: Mechanism of antimicrobial action, synthe-
sis, medical applications, and toxicity effects. International Nano Letters, 32, 1–10.
Prabhu, K. V., Sundaramoorthi, C., and Devarasu, S. 2011. Biosynthesis of silver nanoparticles from
Streptomyces aureofaciens. Journal of Pharmaceutical Research, 4, 820–822.
Pradeep, T. and Anshup 2009. Noble metal nanoparticles for water purification: A critical review,
Thin Solid Films, 517, 6441–6478.
Rai, M., Yadav, A., and Gade, A. 2009. Silver nanoparticles as a new generation of antimicrobials.
Biotechnology Advances, 27, 76–83.
Rautaray, D., Ahmad, A., and Sastry, M. 2004. Biological synthesis of metal carbonate minerals using
fungi and actinomycetes. Journal of Material Chemistry, 14, 2333–2340.
Rickman, D., Luvall, J. C., Shaw, J., Mask, P., Kissel, D., and Sullivan, D. 2003. Precision agriculture:
changing the face of farming. Geotimes, November (feature article).
Sadhasivam, S., Shanmugam, P., and Yun, K. 2010. Biosynthesis of silver nanoparticles by Streptomyces
hygroscopicus and antimicrobial activity against medically important pathogenic microorgan-
isms. Colloids Surfaces B, 81, 358–362.
Samadi, N., Golkaran, D., Eslamifar, A., Jamalifar, H., Fazeli, M. R., and Mohseni, F. A. 2009. Intra/
extracellular biosynthesis of silver nanoparticles by an autochthonous strain of Proteus mirabi-
lis isolated from photographic waste. Journal of Biomedical Nanotechnology, 5, 247–253.
Sanchez-Hervás, M., Gil, J. V., Bisbal, F., Ramón, D., and Martínez-Culebras, P. V. 2008. Mycobiota
and mycotoxin producing fungi from cocoa beans. International Journal of Food Microbiology,
125, 336–340.
Sanjenbam, P., Gopal, J. V., and Kannabiran, K. 2014. Anticandidal activity of silver nanoparticles
synthesized using Streptomyces sp. VITPK1. Journal de Mycologie Médicale, 24, 211–219.
Sastry, M., Ahmad, A., Islam Khan, M., and Kumar, R. 2003. Biosynthesis of metal nanoparticles
using fungi and actinomycete. Current Science, 85, 162–170.
Schrofel, A., Kratošová, G., Safarik, I., Safarikova, M., Raska, I., and Shor, L. M. 2014. Applications of
biosynthesized metallic nanoparticles—A review. Acta Biomaterials, 10, 4023–4042.
Seshadri, S., Prakash, A., and Kowshik, M. 2012. Biosynthesis of silver nanoparticles by marine bac-
terium, Idiomarina sp. PR58–8. Bulletin of Materials Science, 35, 1201–1205.
Shahverdi, A. R., Minaeian, S., Shahverdi, H. R., Jamalifar, H., and Nohi, A. A. 2007. Rapid syn-
thesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological
approach. Process Biochemistry, 42, 919–923.
Shaligram, N. S., Bule, M., Bhambure, R., Singhal, R. S., Singh, S. K., Szakacs, G., and Pandey, A. 2009.
Biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fun-
gal strain. Process Biochemistry, 44, 939–943.
Sharma, N. C., Sahi, S. V., Nath, S., Parsons, J. G., Gardea-Torresde, J. L., and Pal, T. 2007. Synthesis
of plant-mediated gold nanoparticles and catalytic role of biomatrix-embedded. Environmental
Science and Technology, 41, 5137–5142.
Shetty, P. R. and Kumar, Y. S. 2012. Characterization of silver nanoparticles synthesized by using
marine isolate Streptomyces albidoflavus. Journal of Microbiology and Biotechnology, 22, 614–621.
Shivaji, S., Madhu, S., and Singh, S. 2011. Extracellular synthesis of antibacterial silver nanoparticles
using psychrophilic bacteria. Process Biochemistry, 46(9), 1800–1807.
Shivakrishna, P., Krishna, M. R. P. G., and Charya, M. S. 2013. Synthesis of silver nano particles from
marine bacteria Pseudomonas aerogenosa. Octa Journal of Biosciences, 1, 108–114.
Sivalingam, P., Antony, J. J., Siva, D., Achiraman, S., and Anbarasu, K. 2012. Mangrove Streptomyces
sp. BDUKAS10 as nanofactory for fabrication of bactericidal silver nanoparticles. Colloids
Surfaces B, 98, 12–17.
Slawson, R. M., Van Dyke, M. I., Lee, H., and Trevors, J. T. 1992. Germanium and silver resistance,
accumulation and toxicity in microorganisms. Plasmid, 27, 73–79.
Sunkar, S. and Nachiyar, C. V. 2012. Biogenesis of antibacterial silver nanoparticles using the endo-
phytic bacterium Bacillus cereus isolated from Garcinia xanthochymus. Asian Pacific Journal of
Tropical Biomedicine, 2, 953–959.
Thakkar, K. N., Mhatre, S. S., and Parikh, R. Y. 2010. Biological synthesis of metallic nanoparticles.
Nanomedicine: Nanotechnology, Biology and Medicine, 6, 257–262.
Thenmozhi, M., Kannabiran, K., Kumar, R., and Khanna, V. G. 2013. Antifungal activity of
Streptomyces sp. VITSTK7 and its synthesized Ag2O/Ag nanoparticles against medically
important Aspergillus pathogens. Journal de Mycologie Médicale, 23, 97–103.
Tian, J., Wong, K. K., Ho, C. M., Lok, C. N., Yu, W. Y., Che, C. M., and Tam, P. K. 2007. Topical delivery
of silver nanoparticles promotes wound healing. ChemMedChem, 2, 129–136.
Tran, Q. H. and Le, A. T. 2013. Silver nanoparticles: Synthesis, properties, toxicology,applications
and perspectives. Advances in Natural Sciences: Nanoscience and Nanotechnology, 4(3), 033001.
Vala, A. K., Shah, S., and Patel, R. 2014. Biogenesis of silver nanoparticles by marine-derived fungus
Aspergillus flavus from Bhavnagar Coast, Gulf of Khambhat, India. Journal of Marine Biology &
Oceanography, 3(1), 2.
Velmurugan, P., Iydroose, M., Mohideen, M. H. A. K., Mohan, T. S., Cho, M., and Oh, B. T. 2014.
Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evalua-
tion of its antibacterial activity. Bioprocess and Biosystems Engineering, 37, 1527–1534.
Vidyasagar, G. M., Shankaravva, B., Begum, R., and Imrose, R. R. 2012. Antimicrobial activity of
silver nanoparticles synthesized by Streptomyces species JF714876. International Journal of
Pharmaceutical Sciences and Nanotechnology, 5, 1638–1642.
Vigneshwaran, N., Kathe, A. A., Varadarajan, P. V., Nachane, R. P., and Balasubramanya, R. H. 2006.
Biomimetics of silver nanoparticles by white rot fungus, Phaenerochaete chrysosporium. Colloids
Surfaces B, 53, 55–59.
Yakub, I. and Soboyejo, W. O. 2012. Adhesion of E. coli to silver or copper coated porous clay ceramic
surfaces. Journal of Applied Physics, 111(12), 124324.
Zhang, H., Li, Q., Lu, Y., Sun, D., Lin, X., Deng, X., and Zheng, S. 2005. Biosorption and bioreduction
of diamine silver complex by Corynebacterium. Journal of Chemical Technology and Biotechnology,
8, 285–290.
Zonooz, N. F. and Salouti, M. 2011. Extracellular biosynthesis of silver nanoparticles using cell fil-
trate of Streptomyces sp. ERI-3. Scientia Iranica, 18, 1631–1635.
chapter twenty
Nanomaterials
Source of antimicrobial products
Atanu Bhattacharyya, P.M. Gopinath, A. Ranjani,
and Dharumadurai Dhanasekaran
Contents
20.1 Introduction....................................................................................................................... 403
20.2 Utility and beautility of nanomaterials.........................................................................404
20.3 Different forms of nanomaterials and their roles........................................................408
20.4 Titanium dioxide nanoparticles...................................................................................... 410
20.5 Zinc oxide nanoparticles.................................................................................................. 410
20.6 Magnesium oxide and other nanoparticles.................................................................. 411
20.7 Copper oxide nanoparticles............................................................................................ 411
20.8 Iron nanoparticles and aluminum nanoparticles........................................................ 411
20.9 Magnetosomes nanoparticles......................................................................................... 411
20.10 Use of nanomaterials and their risk management....................................................... 413
20.11 Conclusion......................................................................................................................... 414
References...................................................................................................................................... 414
20.1 Introduction
Nanoparticles (NPs) are molecular aggregates having a dimension between 1 and 100 nm.
They possess different physicochemical (strength, electrical, and optical) properties due
to the variation in surface area (Bhattacharyya et al., 2011, 2014). NPs occur in nature,
as v
olcanic dust, lunar dust, and mineral composites. Natural or engineered NPs, also
defined as waste or anthropogenic particles, may be formed as a result of industrial pro-
cesses, like diesel exhaust, coal combustion, and welding fumes. These nanomaterials are
mostly carbon-based (fullerene, single- and multiwalled carbon nanotube) and metal-
based (quantum dots, nanogold, nanozinc, and nanoaluminum) materials (Bhattacharyya
et al., 2011, 2014). These materials are used in several biological processes. A few studies
have focused on the effects and mechanisms of nanomaterials on bacteria (Bhattacharyya,
2009; Xu et al., 2009). The functional aspects of nanomaterials are unique, and these studies
have been reported with the aim to provide further insight of bacteria and nanomaterials
(Bhattacharyya et al., 2007, 2014). Nanoscale metal oxides like TiO2, ZnO, and Al2O3 den-
drimers (nanosized polymers built from branched units) are performed with atom–atom
interaction (Bhattacharyya et al., 2014).
Currently, scientists are interested in materials that are effective at the nanoscale
such as gold and silver because of their natural and chemical characteristics.
403
The introduction of inorganic nanomaterials in this century is a unique phenomenon,

and their novel characteristic features are mentioned earlier. The inorganic nanoma-
terials exhibit well-adopted physical, chemical, and biological properties, though all
the mentioned properties may vary in size based on their character as well as on spe-
cific pH and temperature. In the biological field, nanomaterials are very much adopted
due to their specific selectivity toward the targeted materials in the biological system.
Moreover, recent studies clearly denote that the prepared metal oxide nanomaterials
have increased antibacterial activity (Hamouda et al., 1999; Pal et al., 2007). Several
experimental data are coming through that can predict in an effective way that inor-
ganic metal silver is an antimicrobial agent (Table 20.1). These mentioned inorganic
metals exhibit a very robust antibacterial activity (Novak and Feldheim, 2000; Wiley
et al., 2005; Yamanaka et al., 2005). Now, it is well known that the compound and sim-
ple nature of silver nanomaterials are the reason for the unique bactericidal activity
(Pal et al., 2007; Gopinath et al., 2015). Moreover, it should be remembered that random
use of silver nanoparticles (AgNPs) in various fields has some possibility of toxicity in
human beings as well as in the environment or in the ecosystem. The toxicity and cel-
lular uptake experiments of several NPs clearly denote that the carbohydrate coating
on silver nanoparticle modulates both oxidative stress and cellular uptake, and it can
expose some toxicity in the physiological system. In this context, it has been observed
that the bioactivity of AgNPs and other nanomaterials can change by using a carbohy-
drate coat (Kennedy et al., 2014). Different types of bacterial cells are multidrug toler-
ant and, therefore, able to survive antibiotic treatment. However, recent technological
advances in microfluidics and reporter genes have improved this scenario. Here, we
summarize recent progress in the field, revealing the ubiquitous bacterial stress alarm
one ppGpp as an emerging central regulator of multidrug tolerance and persistence,
both in stochastically and environmentally induced persistence. In several different
organisms, toxin–antitoxin modules function as effectors of ppGpp-induced persis-
tence (Maisoneuve and Gerdes, 2014). Therefore, the effects of different NPs on several
biofilms and also on several bacteria have been dealt with in this chapter.
20.2 Utility and beautility of nanomaterials

Dwarf nanomaterial is an advanced scientific technique in the twenty-first century.
Nanotechnology is becoming a revolutionary feild in its integration with the green chem-
istry approach. Several strategies of nanomaterials involved in strain selection, cultivation
modes, recombinant gene expression, metabolic engineering, and protein redesign and
reengineering and its predictive modeling will allow creating some utility in nanobioreac-
tor process. A new nanobiotechnology arena with a high potential impact in many fields
is gradually developing. Nanotechnology is becoming a new field of increasing research
and industrial interest since 1980s. Nanotechnology can be defined as the manipulation
of atom-by-atom interactions in nature. It can take part in the processes of chemical and
biological functions. In general, the nano-object properties depend on chemical composi-
tion, but also on size, shape, composition, and their environment (like pH and temperature)
including their spatial distribution. It is clear that synthesis techniques can affect consider-
ably the properties of the nano-objects. The synthesis techniques can be categorized into
top-down and bottom-up phenomena. The top-down techniques work with the material in
its bulk form, and size reduction of nanoscale is made via specialized ablations (e.g., lithog-
raphy, thermal decomposition, laser ablation). In this context, engineered nanomaterials
have received a particular attention for their positive impact in improving, among others,
Chapter twenty: Nanomaterials 405
Table 20.1 Antimicrobial activity of metal nanoparticles

Nanoparticle type Size (Average) Organism tested References
ZnO 13 nm Staphylococcus aureus Reddy et al. (2007)
ZnO 60 nm S. aureus Jones et al. (2008)
ZnO 40 nm S. aureus, Escherichia Nair et al. (2009)
coli
ZnO 12 nm E. coli Padmavathy and
Vijayaraghavan (2008)
ZnO ions N/A Pseudomonas McCarthy et al. (1992)
aeruginosa, S. aureus,
Candida albicans
Silver 21 nm E. coli, Vibrio cholerae, Morones et al. (2005)
Salmonella typhi,
P. aeruginosa
Silver Triangles (50 nm) E. coli Pal et al. (2007)
Silver 12 nm E. coli Sondi and Salopek-Sondi
(2004)
Silver 13.5 nm S. aureus, E. coli Kim et al. (2007)
Cu 100 nm E. coli, Bacillus subtilis Yoon et al. (2007)
Fe3O4 9 nm S. aureus Tran et al. (2010)
Fe3O4 8 nm Staphylococcus Taylor and Webster
epidermidis (2009)
Al2O3 11 nm E. coli Simon-Deckers et al.
(2009)
Al2O3 60 nm E. coli, B. subtilis, Jiang et al. (2009)
Pseudomonas
fluorescens
TiO2 17 nm E. coli Simon-Deckers et al.
(2009)
SiO2 20 nm E. coli, B. subtilis, Jiang et al. (2009)
P. fluorescens
Chitosan 40 nm E. coli, S. aureus Qi et al. (2004)
Carboxyl-grafted SPIONs 10–20 nm S. aureus Subbiahdoss et al. (2012)
APTES-grafted SPIONs 10–20 nm
PEGylated SPIONs 10–20 nm
Ag-coated SPIONs 15–20 nm Khan (2012)
Ag–Au-coated SPIONs 20–30 nm
Au-coated SPIONs 25–40 nm
ZnO <100 nm Halophilic bacterium Sinha et al. (2011)
sp. EMB4
Ag <100 nm
Ag caron complex-l- 700–800 nm Vancomycin-resistant Leid et al. (2012)
tyrosine polyphosphate Enterococcus
NP(SCC23-LTP NPs)
ZnO <100 nm B. subtilis Sinha et al. (2011)
Ag <100 nm
Ag 2–4 nm Ruparelia et al. (2008)
CuO 8–10 nm
(Continued)
Table 20.1 (Continued) Antimicrobial activity of metal nanoparticles

Nanoparticle type Size (Average) Organism tested References
Al2O3 40–70 nm Jiang et al. (2009)
TiO2 40–60 nm
Cu-doped TiO2 20 nm Mycobacterium Wu et al. (2010)
nanoparticles smegmatis
Ag Caron complex-l- 800 nm Klebsiella pneumoniae Juan et al. (2010)
tyrosine polyphosphate
NP (SCC23-LTP NPs)
Ag 43 nm Khan et al. (2011)
NO 10–15 nm Friedman et al. (2011)
NO 8–15 nm P. aeruginosa Friedman et al. (2011)
NO-releasing MAP3 80–100 nm Hetrick et al. (2009)
(N-methyl amino
propyltrimethoxysilane)
Si NPs
TiO2 10–25 nm Tsuang et al. (2008)
Ag 1–10 nm Morones et al. (2005)
ZnO 10–20 nm Feris et al. (2009)
ZnO 25–40 nm Salmonella Kumar et al. (2011)
typhimurium
TiO2 40–60 nm
Cu-doped TiO2 NPs 20 nm Shewanella oneidensis Wu et al. (2010)
MR-1
Ag 10 nm Pseudomonas putida Gajjar et al. (2009)
KT2442
CuO 25–40 nm
ZnO 50–70 nm
TiO2 <25 nm Cupriavidus Simon-Deckers et al.
metallidurans (2009)
Al2O3 <25 nm
Multiwalled carbon <25 nm
nanotubes
Ag 16–70 nm Enterobacter sp. ANT 02, Gopinath et al. (2015)
P. aeruginosa ANT 04,
K. pneumoniae ANT 03,
and E. coli ANT 01
Ag — S. typhi, E. coli, Dhanasekaran et al.
Klebsiella sp., (2013)
Pseudomonas sp.,
Enterobacter sp.,
Proteus sp., S. aureus,
S. paratyphi
consumer products, pharmaceutics, cosmetics, transportation, energy, and also agriculture.

It is the beauty of nanomaterials (Cauerhff and Castro, 2013).
Moreover, after Richard Feynman delivered his famous lecture, “There’s plenty of room
at the bottom Nature Nanotechnology looks at its influence on subsequent developments in
nanoscience and technology” Feynman would like to consider the atom-by-atom reactions.
Now, let us consider some examples; suppose that many molecules are in a specific tank, in
order to double the density; having the same speed and exhibiting the same temperature
where they belong. Then, to a close approximation, the number of collisions will be doubled,
and since each will be just as “energetic” as before, the pressure is proportional to the density.
If we consider the true nature of the forces between the atoms, naturally, we would expect a
slight decrease in pressure because of the attraction between the atoms and the pressure is
proportional to the density. In such a way, complex molecules are produced. Thus the com-
plex materials are broken down with the specific instruments for observing whether the said
materials are nanomaterials or not. The nanomaterials possess three important characters:
increases in electrical potential and chemical reaction and development of magnetic power.
These can all be observed through SEM (Figure 20.1), TEM, and also atomic force microscope
(AFM) (Figure 20.2). The scanning tunneling microscope and its offspring, the AFM, are
20 kv ×5,000 5 μm 10 38 SEI 20 kv ×10,000 1 μm 10 38 SEI

(a) (b)
Figure 20.1 Scanning electron microscope image of silver nanoparticles synthesized using fungal
extract. It reveals that the particles were roughly spherical to oval in nature with a little aggregation.
The aggregation of AgNPs occurred during drying process: (a) 5,000x magnification and (b) 10,000x
magnification. (Adapted from Gopinath, P.M. et al., Asian J. Pharm. Sci., 10(2), 138, 2015.).
2 μm
12.67 nm
Z: 12.7 nm
1 μm
0.00 nm
Y:
2.0
μm
X: 2.0 μm
m
X: 2.0 μ Y: 2.0 μm
0 μm
0 μm 1 μm 2 μm Z: 12.7 nm
(a) (b)
Figure 20.2 Atomic force microscope (AFM) images of AgNPs: (a) 2D view and (b) 3D view. The
depth image of AFM shows the spherical arrangement of silver nanoparticles within the diameter
range of 6.3–12.67 nm (Adapted from Gopinath, P.M. et al., Asian J. Pharm. Sci., 10(2), 138, 2015.)
synonymous with nanotechnology, and one might assume that it was inevitable that nano-
technology became possible because of these three instruments (Toumey, 2010; Mody, 2011).
After the discovery of nanomaterials, nanotechnology gradually developed. The pro-
posed technology was adopted in several applications in different areas including bio-
logical sciences and also medical sciences, which may be considered as the beautility of
nanoscience (Yin et al., 2013; Ezzat et al., 2014).
20.3 Different forms of nanomaterials and their roles

Nanotechnology is rapidly growing with NPs produced and utilized in a wide range of
commercial products throughout the world. For example, silver nanoparticles (AgNPs)
are used in electronics, biosensing, clothing, food industry, paints, sunscreens, cosmetics,
and medical devices. In nature, though there are several nanomaterials, we would like
to examine some important nanomaterials and their roles. One of the important NPs is
the silver nanoparticle. A large number of in vitro studies indicate that AgNPs are toxic
to mammalian cells derived from the skin, liver, lung, brain, vascular system, and repro-
ductive organs. Interestingly, some studies have shown that this particle has the poten-
tial to induce genes associated with cell cycle progression, DNA damage, and apoptosis
in human cells at noncytotoxic doses. Cytotoxicity occurs due to random use of silver
nanomaterials (Ahamed et al., 2010). Nanoscience has been established as a new interdis-
ciplinary science that can be defined as a whole knowledge on fundamental properties of
nanosized materials and thus observed as a tremendous effect on biological and chemical
applications (Makwana et al., 2014). Inorganic NPs find applications in therapeutic agents,
electrochemical biosensors, and as catalysts for removal of water pollutants. Moreover,
silver nanoparticles have gained significant interest over the years due to their remark-
able optical properties and have been used as sensors for various metal ions (Makwana
et al., 2014). Silver nanoparticles showed higher antimicrobial efficiency compared to silver
salts because of their extremely large surface area, with diameters generally smaller than
100 nm containing 20–15,000 silver atoms, providing better contact with microorganisms
(Makwana et al., 2014).
Nanosized particles/fillers, both inorganic and organic materials, have unique chemi-
cal, physical, and biological functions and have been extensively studied as biomaterials
or biofunctional materials. Nanocomposite hydrogels, which combine the advantages of
both nanofillers and hydrogel matrices, may result in improved mechanical and biological
properties and find potential biomedical applications. This chapter reviews recent devel-
opments in the synthesis, preparation, and characterization of nanocomposite hydrogels
and their biomedical applications, such as drug delivery matrices and tissue engineering
scaffolding (Song et al., 2015). It is a known fact that silver nanoparticles possess one posi-
tive charge. Naturally, this positive charge may act on negatively charged particles. Thus,
this property of silver nanoparticles can exhibit one antibacterial property (Liu et al., 2010).
Silver nanoparticles always perform their size- and surface-area-dependent functions on
bacterial biofilms. These processes can save fruits, vegetables, herbs, breads, cheeses, soups,
sauces, meats, etc., from bacterial contamination (Patil et al., 2011). The growth of microbes
on textiles during use and storage negatively affects the wearer as well as the textile itself.
The detrimental effects can be controlled by durable antimicrobial finishing of the textile
using broad-spectrum biocides or by incorporating the biocide into synthetic fibers during
extrusion. The application methods of antimicrobial agents and some of the most recent
developments in antimicrobial treatments of textiles use various active agents such as sil-
ver, quaternary ammonium salts, polyhexamethylene biguanide, triclosan, chitosan, dyes
and regenerable N-halamine compounds, and peroxy acids (Gao and Cranston, 2008).
Surface-enhanced Raman scattering schemes have been employed to detect and identify
small molecules like nucleic acids, lipids, peptides, and proteins for in vivo cellular sensing
(Bantz et al., 2011).
The therapeutic use of gold can be traced back to Chinese medical practices in
2500 BC. Red colloidal gold is still used in Indian Ayurveda medicine for rejuvenation and
revitalization during old age under the name of Swarna Bhasma (“Swarna” meaning gold,
“Bhasma” meaning ash). Gold also has a long history of use in the Western world as nerving,
a substance that could revitalize people suffering from nervous conditions. In the sixteenth
century, gold was recommended for the treatment of epilepsy. In the beginning of the
nineteenth century, gold was used in the treatment of syphilis. Following the discovery of
the bacteriostatic effect of gold cyanide toward the tubercle bacillus by Robert Koch, gold-
based therapy for tuberculosis was introduced in the 1920s (Daniel and Astruc, 2004). The
major clinical uses of gold compounds are in the treatment of rheumatic diseases including
psoriasis, juvenile arthritis, palindromic rheumatism, and discoid lupus erythematosus.
Au particles are particularly and extensively exploited in organisms because of their
biocompatibility (Li et al., 2014). Gold nanoparticles (AuNPs) generally are considered to be
biologically inert but can be engineered to possess chemical or photothermal functionality.
In near-infrared (NIR) irradiation, Au-based nanomaterials, Au nanospheres, Au nano
cages, and Au nanorods with characteristic NIR absorption can destroy cancer cells and
bacteria via photothermal heating (Thirumurugan and Kaur, 2013). Au-based NPs can
be combined with photosensitizers for photodynamic antimicrobial chemotherapy. Au
nanorods conjugated with photosensitizers can kill methicillin-resistant Staphylococcus
aureus (MRSA) by photodynamic antimicrobial chemotherapy and NIR photothermal
radiation (Daniel and Astruc, 2004; Li et al., 2014). A hydrophilic photosensitizer, toluidine
blue O, was conjugated on the surface of Au nanorods for photodynamic antimicrobial
chemotherapy. Au nanorods served as both photodynamic and photothermal agents, and
they inactivated MRSA. The combined effect of photodynamic antibacterial chemotherapy
(PACT) and hyperthermia has enhanced the antimicrobial effect of AuNP. The study
clearly showed that gold nanorods conjugated with a hydrophilic photosensitizer
such as toluidine blue O act as dual-function agents in photodynamic inactivation and
hyperthermia against MRSA. Light-absorbing AuNPs conjugated with specific antibodies
have also been exploited to photothermally kill S. aureus. AuNPs have attracted the interest
of scientists for over a century, but research in this field has considerably accelerated
since 2000 with the synthesis of numerous 1D, 2D, and 3D shapes as well as hollow AuNP
structures. Recent studies have focused on functionalizing AuNPs as photothermal agents
for hyperthermically killing pathogens (Li et al., 2014). The cancer biomarker can be
detected by gold plasmatic nanodevices (Perozziello et al., 2014).
The efficacy of the antibacterial activity of AuNPs can be increased by adding anti-
biotics. The antimicrobial activity of the antibiotic vancomycin was enhanced by coating
with AuNP against vancomycin-resistant Enterococci (Li et al., 2014). The coating of amino-
glycoside antibiotics with AuNPs has an antibacterial effect on a range of gram-positive
and gram-negative bacteria (Table 20.1). Cofactor (a second-generation β-lactam antibiotic)–
reduced AuNPs have potent antimicrobial activity on both gram-positive (S. aureus) and
gram-negative bacteria (Escherichia coli) compared to cofactor and AuNPs alone. Further,
the AuNPs generate holes in the cell wall, resulting in the leakage of cell contents and cell
death. It is also possible that AuNPs bind to the DNA of bacteria and inhibit the uncoiling
and transcription of DNA (Li et al., 2014). Recently, bimetallic NPs have received consider-
able attention for their unique optical, magnetic, and catalytic properties, which are very
different from those of their monometallic NP components. Among the bimetallic NPs,
gold–palladium (Au–Pd) is one of the most attractive systems because of its promising use
as a catalyst in CO oxidation, vinyl acetate monomer synthesis, hydrodechlorination of
CClF2, hydrogenation of hydrocarbon, cyclotrimerization of acetylene, and others for bio-
logical application (Ding et al., 2010). Moreover, nanopores can help in the DNA analysis
of gold nanodrills.
AuNPs can be used to coat a wide variety of surfaces, for instance, implants and fabrics
for treatment of wounds and glass surface maintenance. Thus, they help to maintain the
glass hygienic conditions in homes, in hospitals, and also in other places. As they possess a
high surface area, they accelerate biological reactions, and thus their possibility to disinfect
has been observed (Mironava et al., 2013). It has been observed that the conjugant of Fe3O4
and AuNPs can inhibit the multiplication of E. coli (Chatterjee et al., 2011).
20.4 Titanium dioxide nanoparticles

TiO2 exhibits its photocatalytic activity with strong oxidizing power when illuminated
with UV light at wavelength of less than 385 nm. TiO2 particles catalyze the killing of
bacteria on illumination by near-UV light (Stepanov et al., 2013; Sagadevan et al., 2014;
Vasantharaja et al., 2015). There are also studies on bactericidal activity of nitrogen-doped
metal oxide nanocatalysts on E. coli biofilms and on the photocatalytic oxidation of biofilm
components on TiO2-coated surfaces (Table 20.1). The TiO2 photocatalyst acts as an alter-
native means of self-disinfecting materials. Though toxic effects are there, of course, that
depends on the dose and the surface area of the said NP (Vasantharaja et al., 2015).
20.5 Zinc oxide nanoparticles

Nanotechnology has played an extremely important role in the design, synthesis, and char-
acterization of various new and novel energy materials and catalysts for processing fuels
from fossil fuel resources such as coal, petroleum, and natural gas. Today, fossil fuels still
account for 90% of the world’s energy consumption, and their use is expected to peak around
the year 2050 (Liu et al., 2010). Thus, the use of nanotechnology to develop a suite of sustain-
able energy production schemes is one of the most important scientific challenges of the
twenty-first century. The challenge is to design, synthesize, and characterize new functional
nanomaterials with controllable sizes, shapes, and/or structures. Moreover, the goal of nano-
technology is to build nanodevices that are intelligent, multifunctional, exceptionally small,
and extremely sensitive and have low power consumption. When a nanodevice is required
for applications such as in vivo biomedical sensors, a nanoscale power source is required (Liu
et al., 2010; Pan et al., 2010). Nanosized zinc oxide particles are usually used in the cotton and
wool fabrics industries (Becheri et al., 2008). ZnO NPs cross-interact with a critical tumor-
suppressive process and can protect pathways like apoptosis, senescence, and cell cycle
progression. ZnO NPs also induce the endogenous genetic, transcriptomic, and proteomic
landscape of the target cells (Ng et al., 2011). Moreover, the nano–ZnO-coated materials are
being used and exhibit higher activity than fabrics and were significantly higher than the
bulk ZnO (Yadav et al., 2006). ZnO nanowires (NWs) and nanobelts have been widely stud-
ied as a key 1D oxide nanomaterial for numerous applications. Due to the unique piezoelec-
tric and semiconducting coupled properties, a range of novel nanodevices of ZnO have been
developed, such as nanogenerators and 6,9-piezoelectric field effect transistors (Hu et al.,
2010). ZnO NWs have attracted a great deal of interest because of their unique semiconduct-
ing, piezoelectric, biocompatible, and optoelectronic properties, which are fundamental for
their application in electronics, optoelectronics, biology, environmental science, and energy

(Yang et al., 2014). ZnO is a piezoelectric material with a wide direct bandgap of 3.37 eV and
a large exciting binding energy of 60 meV (Hu et al., 2010). It has been demonstrated to have
enormous applications in building up electronic, optoelectronic, electrochemical, and elec-
tromechanical nanodevices, such as UV lasers, light-emitting diodes (Hu et al., 2010), field
emission devices, solar cells, high-performance nanosensors, piezoelectric nanogenerators,
and nanopiezotronics (Xu et al., 2010). Thus, ZnO is used in fabricating flexible fiber nano-
generators that can be used for smart shirts, flexible electronics, and medical applications (Li
and Wang, 2010). It has been reported that the gel-combustion method helps to prepare the
zinc oxide NPs, which exhibit the antimicrobial activity on Klebsiella sp., E. coli, Pseudomonas
aeruginosa, and S. aureus (Ramasami et al., 2015) (Table 20.1).
20.6 Magnesium oxide and other nanoparticles

Magnesium hydroxide, Fe2O3− (Kumari et al., 2012), and other NPs like magnesium oxide
(mesoporous), sulfur dioxide (SO2), nitrogen oxide (NO), and calcium oxide and magnetic
γ-Fe2O3 NPs coated with poly-l-cysteine for chelation of As(III), Cu(II), Cd(II), Ni(II), Pb (II),
and Zn(II) all are metal-binding proteins and can detect biomarker proteins in the physi-
ological system (White et al., 2009).
20.7 Copper oxide nanoparticles

Among all the metal oxides, copper oxide nanomaterials have attracted more attention due
to their unique properties. Cuprous oxide (Cu2O) is a p-type semiconductor with a direct
bandgap of 2.17 eV (Akimoto et al., 2006). In recent years, there is a growing interest to syn-
thesize Cu2O nanostructures not only for the development of synthetic strategies but also
for the examination of their sensing, catalytic, electrical, and surface properties. It has been
suggested that CuO may find potential application as an antimicrobial agent (Table 20.1) as
it can be prepared with an extremely high surface area (Gopalakrishnan et al., 2012).
20.8 Iron nanoparticles and aluminum nanoparticles

The effects of iron oxide and tetroxide NPs over biofilm formation on different biomaterial
surfaces and pluronic-coated surfaces were examined. There was significant reduction in
bacterial adhesion on pluronic-coated surfaces compared to other surfaces. Subsequently,
bacteria were allowed to grow for 24 h in the presence of different concentrations of iron
oxide NPs. A significant reduction in biofilm growth was observed in the presence of the
highest concentration of iron oxide NPs on pluronic-coated surfaces compared to other sur-
faces. Therefore, a combination of polymer brush coating and iron oxide NPs could show
a significant reduction in biofilm formation (Thukkaram et al., 2014). Moreover, aluminum
oxide NPs have a wide range of applications in industrial and personal care products. The
growth inhibitory effect of alumina NPs over a wide concentration range (10–1000 μg/mL)
was observed on E. coli (Thukkaram et al., 2014) (Table 20.1).
20.9 Magnetosomes nanoparticles

Magnetosomes are membranous prokaryotic structures present in magnetotactic bacteria
(MTB). They contain 15–20 magnetite crystals (about 20–110 nm) that together act like a
compass needle to orient MTB in geomagnetic fields. Magnetoaerotaxis of MTB is based
on intracellular membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals, which

are the magnetosomes. The chain-like arrangement of magnetosomes allows adding up
of all the individual magnetic moments, which results in a dipole strong enough to align
the cell within Earth’s weak magnetic field. In the alphaproteobacterium Magnetospirillum
gryphiswaldense, magnetosome biomineralization and chain assembly are under genetic
control. Each magnetite crystal within a magnetosome is surrounded by a lipid bilayer,
and specific soluble and transmembrane proteins are sorted to the membrane. Recent
research has shown that magnetosomes are invaginations of the inner membrane and not
freestanding vesicles (Katzmann et al., 2013). Magnetite-bearing magnetosomes have also
been found in eukaryotic magnetotactic algae, with each cell containing several thousand
crystals. Overall, magnetosome crystals have high chemical purity, narrow size ranges, and
species-specific crystal morphologies and exhibit specific arrangements within the cell.
These features indicate that the formation of magnetosomes is under precise biological con-
trol and is mediated through biomineralization. MTB usually mineralize either iron oxide
magnetosomes, which contain crystals of magnetite (Fe3O4), or iron sulfide magnetosomes,
which contain crystals of greigite (Fe3S4). Magnetosome crystals are typically 35–120 nm
long, which makes them single domain. Single-domain crystals have the maximum pos-
sible magnetic moment per unit volume for a given composition (Komeili et al., 2006).
In the course of routine investigations of seawater mud samples, Blakemore discov-
ered bacteria (1975), which surprisingly showed a magnetotactic behavior, that is, they
were moving within a magnetic field toward a magnetic pole. From the freshwater, he
isolated a type of Spirillum, which he called Aquaspirillum magneto tacticum. The magneto-
taxis is enabled by special organelles, the so-called magnetosomes, which turned out to
be crystals consisting of magnetite (Fe3O4) with a parallelepiped or an orthohexagonal
prismatic structure storing high amounts of iron. The highest amount of iron ever found
was 0.27% in Desulfotomaculum orientis, while E. coli merely contains 0.014% iron (Oberhack
et al., 1987).
Magnetosome bacteria (MB) are generally found in the oxic–anoxic transition zone
and exhibit two distinct types of responses to applied magnetic fields. The first is axial
magnetoaerotaxis whereby the bacteria use the magnetic field as an axis of swimming
with no preference for either pole. Most MB, by contrast, is polar magnetoaerotactic bac-
teria, meaning that they persistently swim toward only one pole of the magnet. The vast
majority of MB in the Northern Hemisphere are north seeking, and those in the Southern
Hemisphere are south seeking. This default swimming pattern of polar MB points them
toward the bottom of their aquatic habitats where the oxygen levels are low (Araujo et al.,
2015). MTB produce intracellular organelles called magnetosomes that are magnetic NPs
composed of magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a lipid bilayer. The synthe-
sis of a magnetosome is through a genetically controlled process in which the bacterium
has control over the composition, direction of crystal growth, and the size and shape of the
mineral crystal. As a result of this control, magnetosomes have narrow and uniform size
ranges, relatively specific magnetic and crystalline properties, and an enveloping biologi-
cal membrane. These features are not observed in magnetic particles produced abiotically,
and thus magnetosomes are of great interest in biotechnology. Most currently described
MTB have been isolated from saline or brackish environments, and the availability of
their genomes has contributed to a better understanding and culturing of these fastidious
microorganisms (Araujo et al., 2015).
Nanometer-sized magnetic particles are of great interest in biotechnology since
they have a large surface area that can be used for anchoring relatively large amounts
of specific molecules and can be easily manipulated using an external magnetic field.
These magnetic particles including magnetosomes can be bound to proteins, cells, viruses,
or genes of interest that can then be subsequently separated using magnetic techniques.
The particles most often used for these types of studies consist of iron oxides, especially
magnetite and maghemite (γ-Fe2O3), that are more stable than iron sulfides such as greigite.
They have been used in various biomedical applications such as immunoassays, cell sepa-
ration, hyperthermia protocols (treatment of cancer by localized heating), drug delivery,
and nuclear magnetic resonance (Ana-Carolina et al., 2015).
20.10 Use of nanomaterials and their risk management

As we have discussed previously, the use of nanomaterials in different sectors vary prop-
erly. It is very amazing that quantum phenomena occur at the scale of single atoms and
small molecules and nanotechnology will bring up new materials with improved and
novel physical, chemical, and biological properties (Duncan, 2005). New architectures
from individual biomolecules and biomacromolecules will possess novel functions. Here
again, we will discuss in short form the uses of important nanomaterials. The silver
nanoparticle is the best one, as its surface-to-volume ratio is high. Moreover, we know
nanoscale materials have been used for decades in applications ranging from window
glass and sunglasses to car bumpers and paints. Now, nanotechnology is part of scientific
disciplines like chemistry, biology, electronics, physics, and engineering (Duncan, 2005).
This technology is also being used in manufacturing, computer chips, medical diagno-
sis, health care, energy, biotechnology, space exploration and security, and so on. Thus,
nanotechnology is expected to have a significant impact on our society. Day by day, the
potential of nanotechnology is gradually applied in medical sciences. We can expect to
see an increase of environmental risk due to the use of engineered nanoscale materials
in nature (Dreher, 2004; Gao et al., 2015). Toxicity aspects of nanotechnology are not suffi-
ciently focused on broader issues of interest to humanity such as resources (water, energy,
and food) and the environment (Renn and Roco, 2006; Xiao et al., 2015). Naturally, the
knowledge of potential human and environmental exposures combined with dose fac-
tor response to toxicity information is more necessary to determine the real or perceived
risk factors of nanomaterials (Renn and Roco, 2006; Gao et al., 2015; Xiao et al., 2015). Risk
analysis is broadly defined here to include risk assessment, risk characterization, risk
communication, risk management, and policy relating to risk. Recent interests include
risks to human health and the environment, both built and natural. We consider in this
chapter the threats from physical, chemical, and biological agents and from a variety
of human activities as well as natural events (Gao et al., 2015; Xiao et al., 2015). We may
assume that nanomaterials may create some toxicity due to inhalation through oral or
dermal routes (Renn and Roco, 2006; Xiao et al., 2015). This key concept of nanotoxicol-
ogy, including the significance of dose, dose rate, dose metric, and biokinetics, is very
essential in the coming decade. This will help us to know the characterization of critical
physicochemical properties of the used NPs, specifically surface properties that influ-
ence their biological/toxicological properties of the cell and biokinetics. If we know the
problems with the use of NPs in the environment, it will help us to solve them in future.
Applications of nanotechnology are already having an impact on a number of products
in the present decade. The nanometer range (generally below 100 nm) particles are spe-
cifically exploited with their functional properties. Thus, with new electronic, magnetic,
optical properties, catalytic activities, solubility, and transport properties, nanosubstances
execute a great role in nature in the third decade. Therefore, clear knowledge-based leg-
islation will establish concise guidelines for the use of nanomaterials.
20.11 Conclusion
Currently, with the advance of nanotechnology, there is a possibility of using nanosen-
sors to track the cold chain and thus make the storage system more efficient. The inser-
tion points for nanotechnology in sensing applications are many. Nanotechnology has the
potential to enable the vision of future sensor technology and sensing systems. The high
surface-to-volume ratio of NWs and other nonmaterials will lead to increased sensitivity
of the transducer in the sensor. Market potentials of nanotechnology in the energy conver-
sion sector will mainly arise in the fields of thin-layer solar cells and fuel cell technology.
Apart from potentially low production costs and a more flexible scalability, thin-layer solar
cells bear the advantage of more consistent performance—even at fluctuating tempera-
tures and suboptimum radiation conditions (angle of incidence, clouds). This opens up
new applications, such as flat roofs or extensive solar plants.
The surface biocides are agents intended to help maintain the hygienic condition
of the food contact surface by preventing or reducing microbial growth and helping
“cleanability.” There should be no preservative effect on the food. Surface biocides may
have a useful function in food-processing equipment. Since nanomaterials have a very
high surface area to mass ratio, materials such as nano–silver zinc oxide or magnesium
oxide may have an effective action as a surface biocide in food contamination. The effect
of polymerization or processing on the size or shape or surface chemistry of NPs has to
be developed for our survival. Now, it has been established that nanomaterials could
elevate the migration of nonnano-ingredients or could cause an undesirable reaction of
the product during the processing and fabrication of food-packaging materials, such as
in human physiology. Still, we do not clearly understand the impact of nanomaterials in
waste disposal streams. More research is needed to understand risks for the sake of sus-
tainable use of nanotechnology in different fields.
Acknowledgments
We thank Dr. H. Raja Naika, Department of Studies and Research in Environmental Science,
Bharat Ratna; Prof. C.N.R. Rao Block, Tumkur University, India; and Dr. K. Palanichelvam,
Department of Biotechnology, Kalasalingam University, Krishnankoil, Tamil Nadu, India,
for their encouragement in writing this article.
References
Ahamed, M., AlSalhi, M.S., and Siddiqui, M.K.J. 2010. Silver nanoparticle applications and human
health. Clinica Chimica Acta, 411(23), 1841–1848.
Akimoto, K., Ishizuka, S., Yanagita, M., Nawa, N., Paul, G.K. and Sakurai, T. 2006. Thin film deposi-
tion of Cu2O and application for solar cells. Solar Energy, 80, 715–722.
Araujo, A.C.V., Abreu, F., Silva, K.T., Bazylinski, D.A., and Lins, U. 2015. Magnetotactic bacteria as
potential sources of bioproducts. Marine Drugs, 13(1), 389–430.
Bantz, K.C., Meyer, A.F., Wittenberg, N.J., Im, H., Kurtuluş, Ö., Lee, S.H., and Haynes, C.L.
2011. Recent progress in SERS biosensing. Physical Chemistry Chemical Physics, 13(24),
11551–11567.
Becheri, A., Dürr, M., Nostro, P.L., and Baglioni, P. 2008. Synthesis and characterization of zinc oxide
nanoparticles: Application to textiles as UV-absorbers. Journal of Nanoparticle Research, 10(4),
679–689.
Bhattacharyya, A. 2009. Nanoparticles-from drug delivery to insect pest control. Akshar, 1(1), 1–7.
Bhattacharyya, A., Barik, B., Kundu, P., Mandal, D.N., Das, A., Sen, P., Rao, C.V., and Mandal, S.
2007. Bioactivity of nanoparticles and allelochemicals on stored grain pest-Sitophilus oryzae (L.)
(Coleoptera: Curculionidae). 27th Annual Session of the Academy of Environmental Biology and
National Symposium of Biomarkers of Environmental Problems, October 26–28, 2007, Jointly orga-
nized by Department of Zoology and Department of Environmental Sciences, Charan Singh
University, Meerut, Uttar Pradesh, India, 28pp.
Bhattacharyya, A., Chandrasekhar, R., Chandra, A.K., Epidi, T.T., and Prakasham, R.S. 2014.
Application of nanoparticles in sustainable agriculture: Its current status. International Book
Mission, Academic Publisher, Manhattan, KS, pp. 429–448.
Bhattacharyya, A., Datta, P.S., Chaudhuri, P., and Barik, B.R. 2011. Nanotechnology—A new frontier
for food security in socio economic development. Disaster, Risk and Vulnerability Conference
2011 School of Environmental Sciences, Mahatma Gandhi University, Kerala, India in association
with the Applied Geoinformatics for Society and Environment, Stuttgart, Germany, March 12–14,
2011, pp. 116–120.
Blakemore, R. 1975. Magnetotactic bacteria. Science, 190(4212), 377–379.
Cauerhff, A. and Castro, G.R. 2013. Bionanoparticles, a green nanochemistry approach. Electronic
Journal of Biotechnology, 16(3), 11–11.
Chatterjee, S., Bandyopadhyay, A., and Sarkar, K. 2011. Effect of iron oxide and gold nanoparticles on
bacterial growth leading towards biological application. Journal of Nanobiotechnology, 9(34), 1.
Daniel, M.C. and Astruc, D. 2004. Gold nanoparticles: Assembly, supramolecular chemistry, quan-
tum-size-related properties, and applications toward biology, catalysis, and nanotechnology.
Chemical Reviews, 104(1), 293–346.
Dhanasekaran, D., Latha, S., Saha, S., Thajuddin, N., and Panneerselvam, A. 2013. Extracellular bio-
synthesis, characterisation and in-vitro antibacterial potential of silver nanoparticles using
Agaricus bisporus. Journal of Experimental Nanoscience, 8(4), 579–588.
Ding, Y., Fan, F., Tian, Z., and Wang, Z.L. 2010. Atomic structure of Au−Pd bimetallic alloyed
nanoparticles. Journal of the American Chemical Society, 132(35), 12480–12486.
Dreher, K.L. 2004. Health and environmental impact of nanotechnology: Toxicological assessment
of manufactured nanoparticles. Toxicological Sciences, 77, 3–5.
Duncan, R. 2005. The dawning era of polymer therapeutics. Nature Reviews Drug Discovery, 2(5),
347–360.
Ezzat, A., Abdelhamid, A.O., Amin, A.I., El Azeem, A., Amal, S., Fouda, M.F.R., and Mohammed,
D.M. 2013. Biochemical studies on the effect of nano particles of some nutrients on apoptosis
modulation of breast cancer cells in experimental animals. Journal of Applied Sciences Research,
9(1), 658–665.
Feris, K., Otto, C., Tinker, J., Wingett, D., Punnoose, A., Thurber, A., and Pink, D. 2009. Electrostatic
interactions affect nanoparticle-mediated toxicity to gram-negative bacterium Pseudomonas
aeruginosa PAO1. Langmuir, 26(6), 4429–4436.
Friedman, A., Blecher, K., Sanchez, D., Tuckman-Vernon, C., Gialanella, P., Friedman, J.M.,
Martinez, L.R., and Nosanchuk, J.D. 2011. Susceptibility of gram-positive and -negative bacte-
ria to novel nitric oxide-releasing nanoparticle technology. Virulence, 2, 217–221.
Gajjar, P., Pettee, B., Britt, D.W., Huang, W., Johnson, W.P., and Anderson, A.J. 2009. Antimicrobial
activities of commercial nanoparticles against an environmental soil microbe, Pseudomonas
putida KT2440. Journal of Biological Engineering, 3(9), 1–13.
Gao, Y. and Cranston, R. 2008. Recent advances in antimicrobial treatments of textiles. Textile
Research Journal, 78(1), 60–72.
Gao, Y., Sarfraz, M.K., Clas, S.D., Roa, W., and Löbenberg, R. 2015. Hyaluronic acid-tocopherol suc-
cinate-based self-assembling micelles for targeted delivery of rifampicin to alveolar macro-
phages. Journal of Biomedical Nanotechnology, 11(8), 1312–1329.
Gopalakrishnan, K., Ramesh, C., Ragunathan, V., and Thamilselvan, M. 2012. Antibacterial
activity of Cu 2O nanoparticles on E. coli synthesized from tridax procumbens leaf extract
and surface coating with polyaniline. Digest Journal of Nanomaterials and Biostructures, 7,
833–839.
Gopinath, P.M., Narchonai, G., Dhanasekaran, D., Ranjani, A., and Thajuddin, N. 2015. Mycosynthesis,
characterization and antibacterial properties of AgNPs against multidrug resistant (MDR)
bacterial pathogens of female infertility cases. Asian Journal of Pharmaceutical Sciences, 10(2),
138–145.
Hamouda, T., Hayes, M., Cao, Z., Tonda, R., Johnson, K., Craig, W., Brisker, J., and Baker. J. 1999.
A novel surfactant nanoemulsion with broad-spectrum sporicidal activity against Bacillus
species. The Journal of Infectious Diseases, 180, 1939–1949.
Hetrick, E.M., Shin, J.H., Paul, H.S., and Schoenfisch, M.H. 2009. Anti-biofilm efficacy of nitric oxide-
releasing silica nanoparticles. Biomaterials, 30(14), 2782–2789.
Hu, Y., Chang, Y., Fei, P., Snyder, R.L., and Wang, Z.L. 2010. Designing the electric transport charac-
teristics of ZnO micro/nanowire devices by coupling piezoelectric and photoexcitation effects.
ACS Nano, 4(2), 1234–1240.
Jiang, W., Mashayekhi, H., and Xing, B. 2009. Bacterial toxicity comparison between nano-and
micro-scaled oxide particles. Environmental Pollution, 157(5), 1619–1625.
Jones, N., Ray, B., Ranjit, K.T., and Manna, A.C. 2008. Antibacterial activity of ZnO nanoparticle
suspensions on a broad spectrum of microorganisms. FEMS Microbiology Letters, 279(1), 71–76.
Juan, L., Zhimin, Z., Anchun, M., Lei, L., and Jingchao, Z. 2010. Deposition of silver nanoparticles on
titanium surface for antibacterial effect. International Journal of Nanomedicine, 5, 261.
Katzmann, E., Eibauer, M., Lin, W., Pan, Y., Plitzko, J.M., and Schüler, D. 2013. Analysis of magne-
tosome chains in magnetotactic bacteria by magnetic measurements and automated image
analysis of electron micrographs. Applied and Environmental Microbiology, 79(24), 7755–7762.
Kennedy, D.C., Orts-Gil, G., Lai, C.H., Müller, L., Haase, A., Luch, A., and Seeberger., P.H. 2014.
Carbohydrate functionalization of silver nanoparticles modulates cytotoxicity and cellular
uptake. Journal of Nanobiotechnology, 12, 59.
Khan, A.U. 2012. Medicine at nanoscale: A new horizon. International Journal of Nanomedicine, 7, 2997.
Khan, Z., Hussain, J.I., Kumar, S., Hashmi, A.A., and Malik, M.A. 2011. Silver nanoparticles: Green
route, stability and effect of additives. Journal of Biomaterials and Nanobiotechnology, 2(04), 390.
Kim, J.S., Kuk, E., Yu, K.N., Kim, J.H., Park, S.J., Lee, H.J., and Cho, M.H. 2007. Antimicrobial effects of
silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 3(1), 95–101.
Komeili, A., Li, Z., Newman, D.K., and Jensen, G.J. 2006. Magnetosomes are cell membrane invagi-
nations organized by the actin-like protein MamK. Science, 311(5758), 242–245.
Kumar, N., Shah, V., and Walker, V.K. 2011. Perturbation of an arctic soil microbial community by
metal nanoparticles. Journal of Hazardous Materials, 190(1), 816–822.
Kumari, M., Rajak, S., Singh, S.P., Kumari, S.I., Kumar, P.U., Murty, U.S., and Rahman, M.F. 2012.
Repeated oral dose toxicity of iron oxide nanoparticles: Biochemical and histopathological
alterations in different tissues of rats. Journal of Nanoscience and Nanotechnology, 12(3), 2149–2159.
Leid, J.G., Ditto, A.J., Knapp, A., Shah, P.N., Wright, B.D., Blust, R., and Cope, E.K. 2012. In vitro anti-
microbial studies of silver carbene complexes: Activity of free and nanoparticle carbene for-
mulations against clinical isolates of pathogenic bacteria. Journal of Antimicrobial Chemotherapy,
67(1), 138–148.
Li, N., Zhao, P., and Astruc, D. 2014. Anisotropic gold nanoparticles: Synthesis, properties, applica-
tions, and toxicity. Angewandte Chemie International Edition, 53(7), 1756–1789.
Li, Z. and Wang, Z.L. 2011. Air/liquid‐pressure and heartbeat‐driven flexible fiber nanogenerators
as a micro/nano‐power source or diagnostic sensor. Advanced Materials, 23(1), 84–89.
Liu, C.J., Burghaus, U., Besenbacher, F., and Wang, Z.L. 2010. Preparation and characterization of
nanomaterials for sustainable energy production. ACS Nano, 4(10), 5517–5526.
Maisoneuve, E. and Gerdes, K. 2014. Molecular mechanisms underlying bacterial persisters. Cell,
157(3):539–48.
Makwana, B.A., Vyas, D.J., Bhatt, K.D., Jain, V.K., and Agrawal, Y.K. 2014. Highly stable antibacte-
rial silver nanoparticles as selective fluorescent sensor for Fe3+ ions. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, 134, 73–80.
McCarthy, T.J., Zeelie, J.J., and Krause, D.J. 1992. The antimicrobial action of zinc ion/antioxidant
combinations. Journal of Clinical Pharmacy and Therapeutics, 17(1), 51–54.
Mironava, T., Hadjiargyrou, M., Simon, M., and Rafailovich, H. 2013. Gold nanoparticles cellular
toxicity and recovery: Adipose derived stromal cells. Nanotoxicology, 8, 1–13.
Mody, C. 2011. Instrumental Community: Probe Microscopy and the Path to Nanotechnology. MIT Press,
Cambridge, MA.
Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T., and Yacaman, M.J.
2005. The bactericidal effect of silver nanoparticles. Nanotechnology, 16(10), 2346.
Nair, S., Sasidharan, A., Rani, V.D., Menon, D., Nair, S., Manzoor, K., and Raina, S. 2009. Role of size
scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast can-
cer cells. Journal of Materials Science: Materials in Medicine, 20(1), 235–241.
Ng, K.W., Khoo, S.P., Heng, B.C., Setyawati, M.I., Tan, E.C., Zhao, X., and Loo, J.S. 2011. The role of the
tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanopar-
ticles. Biomaterials, 32(32), 8218–8225.
Novak, J.P. and Feldheim, D.L. 2000. Assembly of phenylacetylene-bridged silver and gold nanopar-
ticle arrays. Journal of the American Chemical Society, 122, 3979–3980.
Oberhack, M., Süssmuth, R., and Frank, H. 1987. Magnetotactic bacteria from freshwater. Zeitschrift
für Naturforschung, 42, 300–306.
Padmavathy, N. and Vijayaraghavan, R. 2008. Enhanced bioactivity of ZnO nanoparticles—An anti-
microbial study. Science and Technology of Advanced Materials, 9(3), 035004.
Pal, S., Tak, Y.K., and Song, J.M. 2007. Does the antibacterial activity of silver nanoparticles depend
on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli.
Applied and Environmental Microbiology, 73(6), 1712–1720.
Pan, C., Fang, Y., Wu, H., Ahmad, M., Luo, Z., Li, Q., and Zhu, J. 2010. Generating electricity from
biofluid with a nanowire‐based biofuel cell for self‐powered nanodevices. Advanced Materials,
22(47), 5388–5392.
Patil, H.B., Borse, S.V., Patil, D.R., Patil, U.K., and Patil, H.M. 2011. Synthesis of silver nanoparticles
by microbial method and their characterization. Archives of Physical Medicine and Rehabilitation,
2, 153–158.
Perozziello, G., Candeloro, P., Gentile, F., Nicastri, A., Perri, A., Coluccio, M.L., Adamo, A. et al. 2014.
Microfluidics and nanotechnology: Towards fully integrated analytical devices for the detec-
tion of cancer biomarkers. RSC Advances, 4(98), 55590–55598.
Qi, L., Xu, Z., Jiang, X., Hu, C., and Zou, X. 2004. Preparation and antibacterial activity of chitosan
nanoparticles. Carbohydrate Research, 339(16), 2693–2700.
Ramasami, A.K., Naika, H.R., Nagabhushana, H., Ramakrishnappa, T., Balakrishna, G.R., and
Nagaraju, G. 2015. Tapioca starch: An efficient fuel in gel-combustion synthesis of photocata-
lytically and anti-microbially active ZnO nanoparticles. Materials Characterization, 99, 266–276.
Reddy, K.M., Feris, K., Bell, J., Wingett, D.G., Hanley, C., and Punnoose, A. 2007. Selective toxicity of zinc
oxide nanoparticles to prokaryotic and eukaryotic systems. Applied Physics Letters, 90(21), 213902.
Renn, O. and Roco, M.C. 2006. Nanotechnology and the need for risk governance. Journal of
Nanoparticle Research, 8(2), 153–191.
Ruparelia, J.P., Chatterjee, A.K., Duttagupta, S.P., and Mukherji, S. 2008. Strain specificity in antimi-
crobial activity of silver and copper nanoparticles. Acta Biomaterialia, 4(3), 707–716.
Sagadevan, S., Savitha, S., and Preethi, R. 2014. Beneficial applications of nanoparticles in medical
field—A review. International Journal of PharmTech Research, 6(5):1712–1717.
Simon-Deckers, A., Loo, S., Mayne-L’hermite, M., Herlin-Boime, N., Menguy, N., Reynaud, C., and
Carrière, M. 2009. Size-, composition- and shape-dependent toxicological impact of metal oxide
nanoparticles and carbon nanotubes toward bacteria. Environmental Science and Technology,
43(21), 8423–8429.
Sinha, R., Karan, R., Sinha, A., and Khare, S.K. 2011. Interaction and nanotoxic effect of ZnO and
Ag nanoparticles on mesophilic and halophilic bacterial cells. Bioresource Technology, 102(2),
1516–1520.
Sondi, I. and Salopek-Sondi, B. 2004. Silver nanoparticles as antimicrobial agent: A case study on E.
coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science, 275(1), 177–182.
Song, F., Li, X., Wang, Q., Liao, L., and Zhang, C. 2015. Nanocomposite hydrogels and their applica-
tions in drug delivery and tissue engineering. Journal of Biomedical Nanotechnology, 11(1), 40–52.
Stepanov, A.L., Xiao, X., Ren, F., Kavetskyy, T. and Osin, Y.N. 2013. Catalytic and Biological sensitiv-
ity of TiO2 and SiO2 matrices with silver nanoparticles created by ion implantation: A review.
Reviews on Advanced Materials Science, 34, 107–122.
Subbiahdoss, G., Sharifi, S., Grijpma, D.W., Laurent, S., van der Mei, H.C., Mahmoudi, M., and
Busscher, H.J. 2012. Magnetic targeting of surface-modified superparamagnetic iron oxide
nanoparticles yields antibacterial efficacy against biofilms of gentamicin-resistant staphylo-
cocci. Acta Biomaterialia, 8(6), 2047–2055.
Taylor, E.N. and Webster, T.J. 2009. The use of superparamagnetic nanoparticles for prosthetic bio-
film prevention. International Journal of Nanomedicine, 4, 145.
Thirumurugan, T. and Kaur, K. 2013. Biological synthesis and characterization of gold nanoparticles
from pomegranate. International Journal of Future Biotechnology, 2(2), 1–11.
Thukkaram, M., Sitaram, S., and Subbiahdoss, G. 2014. Antibacterial efficacy of iron-oxide nanopar-
ticles against biofilms on different biomaterial surfaces. International Journal of Biomaterials.
http://dx.doi.org/10.1155/2014/716080.
Toumey, C. 2010. 35 atoms that changed the nanoworld. Nature Nanotechnology, 5, 239–241.
Tran, N., Mir, A., Mallik, D., Sinha, A., Nayar, S., and Webster, T.J. 2010. Bactericidal effect of iron
oxide nanoparticles on Staphylococcus aureus. International Journal of Nanomedicine, 5, 277.
Tsuang, Y.H., Sun, J.S., Huang, Y.C., Lu, C.H., Chang, W.H.S., and Wang, C.C. 2008. Studies of photo-
killing of bacteria using titanium dioxide nanoparticles. Artificial Organs, 32(2), 167–174.
Vasantharaja, D., Ramalingam, V., and Reddy, G.A. 2015. Oral toxic exposure of titanium dioxide
nanoparticles on serum biochemical changes in adult male Wistar rats. Biomedical Research, 3, 4.
White, B.R., Stackhouse, B.T., and Holcombe, J.A. 2009. Magnetic γ-Fe2O3 nanoparticles coated with
poly-l-cysteine for chelation of As (III), Cu (II), Cd (II), Ni (II), Pb (II) and Zn (II). Journal of
Hazardous Materials, 161(2), 848–853.
Wiley, B., Sun, Y., Mayers, B., and Xia, Y. 2005. Shape-controlled synthesis of metal nanostructures:
The case of silver. Chemistry: A European Journal, 11, 454–463.
Wu, B., Huang, R., Sahu, M., Feng, X., Biswas, P., and Tang, Y.J. 2010. Bacterial responses to Cu-doped
TiO2 nanoparticles. Science of the Total Environment, 408(7), 1755–1758.
Xiao, S., Castro, R., Rodrigues, J., Shi, X., and Tomás, H. 2015. PAMAM dendrimer/pDNA functional-
ized-magnetic iron oxide nanoparticles for gene delivery. Journal of Biomedical Nanotechnology,
11(8), 1370–1384.
Xu, K., Huang, J., Ye, Z., Ying, Y., and Li, Y. 2009.Recent development of nano-materials used in DNA
biosensors. Sensors, 9, 5534–5557.
Xu, S., Shen, Y., Ding, Y., and Wang, Z.L. 2010. Growth and transfer of monolithic horizontal
ZnO nanowire superstructures onto flexible substrates. Advanced Functional Materials, 20(9),
1493–1497.
Yadav, A., Prasad, V., Kathe, A.A., Raj, S., Yadav, D., Sundaramoorthy, C., and Vigneshwaran, N.
2006. Functional finishing in cotton fabrics using zinc oxide nanoparticles. Bulletin of Materials
Science, 29(6), 641–645.
Yamanaka, M., Hara, K., and Kudo, J. 2005. Bactericidal actions of a silver ion solution on Escherichia
coli, studied by energy-filtering transmission electron microscopy and proteomic analysis.
Applied and Environmental Microbiology, 71, 7589–7593.
Yang, Q., Guo, X., Wang, W., Zhang, Y., Xu, S., Lien, D.H., and Wang, Z.L. 2010. Enhancing sensitivity
of a single ZnO micro-/nanowire photodetector by piezo-phototronic effect. ACS Nano, 4(10),
6285–6291.
Yin, H.T., Zhang, D.G., Wu, X.L., Huang, X.E., and Chen, G. 2013. In vivo evaluation of curcumin-
loaded nanoparticles in a A549 xenograft mice model. Asian Pacific Journal of Cancer Prevention,
14(1), 409–412.
Yoon, K.Y., Byeon, J.H., Park, J.H., and Hwang, J. 2007. Susceptibility constants of Escherichia coli
and Bacillus subtilis to silver and copper nanoparticles. Science of the Total Environment, 373(2),
572–575.
chapter twenty-one
Platinum-based anticancer therapeutics

and their mechanistic aspects
An overview
Bhaskar Biswas
Contents
21.1 Introduction......................................................................................................................... 419
21.2 Role of metal ions in biological system............................................................................ 420
21.2.1 Transition metals in biology.................................................................................. 421
21.3 Platinum drugs in cancer therapy....................................................................................423
21.3.1 Mononuclear platinum drugs...............................................................................423
21.3.2 Di-, tri-, and tetranuclear platinum drugs...........................................................425
21.3.3 Hetero-dinuclear platinum drugs........................................................................ 426
21.3.4 Polymeric platinum drugs..................................................................................... 426
21.3.5 Pt(IV) compounds as anticancer agents.............................................................. 426
21.4 Mechanistic aspects for the platinum compounds as anticancer drugs.................... 428
21.5 Conclusion........................................................................................................................... 429
Acknowledgments.......................................................................................................................430
References......................................................................................................................................430
21.1 Introduction
A significantly mounting interest in the design of synthetic metal compounds as drugs and
diagnostic agents is currently experimental in the area of scientific investigation, appro-
priately termed medicinal inorganic chemistry. Empirical evidence for the effectiveness of
metal-based therapeutics has existed for centuries, and the use of metals and metal com-
plexes in medicine dates back millennia. Investigations in this area focus mostly on the
speciation of metals in biological media based on possible interactions of these metal ions
with diverse microbials and biomolecules, in an effort to contribute to future development
of new antimicrobials, therapeutics, or diagnostic agents. Metallopharmaceuticals used
as anticancer agents, metal-mediated antibiotics, antibacterials, antifungals, antivirals,
antiparasitics, antiarthritics, antidiabetics, and radio-sensitizing agents appear in thera-
peutic medicinal inorganic chemistry (Thomson and Orvig, 2003; Reedijk, 2009; Griffith
et al., 2012).
History shows that metal-based drugs and remedies have been known and used since
very ancient times. For example, silver was employed in the treatment of wounds and
ulcers according to the Greek physician Hippocrates, but its antimicrobial properties had
419
probably been recognized long before because it was used to make vessels for storing
liquids in pure form. The ancient Egyptians also knew how to sterilize water with cop-
per. The medical use of gold can be dated back to 2500 BC in China. However, the new
era of metal-based medicine started almost five decades ago when cisplatin was shown to
inhibit cellular division in Escherichia coli, thereby leading to the first studies of its antitu-
mor activity in rats and its assessment as one of the most powerful drugs for use against
different types of cancer, although many other novel metal-based drugs are promising
and they are attracting growing attention in modern clinical medicine (Figure 21.1). Gold
salts and arsenic compounds have been in use for decades in the treatment of rheuma-
toid arthritis and syphilis, respectively, but studies of cisplatin have definitely shifted the
attention of researchers to the pool of transition “heavy” metals as potential therapeutic
agents (Rosenberg and Vancamp, 1969; Lippard, 1982; Quiroga et al., 2012).
This review will focus on the platinum-based therapeutics with their potential bioac-
tivity and explore the evidences of metal complexes–protein binding relevant to the drug/
diagnostic agent’s mechanisms of action.
21.2 Role of metal ions in biological system

Bioinorganic chemistry is an interdisciplinary subject at the interface of inorganic chemistry
and biology/biochemistry. Medicinal inorganic chemistry includes the study of both non-
essential and essential elements with applications to diagnosis and therapies. Bioinorganic
chemistry is an extremely dynamic field, and the functional roles of selected biological inor-
ganic elements include charge balance and electrolytic conductivity (Na, K, Cl); structure
OAc
O
O O O O
V AcO
O S Au PEt3
O O AcO N
OAc O
Fe
Bis(maltaloto)oxovanadium(IV) Auranofin Ferrocifen

antidiabetic Antiarthritic Experimental anticancer
OMe
OMe
O
MeO N
P O N N
C
O– C C
Tc
N N
N C
O C C OMe
Gd N N
O N
O O
O O
O
OH2 O
O MeO
OMe
Gd based MRI agent Tc based SPECT imaging
Figure 21.1 Some metal-based potential therapeutics in clinical medicine.

Chapter twenty-one: Platinum-based anticancer therapeutics and their mechanistic aspects 421
and templating (Ca, Zn, Si, S); signaling (Ca, B, NO); Bronsted acid–base buffering (P, Si,
CO); Lewis acid–base catalysis (Zn, Fe, Ni, Mn); electron transfer (Fe, Cu); group transfer
such as CH3, O, and S (V, Fe, Co, Ni, Cu, Mo, W); redox catalysis (V, Mn, Fe, Co, Ni, Cu, W,
S, Se); energy storage (H, P, S, Na, K, Fe); and biomineralization (Ca, Mg, Fe, Si, Sr, Cu, P)
(Gielen et al., 2005; Norman and Hambley, 2011; Klein and Hambley, 2009).
Lithium carbonate is a major drug for the treatment of manic episodes and for mainte-
nance therapy in bipolar patients. In multicellular organisms, sodium and calcium are extra-
cellular, while potassium and magnesium are largely intracellular (Thompson, 2011). Calcium
and magnesium are often metal activators in proteins to which they bind with relatively low
affinity. Under appropriate circumstances, these metal ions induce conformational changes
in the protein upon binding, and in doing so, they may transmit a signal, for example, the
firing of neurons by rapid influx of sodium ions across a cell membrane or the regulation
of intracellular functions by calcium-binding proteins such as calmodulin. Bone and teeth
are made from calcium phosphate in the form of a mineral hydroxyapatite. Calcium carbon-
ate biominerals such as calcite and aragonite are used for structural support (Alessio, 2011).
Nerve cells depend on potassium. Without sodium, our cells can not get the nutrients they
need to survive. Sodium also allows our bodies to balance the appropriate amount of water
in our blood. Potassium chloride is used to prevent or to treat low blood levels of potassium
(hypokalemia). Potassium gluconate is needed for normal functioning of cells, nerve con-
duction, muscle contraction, kidney function, and acid–base balance. Losartan (1,2-n-butyl-
4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]-imidazole-5-methanol monopotassium salt)
is a highly selective, orally active, nonpeptide angiotensin II receptor antagonist indicated
for the treatment of hypertension (Ochiai, 2008). Ca maintains the cell shape and integrity
of membranes, exo- and endocytisis, mytosis, and muscle contraction, causing changes in
some proteins/enzymes to modify their function. Calcium is needed by the body for healthy
bones, muscles, nervous system, and heart. Calcium carbonate also is used as an antacid to
relieve heartburn, acid indigestion, and upset stomach. Calcium citrate, calcium chloride,
and calcium gluconate are used to prevent and to treat calcium deficiencies. Mg is needed
for the proper growth, formation, and function of our bones and muscles. Mg also controls
insulin levels in blood and even helps to prevent depression (Noh et al., 2006).
21.2.1 Transition metals in biology

Scientists have shown significant progress in utilization of transition metal complexes as
drugs to treat several human diseases like carcinomas, lymphomas, infection control, anti-
inflammatory, diabetes, and neurological disorders. Transition metals exhibit different oxi-
dation states and can interact with a number of negatively charged molecules. This activity
of transition metals has started the development of metal-based drugs with promising
pharmacological application and may offer unique therapeutic opportunities (Gielen and
Tiekink, 2005). Vanadium compounds at pharmacological doses display relevant biological
actions such as insulin and growth factor mimetic or enhancing effects, as well as osteogenic
and cardioprotective activity. On the other hand, depending on the nature of compounds
and their concentrations, toxicological actions and adverse side effects may also be shown.
The current knowledge and new advances on in vitro and in vivo effects of inorganic and
organically chelated vanadium compounds have been reported (Srivastava and Mehdi,
2005). Vanadium is involved in helping the body convert some foods into energy. It has also
been suggested that diabetics may benefit from vanadium when trying to stabilize blood
sugar levels. Chromium is a mineral that is essential to humans. Chromium helps regulate
sugar levels by interacting with insulin. Chromium picolinate has been used in alternative
medicine as an aid to lowering cholesterol or improving the body’s use of glucose (sugar).
However, chromium supplements are not approved by the FDA for these uses. Manganese
is an essential nutrient involved in many chemical processes in the body, including process-
ing of cholesterol, carbohydrates, and protein. It might also be involved in bone formation.
A number of iron compounds have been found medically useful. For example, ferrous glu-
conate, Fe(C6H11O7)2·2H2O, and ferric pyrophosphate, Fe4(P2O7)xH2O, are among the com-
pounds frequently used to treat anemia. Various ferric salts, which act as coagulants, are
applied. Iron compounds include hemoglobin, which keeps our blood red. Other essential
iron compounds include myoglobin. Iron atoms help join organic molecules (derived from
quinoxaline), forming compounds that act as bactericides (killing bacteria) or bacteriostatic
agents (preventing bacteria from reproducing) (Tarallo et al., 2010). Ferrous sulfate pro-
vides the iron needed by the body to produce red blood cells. Ferrous sulfate comes as
regular, coated, and extended-release (long-acting) tablets; regular and extended-release
capsules; and oral liquid (syrup, drops, and elixir) to take by mouth. Cobalt is essential
in small amount for proper nutrition. Cobalt contained in vitamin B12 is important in pro-
tein formation and DNA regulation. Cobalt-60, a radioactive isotope, is used as commercial
source of high-energy radiation in medicine to destroy cancerous tissue. Cobalt-57 is also
used in medicine. It can be used to work out how much vitamin B12 is being taken in by
the human body. Cobalt-containing drugs are used as cyanide antidote. Cyanide ions will
bind to cobalt, which can be supplied in the form of either hydroxocobalamin or dicobalt
edetate. Cyanide will also bind to methemoglobin formed after administration of sodium
nitrite. Historic uses of copper compounds in medicine are available. Thyroid and immune
system health are crucially dependent on copper, and copper deficiency is the most impor-
tant factor in the development of hyperthyroidism. Women need more copper than men
because copper is required for the production of the enzymes that convert progesterone
into estrogens. However, more zinc is required by men because zinc helps in the forma-
tion of enzyme that converts progesterone into testosterone (Sorenson et al., 1985). Many
people wear copper bands to help them with inflammatory disease, such as arthritis. Much
of our “dietary” copper actually comes from copper pipes, utensils, and cookware. Copper
is important as an electron donor in various biological reactions. Potential benefits of copper
gluconate include helping reduce high cholesterol levels in humans, osteoporosis, wound
healing, cardiac arrhythmia, hypoglycemia, peripheral vascular disease, osteoarthritis, and
rheumatoid arthritis. Evolution has kept stores of copper and iron in excess during the
reproductive years because they are so vital to life. But the oxidant damage from these
excess stores of metals builds up as we age, and natural selection ceases to act after about
age 50 since diseases after that do not contribute to reproductive fitness. Diseases of aging
such as Alzheimer’s disease, other neurodegenerative diseases, arteriosclerosis, diabetes
mellitus, and others may all be contributed to by excess copper and iron. Excess copper
along with high-fat diet leads to cognition loss at over three times the normal rate. Inorganic
copper in drinking water and in supplements is handled differently than food copper and is
therefore more toxic (Dabrowiak, 2010). Zinc is considered to be one of the most important
elements of a healthy immune system and is also needed for the growth and repair of tis-
sues throughout our bodies. Zinc oxide topically applied to the skin is used to treat diaper
rash, minor burns, severely chapped skin, or other minor skin irritations. Zinc–amino acid
chelate provides better absorption (60%–70%), better bioavailability, and better tolerance
than zinc salts (Navarro et al., 2010). Gallium mimics ferric iron. Gallium, in the form of
intravenously administered gallium nitrate (Ganite®), was approved by the U.S. FDA in
1991 for the treatment of cancer-related hypercalcemia, a disorder that occurs in about 10%–
20% of cancer patients. Molybdenum is found in all tissues of the human body but tends
to be most concentrated in the liver, kidneys, skin, and bones. Molybdenum is important
for transforming sulfur into a usable form in humans. It is required for the proper function
of several chemicals in the human body. Metal ions and protein interaction is important
regarding binding, stability, and folding. Fe, Cu, and Zn are strongly associated with pro-
teins and form the so-called metalloproteins. For example, ferritin stores iron in the body.
Metal cofactors can help catalyze unique chemical reactions and perform specific physi-
ological functions. Fe3+/Fe2+ and Cu2+/Cu+ redox couples play critical roles as cofactors for
electron transfer reactions in the catalysis of redox reactions. Some metal ions found deeply
buried within proteins interact with the protein and help insure the optimal protein struc-
ture and contribute to the stability and appropriate acid–base behavior necessary for the
physiological function. For example, the Zn2+ ions in Zn fingers are necessary for the adop-
tion of the proper shape of the protein, which allows it to interact with DNA (Tamasi, 2010).
21.3 Platinum drugs in cancer therapy

As cancer remains a major killer in the developed world, a broad spectrum of novel and
exciting approaches are being developed and tested. The importance of metal compounds
in medicine is undisputed, which can be judged by their use in the treatment of various
diseases. In terms of antitumor activity, a wide range of compounds of both transition
metal and main group elements have been investigated for efficacy. The existence of a
relationship between cancer and metals is widely acknowledged by researchers. Synthetic
metal complexes appear to provide a rich platform for the design of novel anticancer drugs.
The documented history of noble metal-based drugs started with the chance discov-
ery of cisplatin (cis-diamminedichloroplatinum(II)) (Rosenberg et al., 1965) as one of the
most powerful chemotherapeutic agents against ovarian and testicular cancers, although
gold salts had been tested against tuberculosis and were employed as antirheumatics in
1929 (Chen, 2006). Since then, a vast library of metal complexes have been synthesized
and applied in the p harmacological field, mostly as anticancer agents, but also as anti-
inflammatory, antibacterial, antirheumatic, and antimalarial drugs. In particular, they
have many applications against tumors, which are based mainly on the strong interactions
between metals and DNA. Cisplatin is routinely used for the treatment of testicular and
ovarian cancers and is increasingly used against other tumors, such as cervical, bladder,
and head/neck tumors (Kelland, 2007).
21.3.1 Mononuclear platinum drugs

Cisplatin contains a square-planar platinum(II) center coordinated to two ammonia
ligands and two chloride ligands with a cis-ligand conformation. Its activity was discov-
ered by chance in an experiment looking at the effect of electric fields on the growth of bac-
teria and using platinum electrodes. It is a testament to the tenacity of Rosenberg and his
coworkers that the active compound was identified and the chance observation led to such
a powerful drug (Rosenberg, 1999). Other few related mononuclear platinum complexes
such as carboplatin, nedaplatin, lobaplatin, and oxaliplatin are among the most widely
used cancer therapeutic agents (Figure 21.2). Among the platinum drugs, cisplatin was
considered as first-generation anticancer drug, while changing of chlorine atoms by ligand
produced second-generation anticancer drug, and when ammonium ligands are replaced
by different ligands, third-generation drugs are produced.
While cisplatin is among the most effective anticancer agents in the armory of drugs
available to cancer clinicians, this broad-spectrum cytotoxic is not without its drawbacks
O O
H3N CI H3N O H 3N O
Pt Pt Pt
H 3N CI H 3N O H3N O
O
Cisplatin (1st generation) Carboplatin (2nd generation) Nedaplatin (2nd generation)
H2 O
N O
O O
O H2N
Pt Pt
N O O N
O H2 O
H2
O
Oxaliplatin (3rd generation) Heptaplatin (3rd generation)
O OAc
NH2 O H3N CI
Pt Pt CI
N
NH2 O H 2N CI
CH3 Pt
OAc
L
C6H11 CI
Lobaplatin (3rd generation) Satraplatin (oral drug) Transplatin (L = py, NH3, dmso)
Figure 21.2 Structures of the mononuclear platinum drugs.
(Lippert, 1999). Side effects include nephrotoxicity, nausea, vomiting, and loss of sensation
in the extremities (though not the hair loss associated with other chemotherapy agents).
These are thought to arise through a combination of the nonspecificity of the drug and
resulting damage in tissues other than the tumor and platination of the sulfur residues
on proteins by the soft platinum(II) center. The change of leaving group does reduce the
activity of the agent somewhat, however. While it is as effective in ovarian cancer, it is less
potent against testicular, head, and neck cancers. Correspondingly, the side effects are less
severe. Consequently, cisplatin has tended to remain the agent of choice, with carboplatin
used when there is a clinical need to minimize the platinum drug side effects because of
other medical conditions. Alongside cisplatin and carboplatin, three other very similar
drugs (Figure 21.2) have appeared, which have been approved for use in specific coun-
tries: nedaplatin (Japan; Shionogi and Co. Ltd.), heptaplatin (South Korea; SK Pharma),
and lobaplatin (China). Of these, nedaplatin combines the {cis-Pt(NH3)2} active fragment
with a different bidentate leaving group (and thus is a direct analog of cisplatin and carbo-
platin), while heptaplatin and lobaplatin link the amines into a bidentate ligand structure
and use a dicarboxylate leaving group. Although, no dramatic clinical benefits have been
described for these drugs over cisplatin.
Early on, in the studies of cisplatin, it was recognized that the trans-isomer (trans-
platin) was inactive, and thereafter, the need for a cis-geometry at platinum rapidly
became a dogma. However, this dogma (as the other design rules) has more recently been
shown to be invalid, and three distinct classes of trans-compounds have shown to pos-
sess anticancer activity (Natile and Coluccia, 2001): trans-compounds containing pyridine
ligands (developed by Farrell et al., 1989), trans-compounds containing an alkylamine and
an isopropylamine (Montero et al., 1999), and trans-compounds containing iminoether
ligands (developed by Coluccia et al., 1993) (Figure 21.2). These three classes of agents show
potencies similar to that of cisplatin and, perhaps more importantly, are active against cis-
platin-resistant cell lines. The DNA lesions formed by a trans-platinum agent will be inher-
ently different from those formed by a cis-agent. Indeed, these trans-agents preferentially
form monofunctional adducts with the DNA or interstrand cross-links, rather than the
1,2-intrastrand cross-link preferred by cisplatin. Thus, their molecular-level interactions
with DNA are different, and hence their activity profiles differ. Despite their promising
activity, representatives of these trans-platinum(II) classes have not been evaluated in the
clinic. Although these platinum(II) cytotoxics are fairly nonspecific in their action and
have been in the clinic for three decades, their continuing importance (and the size of the
market) is illustrated by the fact that at least seven further platinum drugs are currently in
commercial development: satraplatin (GPC Biotech and Pharmion), miriplatin (Dainippon
Sumitomo Pharma and Bristol-Myers K.K.), prolindac (Access Pharmaceuticals), BP-C1
(Meabco), cisplatin lipid complex (Transave), aroplatin (Antigenics), and picoplatin
(Poinard) (Kelland, 2007).
21.3.2 Di-, tri-, and tetranuclear platinum drugs

The most dramatic example of platinum(II) agents that break the traditional design rules
are Farrell’s di-, tri-, and tetranuclear platinum compounds (Figure 21.3) in which the metal
centers are linked by flexible diamine chains (Farrell, 2004). The first ideas to add a second
2+ 4+
H2 H2 NH3
N CH2 N NH3 NH3 H2 H2
H3N n N N Pt CI
Pt Pt CI Pt N N
CI NH3 H3N CI H2 H2 NH3
NH3
(A) (B)
(a)
4+
NH3 NH3
NH3
H2N Pt CI
H2N Pt NH2
CI Pt NH2
NH3
NH3 n
NH3 n
(b)
H3N CI 4+
CI NH3
Pt
Pt N
H2 NH3
H3N N
H2 N N H2
N NH3
H 3N H2
N Pt
Pt
H3N CI
CI NH3
(c)
Figure 21.3 (a) Two dinuclear Pt cations (A and B), n = 6; (b) trinuclear Pt cation, n = 5; and (c) tetra-
nuclear, dendritic-type platinum cation.
metal in an anticancer drug were developed primarily by Farrell et al. (2011). In fact, the
observation that nucleobases and nucleotides have more than one binding site already
led to the early suggestion of taking an excess Pt in such binding reactions. The work of
Lippert’s group has subsequently shown that such multiple binding may occur on a rela-
tively large scale (Lippert, 2000), although it seems unlikely that such binding would occur
under physiological conditions. Therefore, a search to chemically linked metal ions, by
bridging ligands with kinetically stable M–L bonds, is required to study such compounds.
The first dinuclear Pt compounds with flexible linkers from the Farrell group showed
a promising activity (Farrell et al., 1995), and quite interesting binding with DNA has been
observed, including hairpin folding after binding (Mellish et al., 1997). The success of the
dinuclear compounds was soon followed by the trinuclear species, with bifunctional DNA
binding (Kabolizadeh et al., 2007). The DNA binding of such compounds is quite interest-
ing and can span long distances, as shown by advanced NMR studies (Zhang et al., 2008).
21.3.3 Hetero-dinuclear platinum drugs

Recently, it is seen that different metal ions with flexible linkers and also a group of dinu-
clear Pt(II) compounds with rigid linkers have been employed as clinical antitumor agent,
and structures are presented in Figure 21.4. Realizing that Cu-phenanthroline compounds
can cut DNA in an oxidative way has combined Cu-phenanthroline with several Pt-amines
using a variety of spacers, primarily aiming for bifunctionality (de Hoog et al., 2007, 2008;
Ozalp‐Yaman et al., 2008). In several cases, a high activity was found, and many of the
newly prepared compounds were shown to be effective DNA cutting agents. Replacing
the Cu part by a fluorescent group in such compounds with relatively flexible spacers had
provided strong evidence for the pathway in the cells of Pt compounds (Molenaar et al.,
2000). However, changing the Cu–Ru, as studied by Schilden et al. (2004) (Figure 21.4), did
not result in any significant anticancer activity, despite DNA binding taking place.
21.3.4 Polymeric platinum drugs

The approach of binding the Pt species for a short time to a polymer (chemically) has been
known for some years, and some applications are close to clinical use. As tumors may
be hyperpermeable toward macromolecules as a result of compromised vasculature, this
“enhanced permeability and retention effect” (the so-called EPR effect) may result in an
increased drug concentration within the tumor tissue (Taube, 1952). This is possible when
the therapeutic agent is coupled to a macromolecular carrier or may be packed inside a
nanosized particle, which after endocytosis will result in drug uptake into the tumor cells
(Figure 21.5). The well-known acrylamide-based drug AP5280 with a peptide spacer is
given for illustration (Bouma et al., 2002; van Zutphen and Reedijk et al., 2005).
21.3.5 Pt(IV) compounds as anticancer agents

Pt(IV) compounds activated by visible light Pt(IV) complexes are normally less cytotoxic
than their divalent counterparts, so they are considered to be prodrugs and they are assumed
to provide Pt(II)-active species via a mechanism known as “activation by r eduction.” New
and interesting Pt(IV) prodrugs include those activated by light, which generally have a
trans-configuration, and they bear two amines (also mixed) (Bednarski et al., 2013; Cubo
et al., 2010), two imines (Cubo et al., 2010), two azido ligands (Ruhayel et al., 2011), or a mixed
diazido-amine system (Zhao et al., 2013). They are normally inactive or slightly active in the
X
H 3N N N NH3
Pt Pt
OH
H3N NH3
[cis-Pt(NH2R)2]2(μ-OH)(μ-azolate)]2+ X = CH or N
(a)
1+
N
NH2
N O N
CI Cu
N Pt
O H
N n CI CI
N
Heterodinuclear Pt–Cu compound; n = 1– 4

3+
N N
N
N Ru N O O O N Pt CI
N N
N
Heterodinuclear Pt–Ru compound

(b)
Figure 21.4 (a) Homo- and (b) hetero-dinuclear cationic Pt drugs with flexible and rigid linkers.
ONa O
O O
O
CH2 O
H H N Pt
H 3C N N NH3
N N
H H H3N
O O O m
CH2 O
H3C
N
H
OH n
Figure 21.5 Polymeric Pt drug containing amide spacer [AP-5280; n > m].
dark, but they are selectively activated under UV and/or visible light. Their efficiency is
increased by replacing one or two NH3 ligands with pyridine, which can reach up to 50–65
times that of cisplatin when measured in the same conditions (Zhao et al., 2013).
21.4 Mechanistic aspects for the platinum

compounds as anticancer drugs
Differences in coordination geometry, binding preferences according to the hard and soft
acids and bases principle, important redox activity, kinetics of ligand exchange reactions,
or even the simple capacity of replacement of essential metals form the chemical basis for
a diversity of pharmacologically relevant interactions with biomolecules (Kostova, 2010).
The metal, its oxidation state, the number and types of coordinated ligands, and the coor-
dination geometry of the complexes can provide a variety of properties. On the other side,
the ligands not only control the reactivity of the metal but also play critical roles in deter-
mining the nature of interactions involved in the recognition of biological target sites such
as DNA, enzymes, and protein receptors. These variables provide enormous potential
diversity for the design of metallodrugs (Chaires and Waring, 2001; Lippard, 1982).
The mode of action is now widely accepted to be through the drug’s interaction with
DNA (Barton, 1994). The compound is administered by injection into the bloodstream and
is believed to remain in its neutral state until after it crosses the cell membrane where one
or both chlorides are displaced by aqua ligands (the chloride concentration being lower
inside than outside the cell) affording cationic compounds. These cationic aqua derivatives
react with the bases on DNA, most commonly with the N7 of purine bases (with guanine
favored over adenine), which displace the aqua/chlorido ligands. A bifunctional adduct
is formed between the {cis-Pt(NH3)2} unit and two adjacent bases on the same strand (the
1,2-intrastrand GG adduct accounts for >70% of all adducts formed; 1,2-intrastrand AG
adducts are the next most common at around 20% of all lesions; and 1,3-adducts and
monoadducts are much less common). The platinum center is located in the DNA major
groove, and the effect of the platinum coordination to two adjacent bases is to bend (kink)
the DNA by around 45°, toward the site of platination (Figure 21.6) (Reedijk, 2011). This
bent DNA structure is then recognized by nuclear high-mobility group (HMG) proteins
that bind and are believed to protect the lesion from DNA repair.
Oxaliplatin and some other second- and third-generation platinum drugs are active
against some cisplatin-resistant cancers. The predominant DNA adducts formed by oxali-
platin are 1,2-intrastrand GG adducts analogous to those formed by cisplatin. The DNA
lesions formed by a trans-platinum agent will be inherently different from those formed
by a cis-agent. Indeed, these trans-agents preferentially form monofunctional adducts with
H3N
H3N
Pt
H3N N
Pt N
H3N Pt
H3N
H3N
Figure 21.6 Distortion of DNA after binding with cisplatin and dinuclear Pt compounds.
OH
H2 H2
N N3 N OH
Pt Pt
N N3 N OH
H2 2N3 3N2 H2
OH
Figure 21.7 Photochemical activation on Pt drug.
the DNA or interstrand cross-links, rather than the 1,2-intrastrand cross-link preferred by
cisplatin (Farrer et al., 2009). Thus, their molecular-level interactions with DNA are differ-
ent, and hence their activity profiles differ. Despite their promising activity, representa-
tives of these trans-platinum(II) classes have not been evaluated in the clinic.
The most dramatic example of platinum(II) agents that break the traditional design
rules are di-, tri-, and tetranuclear platinum compounds. These platinum complexes
mainly form bifunctional long-range DNA adducts (both inter- and intrastrand cross-
links). In addition, a DNA conformational change is induced from a right-handed (B) to a
left-handed (Z) DNA double helix. This induction of Z-DNA is irreversible and is associ-
ated with the cross-linking (Farrell et al., 1995).
With close inspection on homo- and hetero-dinuclear platinum drugs, it is seen that
upon loss of the leaving OH group, each Pt would be able to bind at a guanine-N7, which
would result in a very small kink of double-stranded DNA when the two G bases would be
adjacent. The DNA binding of these dinuclear rigid compounds was as expected, result-
ing in a very small distortion of the helix only (Zhang et al., 2008), and the activity of the
compounds in several cancer cell lines was found to be an order of magnitude better than
that of cisplatin (de Hoog et al., 2007). Realizing that Cu-phenanthroline compounds can
cut DNA in an oxidative way, de Hoog has combined Cu-phenanthroline with several
Pt-amines using a variety of spacers, primarily aiming for bifunctionality. In several cases,
a high activity was found, and many of the newly prepared compounds were shown to
be effective DNA cutting agents. Changing the Cu–Ru, as studied by van der Schilden
et al. (2004), did not result in any significant anticancer activity, despite DNA binding
taking place.
It has been demonstrated that light at the appropriate wavelength will cause the
dissociation of one or more ligands, thereby giving different active Pt(II) photoprod-
ucts, but rarely Pt(IV) species (Figure 21.7). The formation of a cytotoxic azydil radical
has also been suggested for complexes containing the azide ion. Their mechanisms
of action are still under study, but DNA appears to be the target involved most often,
where its damage cannot be recognized by HMGB1 protein, in contrast to cisplatin-type
lesions (Zhao et al., 2013). Cell death via nonapoptotic pathways has also been recog-
nized (Hambley et al., 2009).
21.5 Conclusion
The action of metal complexes in living organisms are expected to differ, in general, from
the action of nonmetal containing agents and may offer unique research, diagnostic, or
therapeutic opportunities. The key to the continuing rapid evolution of the field now lies
in its integration with the emerging postgenomic knowledge and technologies from fields,
including systems biology, genomics, proteomics, and structural biology. Also underde-
veloped, but less directly related to DNA binding, is the control of the toxic side effects;
development of the coordination chemistry of Pt compounds with rescue and protective
agents (usually S-donor ligands) and especially the reactions of these compounds with
other cellular components and their cell wall transport needs serious attention. More
directly related to DNA binding of metal compounds is the migration of Pt units along the
DNA chain. Finally, it should be mentioned again that some of the new compounds pos-
sessing chemical and biological properties related to those of cisplatin might be very active
but show weak binding or no binding at all to DNA. Many other metal compounds may
be shown to be active in cancer treatment and may primarily interact with other biological
targets.
In the long term, “individualized” medical treatments with optimized drugs and
clinical regimes tailored to the individual can be envisaged. Harnessing this knowledge
and responding to and taking advantage of this new environment require teams commit-
ted to integrating chemistry and biology research and knowledge. These advances will
revolutionize the field of metallodrugs, taking it far beyond its origins in simple platinum
compounds toward sophisticated and modular molecular designs with different and pre-
dicted modes of action.
Acknowledgments
B. Biswas gratefully acknowledges the financial support by the Department of Science
and Technology (DST), New Delhi, India, under FAST TRACK SCHEME for YOUNG
SCIENTIST (No. SB/FT/CS-088/2013 dtd. 21/05/2014). B. Biswas sincerely thanks University
Grant Commission, New Delhi, India (F. No. PSW-84/12-13(ERO) dated 05/02/2013) for
financial support.
References
Alessio, E. (Ed.). 2011. Bioinorganic Medicinal Chemistry. Wiley-VCH, Weinheim, Germany.
Barton, J.K. 1994. Metal/nucleic acid interactions. In Bioinorganic Chemistry (Bertini, I., Gray, H.B.,
Lippard, S.J., and Valentine, J. S., eds.). University Science Books, Mill Valley, CA.
Bednarski, P.J., Korpis, K., Westendorf, A.F., Perfahl, S., and Grünert, R. 2013. Effects of light-activated
diazido-PtIV complexes on cancer cells in vitro. Dalton Trans. 40:5342–5351.
Bouma, M., Nuijen, B., Stewart, D.R., Rice, J.R., Jansen, B.A.J., Reedijk, J., and Beijnen, J.H. 2002.
Stability and compatibility of the investigational polymer-conjugated platinum anticancer
agent AP 5280 in infusion systems and its hemolytic potential. Anti-Cancer Drugs 13(9):915–924.
Chaires, J.B. and Waring, M.J. 2001. Drug–Nucleic Acid Interactions, vol. 340. Academic Press, San
Diego, CA.
Chen, H. 2006. Atlas of Genetic Diagnosis and Counselling. Humana Press, Totowa, NJ.
Coluccia, M., Nassi, A., Loseto, F., Boccarelli, A., Mariggio, M.A., Giordano, D., and Natile, G. 1993.
A trans-platinum complex showing higher antitumor activity than the cis congeners. J. Med.
Chem. 36:510.
Cubo, L., Pizarro, A.M., Quiroga, A.G., Salassa, L., Navarro-Ranninger, C., and Sadler, P.J. 2010.
Photoactivation of trans platinum diamine complexes in aqueous solution and effect on reac-
tivity towards nucleotides. J. Inorg. Biochem. 104:909–918.
Dabrowiak, J.C. 2010. Metals in Medicine. John Wiley & Sons, Hoboken, NJ.
de Hoog, P., Boldron, C., Gamez, P., Sliedregt-Bol, K., Roland, I., Pitié, M., and Reedijk, J. 2007. New
approach for the preparation of efficient DNA cleaving agents: Ditopic copper-platinum com-
plexes based on 3-Clip-Phen and cisplatin. J. Med. Chem. 50:3148–3155.
de Hoog, P., Pitié, M., Amadei, G., Gamez, P., Meunier, B., Kiss, R., and Reedijk, J. 2008. DNA cleavage
and binding selectivity of a heterodinuclear Pt–Cu(3-Clip-Phen) complex. J. Biol. Inorg. Chem.
13:575–586.
Farrell, N. et al., 1989. Cytostatic trans-platinum (II) complexes. J. Med. Chem. 32:2240.
Farrell, N. 1995. DNA binding and chemistry of dinuclear platinum complexes. Comments Inorg.
Chem. 16:373–389.
Farrell, N. 2004. Polynuclear platinum drugs. Metal Ions Biol. Syst. 41:251–296.
Farrell, N. 2011. Solution studies of dinuclear polyamine-linked platinum-based antitumour
complexes. Dalton Trans. 40:4147–4154.
Farrer, N.J., Woods, J.A., Munk, V.P., Mackay, F.S., and Sadler, P.J. 2009. Photocytotoxic trans-diam(m)
ine platinum(IV) diazido complexes more potent than their cis isomers. Chem. Res. Toxicol.
23:413–421.
Gielen, M. and Tiekink, E.R.T. (Eds.). 2005. Metallotherapeutic drugs and metal-based diagnostic
agents. The Use of Metals in Medicine (Graves, M.G., ed.). Wiley, Chichester, U.K.
Griffith, D.M. et al. 2012. The prevalence of metal-based drugs as therapeutic or diagnostic agents:
Beyond platinum. Dalton Trans. 41:13239–13257.
Kabolizadeh, P., Ryan, J., and Farrell, N. 2007. Differences in the cellular response and signaling path-
ways of cisplatin and BBR3464 ([{trans-PtCl (NH3)2} 2μ-(trans-Pt(NH3)2(H2N(CH2)6-NH2)2)]4+)
influenced by copper homeostasis. Biochem. Pharmacol. 73:1270–1279.
Kelland, L.R. 2007. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer
7:573–584.
Klein, A. and Hambley, W. 2009. Platinum drug distribution in cancer cells and tumors. Chem. Rev.
109:4911–4920.
Kostova, I. 2010. Metal-containing drugs and novel coordination complexes in therapeutic antican-
cer applications—Part II. Anti-Cancer Agents Med. Chem. 10:352–353.
Lippard, S.J. 1982. New chemistry of an old molecule: cis-[Pt(NH3)2Cl2). Science 218:1075–1082.
Lippert, B. (Ed.). 1999. Cisplatin, Chemistry and Biochemistry of a Leading Anticancer Drug. Wiley-VCH,
Weinheim, Germany.
Lippert, B. 2000. Multiplicity of metal ion binding patterns to nucleobases. Coord. Chem. Rev.
200:487–516.
Mellish, K.J., Qu, Y., Scarsdale, N., and Farrell, N. 1997. Effect of geometric isomerism in dinuclear
platinum antitumour complexes on the rate of formation. Nucleic Acids Res. 25:1265–1271.
Molenaar, C., Teuben, J.M., Heetebrij, R.J., Tanke, H.J., and Reedijk, J. 2000. New insights in the cel-
lular processing of platinum antitumor compounds, using fluorophore-labeled platinum com-
plexes and digital fluorescence microscopy. J. Biol. Inorg. Chem. 5:655–665.
Montero, E.I., Díaz, S., González-Vadillo, A.M., Pérez, J.M., Alonso, C., and Navarro-Ranninger, C.
1999. Preparation and characterization of novel trans-[PtCl2(amine)(isopropylamine)] com-
pounds: Cytotoxic activity and apoptosis induction in ras-transformed cells. J. Med. Chem.
42:4264.
Natile, G. and Coluccia, M. 2001. Current status of trans-platinum compounds in cancer therapy.
Coord. Chem. Rev. 216–217:383.
Navarro, M., Gabbiani, C., Messori, L., and Gambino, D. 2010. Metal-based drugs for malaria, try-
panosomiasis and leishmaniasis: Recent achievements and perspectives. Drug Discov. Today
15:1070–1078.
Noh, J.Y., Chino, T, and Ito, K. 2006. Treatment with inorganic iodine for Graves’ hyperthyroidism.
Nippon Rinsh 64:2269–2273.
Norman, J.F. and Hambley, T.W. 2011. Targeting strategies for metal-based therapeutics. In Alessio, E.
(Ed.). Bioinorganic Medicinal Chemistry. Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim,
Germany.
Ochiai, E. 2008. Bioinorganic Chemistry: A Survey, Medical Applications of Inorganic Compounds,
John Wiley & Sons Ltd., England, U.K.
Ozalp-Yaman, Ş., de Hoog, P., Amadei, G., Pitié, M., Gamez, P., Dewelle, J., and Reedijk, J. 2008.
Platinated copper (3-clip-phen) complexes as effective DNA-cleaving and cytotoxic agents.
Eur. J. Inorg. Chem. 14:612–619.
Quiroga, A.G. 2012. Understanding trans platinum complexes as potential antitumor drugs beyond
targeting DNA. J. Inorg. Biochem. 114:106–112.
Reedijk, J. 2009. Platinum anticancer coordination compounds: Study of DNA binding inspires new
drug design. Eur. J. Inorg. Chem. 2009:1303–1312.
Reedijk, J. 2011. Increased understanding of platinum anticancer chemistry. Pure Appl. Chem.
83:1709–1719.
Rosenberg, B. 1999. In Lippert, B. (Ed.). Cisplatin, Chemistry and Biochemistry of a Leading Anticancer
Drug. Bioinorganic Chemistry: A Survey, Wiley-VCH, Weinheim, Germany.
Rosenberg, B. and Van Camp, L. 1969. Platinum compounds: A new class of potent antitumour
agents. Nature 222:385–386.
Rosenberg, B., Van Camp, L., and Krigas, T. 1965. Inhibition of cell division in Escherichia coli by
electrolysis products from a platinum electrode. Nature 205:698–699.
Ruhayel, R.A., Zgani, I., Berners-Price, S.J., and Farrell, N.P. 2011. Solution studies of dinuclear poly-
amine-linked platinum-based antitumour complexes. Dalton Trans. 40:5342–5351.
Schilden, K., Garcia, F., Kooijman, H., Spek, A.L., Haasnoot, J.G., and Reedijk, J. 2004. A highly
flexible dinuclear ruthenium (ii)-platinum (ii) complex: Crystal structure and binding to
9-ethylguanine. Angew. Chem. Int. Ed. 43:5668–5670.
Sorenson, J.R.J. 1985. Historic uses of copper compounds in medicine. Trace Elem. Med., 2:80–87.
Srivastava, A.K. and Mehdi, M.Z. 2005. Insulino-mimetic and antidiabetic effects of vanadium com-
pounds. Diabet. Med. 22:2–13.
Tamasi, G. 2010. Metal-oxicam coordination compounds: Structure, biological activity and strate-
gies for administration. Open Crystallogr. J. 3:41–53.
Tarallo, M.B., Urquiola, C., Monge, A., Costa, B.P., Ribeiro, R.R., Costa-Filho, A.J., and Gambino, D.
2010. Design of novel iron compounds as potential therapeutic agents against tuberculosis.
J. Inorg. Biochem. 104:1164–1170.
Taube, H. 1952. Rates and mechanisms of substitution in inorganic complexes in solution. Chem.
Rev. 50:69–78.
Thompson, K.H. 2011. Medicinal inorganic chemistry: Metallotherapeutics for chronic diseases.
Scott, R.A. (Ed.), Encyclopedia of Inorganic Chemistry, John Wiley & Sons, Ltd, Athens, GA.
Thompson, K.H. and Orvig, C. 2003. Boon and bane of metal ions in medicine. Science 300:936–939.
Van der Schilden, K., Garcìa, F., Kooijman, H., Spek, A.L., Haasnoot, J.G., and Reedijk, J. 2004.
A highly flexible dinuclear ruthenium(II)–platinum(II) complex: Crystal structure and bind-
ing to 9‐ethylguanine. Angew. Chem. Int. Ed. 43:5668–5670.
van Rijt, S.H. and Sadler, P.J. 2009. Current applications and future potential for bioinorganic chem-
istry in the development of anticancer drugs. Drug Discov. Today 14:1089–1097.
van Zutphen, S. and Reedijk, J. 2005. Targeting platinum anti-tumour drugs: Overview of strategies
employed to reduce systemic toxicity. Coord. Chem. Rev. 249:2845–2853.
Wang, X. 2010. Fresh platinum complexes with promising antitumor activity. Anti-Cancer Agents
Med. Chem. 10:396–411.
Zhang, J., Thomas, D.S., Berners-Price, S.J., and Farrell, N. 2008. Effects of geometric isomerism and
anions on the kinetics and mechanism of the stepwise formation of long‐range DNA inter-
strand cross‐links by dinuclear platinum antitumor complexes. Chem. Eur. J. 14(21):6391–6405.
Zhao, Y., Woods, J.A., Farrer, N.J., Robinson, K.S., Pracharova, J., Kasparkova, J., and Sadler, P.J. 2013.
Diazido mixed‐amine platinum(IV) anticancer complexes activatable by visible‐light form
novel DNA adducts. Chem. A Eur. J. 19(29):9578–9591.
section five
Narrow-spectrum antimicrobials
chapter twenty-two
Marine actinobacteria as
potential drug storehouses
A future perspective on
antituberculosis compounds
N. Tamilselvan, Ernest David, Dharumadurai Dhanasekaran,
and Kumar Saurav
Contents
22.1 Introduction......................................................................................................................... 435
22.1.1 Natural products..................................................................................................... 436
22.1.2 Marine microorganisms as a source of natural drugs...................................... 436
22.2 Marine actinomycetes: a new source for bioactive metabolites................................... 437
22.1.2 Antibacterial............................................................................................................ 438
22.2.2 Antifungal................................................................................................................440
22.2.3 Anticancer................................................................................................................440
22.2.4 Antimalarial............................................................................................................444
22.2.5 Enzyme inhibitory..................................................................................................444
22.2.6 Miscellaneous..........................................................................................................445
22.3 Antituberculosis compound from marine organisms..................................................445
22.3.1 Antitubercular compounds produced by actinomycetes................................. 447
22.3.2 Mechanism of action of available drugs against TB bacteria...........................448
22.3.3 Marine-derived active lead compound as TB drug discovery......................... 451
References...................................................................................................................................... 451
22.1 Introduction
For billions of years, certain bacteria and fungi have produced chemical substances to pro-
tect them from attack from other microorganisms. Those used in clinical medicine today
are referred to as “antibiotics” or “antimicrobial agents.” As a survival mechanism, other
microbes have developed mechanisms for resisting the toxic effect of antimicrobials.
“Antimicrobial resistance” is thus an ancient phenomenon encoded on resistance genes
passed down through microbial lineages. Susceptible strains can become resistant either
through mutations in existing genes or by acquiring a resistance gene from another organ-
ism that is already resistant. This is the first step in the emergence of “new resistance.”
Although for most organisms the sudden appearance of new resistance is rare, this is not
the case for all pathogens. For example, in patients with tuberculosis (TB) or HIV infection,
new mutations in susceptible strains can occur within a patient, especially when therapy is
435
suboptimal. The emergence of resistant strains during therapy greatly increases the risk of a
poor clinical outcome, including death. Consequently, effective treatment is absolutely criti-
cal to avoid the development of resistance during treatment. Inadequate infection control in
hospitals and the lack of effective public health measures to control infections in the com-
munity are factors that are contributing to this problem, particularly in developing country
settings where the burden of infection is high and the choices for therapy are limited.
The discovery of new drugs for systemic infections is a major challenge in infectious
disease research. Despite some major advancement toward drug discovery, the continuing
increase in the incidence of infections together with the gradual rise in resistance high-
lights the need to find novel compounds with divergent mechanisms of action. Hence, con-
siderable research is being directed to isolate new compounds with defined mechanisms
of action that can serve as templates for further medicinal chemistry modifications.
22.1.1 Natural products

Natural products are chemical compounds derived from living organisms, for e xample,
plants, animals, and microorganisms. They can be defined as chemical compounds i solated
or derived from primary or rather secondary metabolism of organisms concerned natu-
rally. Nature acts as a prominent reservoir for new and novel therapeutics. By employing
sophisticated techniques under various screening programs, the rate of discovery of natu-
ral compounds exceeds one million so far, out of which 22,500 biologically active com-
pounds that have been extracted are from microbes: 45% are produced by actinobacteria,
38% by fungi, and 17% by unicellular bacteria (Demain and Sanchez, 2009). Members of
the class Actinobacteria especially Streptomyces spp. have long been recognized as pro-
lific sources of useful bioactive metabolites, providing more than 85% of the naturally
occurring antibiotics discovered to date and continuing as a rich source of new bioactive
metabolites (Berdy, 2005). Unfortunately, the emergence of drug-resistant pathogens and
the increase in diseases affecting the immune system have greatly intensified the need to
investigate new bioactive metabolites for potential pharmaceutical and industrial applica-
tions. Over the past 75 years, natural product derived compounds have led to the discov-
ery of many drugs to treat numerous human diseases (Grabley, 1999).
22.1.2 Marine microorganisms as a source of natural drugs

The oceans cover more than 70% of the Earth’s surface, and little is known about the
microbial diversity of marine sediments, which is an inexhaustible resource that has not
been fully exploited. Marine extremophiles serves as valuable natural resource for novel
products such as antibiotics, antitumor agents, and other therapeutic substances (Amador
et al., 2003). Natural products isolated from microorganisms have been the source of most
of the antibiotics developed to date for various diseases. Some of them are erythromycin,
vancomycin, and streptomycin, which are used for treating bacterial infections (Kelecom,
2002). Microbial natural products are very important not only for their potent therapeu-
tic properties but also for the fact that they possess pharmacokinetic properties required
for the clinical development. Marine microbes are particularly attractive because (1) they
have not been extensively exploited as their terrestrial counterparts and (2) they have high
potency required for bioactive compounds to be effective in the marine environment, due
to the diluting effect of seawater. Most of the marine compounds that have been success-
fully screened and structurally elucidated so far originated from microorganisms, espe-
cially from bacteria. It has been shown that marine bacteria produce bioactive substances
Chapter twenty-two: Marine actinobacteria as potential drug storehouses 437
that are different from known compounds from terrestrial bacteria (Fenical, 1993). It is
estimated that less than 1% of potentially useful chemicals from marine environment
have been screened so far, with microbial products representing approximately 1% of the
total number. Exploration of microbial secondary metabolites has led to the discovery of
hundreds of biologically active compounds that possess antibiotic, antitumor, and other
pharmacological activities that are currently being used for the treatment of various dis-
eases in humans, animals, and plants. Natural products with antibiotic activity that come
from bacteria or fungi are almost always products of secondary metabolic pathways. The
focus on the physiology and the potential of bioactive substances of noncultivable marine
microorganisms are of current problem and pose a great challenge to researchers to culti-
vate and isolate novel secondary metabolites for therapeutic applications.
22.2 Marine actinomycetes: a new source

for bioactive metabolites
Actinobacteria or actinomycetes are a group of gram-positive bacteria with high G + C
ratio. Actinobacteria are widely distributed in terrestrial and aquatic ecosystems, espe-
cially in soil, where they play a crucial role in the recycling of refractory biomaterials by
decomposing complex mixtures of polymers in dead plant, animal, and fungal materials.
They are important in soil biodegradation and humus formation by recycling the nutrients
associated with recalcitrant polymers such as keratin, lignocelluloses, and chitin. They
also produce several volatile substances like geosmin responsible of the characteristic
“wet earth odor.” They also exhibit diverse physiological and metabolic properties, such
as the production of extracellular enzymes (Olano et al., 2009). In 1940, Waksman discov-
ered actinomycin from soil bacteria. He was awarded with a Nobel Prize for his discovery
in 1952. Since then, hundreds of naturally occurring antibiotics have been discovered in
actinomycetes, especially from the genus Streptomyces (Stackebrandt et al., 1997). Marine
actinomycetes exhibit very different 16S rDNA sequences compared to their terrestrial
counterparts. As a result, marine actinomycetes produce many novel metabolites that may
be biologically active and a potential source of new anti-infective drugs (Lam, 2006). The
order Actinomycetales, commonly called as actinomycetes, represent one of the most stud-
ied and exploited classes of bacteria for their ability to make a wide range of biologically
active metabolites (Ikeda et al., 2003). The actinobacteria play an important role among the
marine bacterial communities, because of its diversity and ability to produce novel chemi-
cal compounds of high commercial value (Amador et al., 2003; Hopewood, 2007).
Though more than 50% of the microbial antibiotics discovered so far originate from
actinomycetes, only Streptomyces and Micromonospora account for most of these compounds
(Berdy, 2005). It was long been known that actinomycetes can be recovered from the sea,
and more recently, it was isolated from the deepest known ocean trenches (Pathom-Aree
et al., 2006). It was reported that common soil-inhabiting actinomycetes can be readily
recovered from marine samples (especially samples collected close to land), but the exis-
tence of distinct marine populations was clear in 1984 with the taxonomic description
of the first marine actinomycete, Rhodococcus marinonascens (Helmke and Weyland, 1984).
More recently, at least three marine genera (Maldonado et al., 2005) have been described,
including the genus Salinispora, which requires seawater and sodium for growth. Sampling
for marine actinomycetes began in the late 1960s, but in 2005, the first seawater-obligate
marine actinomycete (Salinispora spp.) was described (Maldonado et al., 2005). The culti-
vation of Salinispora spp. was originally reported in 1991 (Jensen et al., 1991), but at that
time, their taxonomic significance was unaware. Approximately 10 years later, after the
application of DNA sequence-based methods to infer evolutionary (phylogenetic) relation-

ships, it became clear that these bacteria are distinct from their terrestrial counterparts
(Mincer et al., 2002). Inspired by this finding and by the unusual metabolites observed
during initial chemical studies of Salinispora spp., studies were broadened to other marine-
derived actinomycetes, and several strains were cultured within at least six actinomycete
families, including many of them seem to represent new taxa. These efforts have corrobo-
rated the existence of marine actinomycetes, and subsequent chemical studies have shown
that these strains are also an excellent source of unprecedented secondary metabolites.
Marine actinomycetes have been a rich source of unique and biologically active metab-
olites. Although heavily studied over the past three decades, actinomycetes continue to
prove themselves as reliable sources of novel bioactive compounds. Among the well-
characterized pharmaceutically relevant microorganisms, actinomycetes remain a major
source of novel, therapeutically relevant natural products. They are the providers of novel
structures with unique pharmacological activities that continue to be discovered and
reported from all types of natural product sources. The majority of these compounds dem-
onstrate one or more bioactivities that could provide value to human medicine. Analytical
methods help us in rapid elucidation of structures and make natural products valuable
components of modern drug discovery.
Occurrence of distinct rare genera in the marine ecosystem has been reported with
the taxonomic description of the first marine actinomycetes Rhodococcus marinonascens
(Helmke and Weyland, 1984). Actinomycetes have also been isolated from free swimming
as well as sessile marine vertebrates and invertebrates (Ward and Bora, 2006). Unusual
actinomycetes belonging to Micrococceae, Dermatophilaceae, and Gordoniaceae have
been isolated from sponges (Lam, 2006). Tetrodotoxin-producing actinomycete has been
isolated from puffer fish ovaries (Wu et al., 2005). The organism was found to be most
closely related to Nocardiopsis dassonvillei. Researchers are finding new genera from
marine environments on a regular basis and discovering new metabolite producers
never reported earlier. Actinomycetes genera identified by cultural and molecular tech-
niques from different marine ecological niches include Actinomadura, Actinosynnema,
Amycolatopsis, Arthrobacter, Blastococcus, Brachybacterium, Corynebacterium, Dietzia,
Frankia, Frigoribacterium, Geodermatophilus, Gordonia, Kitasatospora, Micromonospora,
Micrococcus, Microbacterium, Mycobacterium, Nocardioides, Nocardiopsis, Nonomuraea,
Pseudonocardia, Rhodococcus, Saccharopolyspora, Salinispora, Serinicoccus, Solwaraspora,
Streptomyces, Streptosporangium, Tsukamurella, Turicella, Verrucosispora, and Williamsia
(Ward and Bora, 2006). In spite of improvements being made in the cultural methods
for the isolation of rare marine actinomycetes, many of these organisms still remain
uncultivable and have to be identified by using molecular techniques. Metagenomic
methods are useful for characterizing microbes that cannot be cultivated and can also
be used to isolate their genes (Tringe et al., 2005).
22.1.2 Antibacterial
The α-pyrones are six-membered cyclic unsaturated esters that share chemical and physi-
cal properties reminiscent to alkene and aromatic compound. These compounds occur
abundantly in naturally occurring molecules and are responsible for vast range of bio-
logical activities including antibacterial, antifungal, neurotoxic, and phytotoxic properties
(Dickinson, 1993; McGlacken and Fairlamb, 2005) and plant growth-regulating (Kobayashi
et al., 1994; Tsuchiya et al., 1997) antitumor (Suzuki et al., 1997; Kondoh et al., 1999), and HIV
protease-inhibiting activities (Thaisrivongs et al., 1996; Turner et al., 1998; Chen et al., 2007).
Several α-pyrones, for example, fusapyrones (Altomare et al., 2000), gibepyrones A–F
(Barrero et al., 1993), herbarins A–B (Jadulco et al., 2002), styrylpyrones (Rossi et al., 1997;
Kamperdick et al., 2002), nocapyrones E–J (Fu et al., 2011a), actinopyrones A–C (Yano et al.,
1986), pironetins (Kobayashi et al., 1994), and germicidins A–D (Petersen et al., 1993), have
been previously reported with biological activities. For example, nocapyrones E–G (1a–1c)
and germicidins A–D (2–5) were isolated from the actinomycetes Streptomyces viridochro-
mogenes NRRL B-1551 and Nocardiopsis dassonvillei, respectively, which possess moderate
activity against Bacillus subtilis.
Pseudonocardians A–C (Li et al., 2011) (6–8), three new diazaanthraquinone deriva-
tives, were isolated from the strain SCSIO 01299, a marine actinomycete member of the
genus Pseudonocardia, collected from deep-sea sediment of the South China Sea, and poses
antibacterial activities on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC
29212, and Bacillus thuringensis SCSIO BT01, with minimum inhibitory concentration
(MIC) values of 1–4 μg mL−1.
OH
R1
O O R2
R1 O O
R2
1a. R1 H; R2 = CH2CH3 2. R1 = H, R2 = H
1b. R1 H; R2 = CH3 3. R1 = H, R2 = CH3
1c. R1 = OH; R2 = CH2CH3 4. R1 = CH3, R2 = H
5. R1 = CH3, R2 = CH3
N N N N
0 O 0 OH H O
OH O
HO
R HO O
OH
6. R CH3CH2 8
7. R CH3
Recent advancement led to the discovery of several novel secondary metabolites to

circumvent the problem of resistant pathogens, which are no longer susceptible to the
currently used drugs. An endophytic Streptomyces sp. provided the indolosesquiterpenes,
xiamycin B (9), indosespene (10), and sespenine (11) and the known xiamycin A, which had
moderate to strong activity against several bacteria, including MRSA and vancomycin-
resistant E. faecalis (Ding et al., 2011).
OH OH
COOH HOOC
OH H
HN
H
9
OH
H COOH
H N
H H
HN
10 11
A series of chlorinated bisindole pyrroles, lynamicins A–E, was discovered from

marine actinomycete, NPS12745, which was isolated from marine sediment collected
off the coast of San Diego, California, in which it was shown that these substances pos-
sess broad-spectrum activity against both gram-positive and gram-negative organisms.
Significantly, compounds 12–16 were active against drug-resistant pathogens such as
methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium
(McArthur et al., 2008).
H
N R2
R1
12. R1 = R4 = R5 = H, R2 = COOMe, R3 = CI
13. R1 = R5= H, R2 = COOMe, R3 = R4 = CI
R3 CI 14. R1 = R2= H, R3 = R4 = R5 = CI
15. R1 = R2= COOMe, R3 = CI, R4 = R5 = H
R4 N N R5 16. R1 = R2= COOMe, R3 = R4 = R5 = H
H H
22.2.2 Antifungal
Daryamides (17) are cytotoxic and antifungal polyketides isolated from culture broth of
a Streptomyces strain, CNQ-085. These bioactive compounds have been shown to exhibit
moderate cytotoxicity against the human colon carcinoma cell line HCT-116 and moderate
antifungal activities against Candida albicans (Asolkar et al., 2006). Similarly, chandranani-
mycins (18–19), isolated from marine Actinomadura sp. MO48, have been shown to exhibit
antibacterial, anticancer, and antifungal activities (Maskey et al., 2003).
O
H
N O H
N O H
O N
HO O
HO HO O
HO
O
O H2N O
H 2N H2N
17 18 19
The antibiotics antimycin A19 (20) and A20 (21) were isolated from Streptomyces antibi-
oticus with potent activity against C. albicans (Xu et al., 2011).
O O
O OAc
O H O
H O N
N OHCHN O
OHCHN O
OH O
OH O O
O
20 21
22.2.3 Anticancer
Five bipyridine alkaloids named caerulomycins F–J (22–26), along with a phenylpyri-
dine alkaloid caerulomycins K (27), were discovered from Actinoalloteichus cyanogriseus
WH1-2216-6 with cytotoxic effect on cancerous cell lines HL-60, K562, KB, and A549.
Caerulomycin analogues also exhibited potential antimicrobial activity at MIC ranging

from 9.7 to 38.6 µM (Fu et al., 2011b).
R1 OR2 OMe
N
N N OH
R3 N
22. R1 = H, R2 = Me, R3 = CH2OH 27
23. R1 = OMe, R2 = Me, R3 = CH2OH
24. R1 = R2 = H, R3 = CHNOH
25. R1 H, R2 Me, R3 CONHOMe
26. R1 = R2 = H, R3 = CH2NHCOMe
A PCR-based screening approach led to the identification of a deep-sea-derived

Streptomyces sp. SCSIO 03032 capable of producing new bisindole alkaloids spiroindimi-
cins A–D (28–31). Spiroindimicins B–D (29–31) with a [5,5] spiroring exhibited moderate
cytotoxicities against several cancer cell lines (Zhang et al., 2012).
H3CO OCH3 H3CO H

H N
N R2
O O O
R1 N
N
CI
NH HN
CI
CI CI
29. R1 = CH3, R2 = H
28 30. R1 = H, R2 = H
31. R1 = CH3, R2 = COOCH3
Levantilides A (32) and B (33) are isolated from deep marine sediment–derived strain
Micromonospora sp. with moderate antitumor activity against several cell lines (Gartner
et al., 2011).
O OH
X O HO
32. X = H, OH
33. X = O
OH
Saliniquinones A–F (Murphy et al., 2010) (34–39), anthraquinone-γ-pyrones, and aren-

jimycin (Asolkar et al., 2010) (40) isolated from Salinospora arenicola exhibit potent inhibi-
tory activity against HCT-116 cell line.
CI
R2 OH
36. R1 = CH2OH, R2 =
OH O O
R1 37. R1 = CH2OH, R2 =
O
HO
O
34. R1 = CH2OH, R2 = 38. R1 = Me, R2 =
O
35. R1 = CH2OH, R2 = OH
39. R1 = CH2OH, R2 =
O OH O
O OMe
HO
HO
OMe
N
H OH
O O
HO
40 COOMe
Marinomycins A–D (41–44), which are unusual macrodiolides composed of dimeric

2-hydroxy-6-alkenyl-benzoic acid lactones with conjugated tetraene-pentahydroxy
polyketide chains, produced by Marinispora sp. CNQ-140, are isolated from a sediment
sample collected at a depth of 56 m offshore of La Jolla, California. These compounds
inhibit cancer cell proliferation with an average LC50 of 0.2–2.7 μM against the NCI’s 60 can-
cer cell line panel. Marinomycin A showed significant tissue-type selectivity being more
active against human melanoma cell lines LOX IMVI, M14, SK-MEL-2, SK-MEL-5, UACC-
257, and UACC-62 (Kwon et al., 2006).
OH
HO
OH OH OH O O
O O OH OH
OH
HO
41
HO
O
HO O OH OH O O HO
R OH HO
HO OH HO
O OH
HO
HO OH O
HO OH O OH
OH
O 44
42. R = H OH
43. R = Me
The manumycins constitute a class of compounds with antibiotic, cytotoxic, and other
biological activities. It has been reported that manumycin A (45) and its analogues inhibit
Ras farnesyltransferase and the growth of Ki-ras-activated murine fibrosarcoma in mice
(Kouchi et al., 1999).
O
H
N
O
R
OH 45 MeO N
OH
MeO
OH
46. R = H
47. R = Me
O NH
HO O
Piericidins C7 (46) and C8 (47), produced by Streptomyces sp. YM14-060, were iso-
lated from an unidentified ascidian collected at Iwayama Bay, Palau Island. The bio-
logical activity of piericidins was examined using rat glial cells transformed with the
adenovirus E1A gene (RG-E1A-7), Neuro-2a mouse neuroblastoma cells, C6 rat glioma
cells, and 3Y1 rat normal fibroblast. Piericidins C7 and C8 showed selective cytotoxicity
against RG-E1A-7 cells (IC50 of 1.5 and 0.45 nM, respectively) and inhibited the growth
of Neuro-2a cells (IC50 of 0.83 and 0.21 nM, respectively) without cytotoxic cell death. On
the other hand, C6 rat glioma cells and 3Y1 rat normal fibroblast were not affected by
piericidins (Hayakawa et al., 2007). Various other antitumors from marine actinomycetes
are tabulated in Table 22.1.
Table 22.1 Marine actinomycete compounds as antitumors

Source Compound Activity
Streptomyces strain CNQ-085 Daryamides Antitumor
Streptomyces sp. FMA Streptocarbazole A Antitumor
Streptomyces chibaensis AUBN1/7T Resistoflavin Antitumor
Streptomyces sp. 04DH10 Streptochlorine Antitumor
Streptomyces sp. BOSC-022A Barmumycin Antitumor
Streptomyces sp. B6007 Caprolactone Antitumor
Streptomyces aureoverticillatus NPS001583 Aureoverticillactam Antitumor
Streptomyces strain CNH990 Marmycin A Antitumor
Streptomyces strain BL-49-58-005 Indoles Antitumor
Streptomyces strain Mei37 Mansouramycin Antitumor
Marinospora sp. CNQ-140 Marinomycins Antitumor
Actinoalloteichus cyanogriseus WH1-2216-6 Caerulomycins Antitumor
Streptomyces sp. MDG-04-17-069 Tartrolon Antitumor
22.2.4 Antimalarial
Malaria is an arthropod-borne disease prevalent in many developing countries.
Antimalarial drugs are mainly chemically synthesized and extracted from plant source,
but recently, the development of drug-resistant strain possess a great challenge. The source
of novel compounds from marine sources yielded potent antimalarial compounds from
actinomycetes. Β-carboline alkaloids, marinacarbolines A–D (48–51), showed potent activ-
ity against Plasmodium falciparum at varying inhibitory concentrations from 1.9 to 36.03 µM
(Huang et al., 2011).
H O H O
N N
R H
O H OH
N N H N
N O
48. R = OCH3 NH NH
49. R = OH O O
50. R = H 52
51 CI
Secondary metabolite salinosporamide A (52) isolated from Salinispora tropica showed

protective action against the malaria parasite in the erythrocytic stage by inhibiting the
20S proteasome.
22.2.5 Enzyme inhibitory

Benzoxacystol (53–54), a 1,4-benzoxazine-type metabolite obtained from Streptomyces
griseus (deep-sea sediment, Canary Basin), was an inhibitor of glycogen synthase kinase
3b, in addition to displaying weak antiproliferative activity against mouse fibroblast cells
(Nachtigall et al., 2011).
MeO O CI
OH
N O OO
AcHN H Me2N OH
O O
S O O
53
COOH
CI
MeO O
P NHR
H2N 55. R Ac
COOMe 56. R H
54
Fijiolides A (55) and B (56) were isolated from a Nocardiopsis sp. (sediment, Beqa
Lagoon, Fiji). Fijiolide A 17 was a potent inhibitor of the TNF-α-induced transcription fac-
tor NFkB and induced quinone reductase activity, while fijiolide B 18 inhibited NFkB to a
lesser extent and had no effect on quinone reductase activity.
Screening of marine actinobacterial strains for the presence of hydroxy-3-
methylglutaryl-CoA reductase, a key enzyme in the mevalonate (MVA) pathway, identified
a Streptomyces sp., the culture of which yielded three new phenazine-derived isoprenoids,
JBIR-46–48 (57–59) (Izumikawa et al., 2010).
OH
OH
N
N
N
+
, O– N
OH R
OH
57. R = H
58. R = 59
22.2.6 Miscellaneous
Tumescenamides A (60) and B (61) are cyclic peptides from Streptomyces tumescens (sediment,
Big Drop-off, Republic of Palau). Tumescenamide A induced reporter gene expression
under the control of the insulin-degrading enzyme promoter, suggesting promise as a
potential treatment for Alzheimer’s disease (Motohashi et al., 2010a).
NH
H
O O O N
HN n
H O
N O
60. n = 2
61. n = 3
OH
Three new species of Streptomyces sp. (sponge Haliclona sp., Tateyama City, Chiba,
Japan) have been studied. The first species yielded two chlorinated indole-containing tet-
rapeptides, JBIR-34 (62) and JBIR-35 (63), both with weak DPPH activity (Motohashi et al.,
2010b).
O H
N N
OH COOH
N H
R O
OH
CI N 62. R = Me
63. R = H
22.3 Antituberculosis compound from marine organisms

TB has been known to humans since ancient days. TB occurred as an endemic among ani-
mals long before, which later affected humans. The presence of spinal TB was observed
in the fossil bones dated around 8000 BC (Ayyazian, 1993; Basel, 1998; Gregory, 2015).
The confirmed presence of TB in humans was noted in the deformities of the skeletal
and muscular parts of the Egyptian mummies of around 2400 BC (Andreas et al., 1997).
Nevertheless, it could not be determined whether the disease was due to Mycobacterium
bovis or Mycobacterium tuberculosis. Scientific investigation for the evolutionary origins of
the M. tuberculosis complex has concluded that the most recent common ancestor was a
human-specific pathogen, which underwent population congestion. Analysis of myco-
bacterial interspersed repetitive units has allowed dating of the bottleneck to approxi-
mately 40,000 years ago, which corresponds to the period subsequent to the expansion
of Homo sapiens out of Africa. This analysis of mycobacterial interspersed repetitive units
also dated the Mycobacterium bovis lineage as dispersing approximately 6000 years ago,
which may be linked to animal domestication and early farming (Wirth et al., 2008). Prior
to this disease, it has been called by numerous names including consumption (because of
the severe weight loss and the way the infection appeared to consume the patient), phthisis
pulmonalis, scrofula, Pott’s disease, and the white plague (because of the extreme pallor
seen among those infected). In the 1700s and early 1800s, TB prevalence peaked in Western
Europe and the United States and was undoubtedly the largest cause of death. For the
past 100–200 years, it had spread toward Eastern Europe, Asia, Africa, and South America
(Bloom and Murray, 1992). For the past 15,000–20,000 years, M. tuberculosis existed. It has
been found in relics from ancient Egypt, India, and China. The effectiveness of oral thera-
pies was developed in the 1950s that paved a way of optimism, leading to think that TB
would soon be a thing of the past. Even today, the majority of the world’s population has
been exposed and infected with TB, with over 90% in the developing countries, less com-
pared to developed countries. More than eight million new cases are recorded each year
worldwide, and more than two million people are dying from it. Most of the death cases
are recorded from countries that cannot offer modern drug therapy or even simple mod-
ern conveniences. Frightening also is the specter of antibiotic-resistant TB, borne of inad-
equate treatment and rendering cure difficult and prohibitively expensive. The control of
TB remains a global health issue. In the nineteenth century, TB was known as “the captain
of all men of death”—and so it remains (Table 22.2).
Marine natural products (MNP) are the best source of chemical diversity structures
with promising biological activities. The chemical novelties of those compounds possess
novel mechanisms of action. Parental compounds from marine sources are metabolized
into fewer compound through metabolism. The use of these resultant compounds as drugs
may reduce unwanted effects to host functions. The chemical and structural character-
ization of marine natural products is not completed until the drug development process
begins. At that time only the therapeutic values are evaluated. However, full assessment
of pharmacological significance is not studied until after drugs reach the market. After
this major technological process, it’s easy to find out the active lead compound present
in the parental compound that leads to designing the drug. In earlier days, there was
no perfect structure for the active metabolites used for the treatment of infectious dis-
eases; meanwhile, these active metabolites were available in the market. This major tech-
nological advance allows for drug repurposing or repositioning of the existing drugs in
the market to improve the efficacy of available drugs to treat drug resistant pathogens.
A biological transformation method is used to design the active metabolites as drug
with suitable pharmacological process such as physiochemical, pharmacodynamics and
pharmacokinetic properties. Apart from many anti-TB compounds isolated from marine
Table 22.2 Anti-TB compound isolated from marine organisms

S. No. Isolated compound Organism References
1. Pseudopteroxazole Pseudopterogorgia elisabethae Rodriguez (1999)
2. Massetolide A and viscosin Pseudomonas sp. Said et al. (2002)
3. Caprazamycin B Streptomyces sp MK730–62F2 Igarashi et al. (2003)
4. Hexapeptides Tychonema sp. Muller et al. (2006)
brunsvicamides
5. Sansanmycin Streptomyces sp. Xie et al. (2007, 2008)
6. Halicyclamine A Haliclona sp. Arai et al. (2008)
7. Trichoderins Trichoderma sp. Pruksakom et al. (2010)
8. 4-Deoxybostrycin Nigrospora sp. Wang et al. (2013)
9. Caulerpin Caulerpa sp. Chay et al. (2014)
10. Kahalalide Elysia rufescens
11. Lanosterol, nephalsterol Nepthea sp. Iguchi et al. (1989)
12. Cyanthiwigins Myrmekiodermastyx El Sayed et al. (2000)
13. Manadomanzamines Acanthostrongylophora sp. Peng et al. (2003)
14. Saringosterol Lessonia nigrescens Wachter et al. (2001)
15. Ingenamine Pachychalina sp. De Oliveira et al. (2004)
16. Araguspongine C Xestospongia exigua Orabi et al. (2002)
17. Ecteinascidins Ecteinascidia thurstoni Suwanborirux et al. (2002)
18. 12-Deacetoxyscalarin Brachiaster sp. Wonganuchitm et al. (2004)
19-acetate, manolide
25-acetate
organisms (Table 22.2), eight marine drugs have been approved by the FDA or EMEA,
namely cephalosporin C, cytarabine (Ara-C), vidrabine (Ara-A), zincontide (Prialt), omega-
3-acid ethyl esters (Lovaza), ET-743 (Yondelis), E7389 (Halaven), and brentuximabvendo-
tin (SGN-35) (Mayer et al., 2011; Gerwick and Moore, 2012). Some of the MNP were in
clinical trials such as soblidotin (TZT 1027), tasidotin, synthadotin (ILX-651), bryostatin 1,
hemiasterilin (E7974) and pseudopterosin have been completed, a compound (plitidepsin)
in phase III trials, six compounds DMXBAG (GTS-21), plinabulin (NPI 2358), PM00104,
elisidepsin, PM01183, CDX-11 are in phase II trials, and four compounds such as marizo-
mib, PM060184, SGN-75 and ASG-5ME are in phase I trials and have been processed with
multitudinous marine natural products being investigated preclinical as clinical candi-
dates (Liu, 2012). Unfortunately there were no active drugs from natural products available
for Mycobacterium tuberculosis; this makes the new opportunity for medicinal chemists
to find the drugs which could overcome drug resistance.
22.3.1 Antitubercular compounds produced by actinomycetes

Streptomycin was the first drug introduced in the market in 1944 for the treatment of TB,
but almost immediately after the introduction of this drug, many patients were show-
ing resistance to this antibiotic (Youmans et al., 1946; Pyle et al., 1947; Medical Research
Council, 1948). Later, para-aminosalicylate (PAS) was introduced into the market, which
overcame the emergence of drug-resistant TB strains (Medical Research Council, 1950).

Isoniazide, also known as isonicotinylhydrazine or INH was introduced, and the treatment
with isoniazid and streptomycin was even more effective than PAS. To date, many drugs
are available, which are classified into two categories: first-line therapy and second-line
therapy. First-line therapy includes five medications: isoniazid, pyrazinamide (analog of
nicotinamide), ethambutol [(S,S)-2,2(ethylenediamine)di-1-butanol], rifampicin (lipophilic
ansamycine), and streptomycin (aminocyclitol glycoside) (Goldberger, 1988). Second-line
therapy includes cycloserine, c apreomycin, fluoroquinolone, ethionamide, PAS, thioacet-
azone, rifabutin, clofazimine, and macrolide, which are prescribed for drug-resistant cases
(Iseman, 1993). Multidrug-resistant TB (MDR-TB), defined as resistant to at least isonia-
zid and rifampin, was documented in nearly every country from 1994 to 2000 by WHO
International Union against Tuberculosis and Surveillance Project (Gupta and Espinal,
2003). The ability of Mtb to remain dormant or p ersistent within host cells for many years
with the potential to be activated allows the b acterium to escape the activated immune
system of the host (Meena and Rajini, 2010). The latent or persistent TB infection is a major
obstacle in the cure and prevention of TB. Reactivation of latent TB is a high-risk factor
for disease development particularly in immunocompromised individuals such as those
coinfected with HIV (Koul et al., 2011). However, there is an urgent to discover novel anti-
biotics against drug resistance in M. tuberculosis. The two natural antibiotics, lasalocid and
monensin, are isolated mainly from fungal mycelium of Streptomyces cinnamonensis (acti-
nomycetes). These antibiotics belong to the family of the polycyclic carboxylic polyethers.
Monensin and lasalocid and their metal complexes with Tl(I), La(III), and Gd(III) are active
against M. tuberculosis. These ionophores could be considered as potential antitubercular
agents for the future discovery of new drug design.
O
OCH3 H
H3CO HN N
OCH3
O CH3 S
O
O O
H3C O O O O H O
N
H3C O N NH N COOH
O HO H H
N O
CH3 HO H
O N
H 2N
O 67
66 H3CN NCH3
N
O O
H2N O O
HO OH
OH OH
HO
22.3.2 Mechanism of action of available drugs against TB bacteria

There are many compounds with antimicrobial activity as antimicrobial agents. The
antimicrobial agents presented here will be grouped into three major classes, based
on the target microbes: (1) antibacterials, (2) antimycobacterials, and (3) antifungals.
Here, we are summarizing the current information about working models for the mech-
anism of action of antimycobacterial agents. The mechanism of action is categorized
into the following types: inhibition of cell wall synthesis, interference with membrane
integrity, inhibition of nucleic acid synthesis, protein synthesis, inhibition of synthesis
of small molecules, and some other unknown effects. The existing drug therapy may
avail this kind of mechanism; then, only the drug must be effective to kill the patho-
genic M. tuberculosis. There are two types of drug therapy currently available for treat-
ing TB, that is, front-line therapy and second-line therapy. Front-line therapy treats
the TB-affected person with the combination of different drugs such as rifampicin,
isoniazid, ethambutol, and pyrazinamide for 2 months period, followed by rifampicin
and isoniazid for an additional of 4 months. Rifampicin inhibits RNA polymerase, and
the combination of isoniazid and ethambutol collapses the cell wall biosynthesis, but
if these drugs are used singly, there is not much effect. Pyrazinamide specifically stops
the replication of TB bacteria (Bai et al., 2011). Second-line therapies specifically for
MDR-TB strains include fluoroquinolones (nalidixic acid, levofloxacin, ciprofloxacin,
ofloxacin, sparfloxacin, moxifloxacin, and gatifloxacin), bedaquilines (TMC207), and
aminoglycosides (kanamycin, streptomycin, spectinomycin, gentamycin, and hygro-
mycin B). Isoniazid (INH) enters the mycobacterial cell by passive diffusion: movement
of biochemicals and other atomic or molecular substances across the cell membrane
without need of energy. INH is not toxic to the bacterial cell, but it acts as a prodrug
and is activated by the enzyme Kat G, a multifunctional catalase–peroxidase. These
compounds are potent inhibitors of critical enzymes in the biosynthesis of cell wall
lipids and nucleic acids. Some INH-derived reactive species, such as nitric oxide, have
a direct role in inhibiting mycobacterial metabolic enzymes (Timmins and Deretic,
2006). The mechanism of action of ethambutol is not completely understood. It inhibits
arabinosyl transferase enzymes that are involved in the biosynthesis of arabinoglycan
and lipoarabinomannan, which are essential elements within the mycobacterial cell
wall (Belanger et al., 1996). Pyrazinamide’s exact mechanism is not well understood,
and it acts as a prodrug that is converted into the active form, pyrazionic acid, by the
bacterial nicotinamidase/pyrazinamidase. However, it appears in the disruption of
membrane energetic and inhibited cytoplasmic membrane function in M. tuberculosis
(Zhang et al., 2003). Rifampicin involves in the inhibition of DNA-dependent RNA
synthesis caused by strong binding to the β-subunit of the DNA-dependent RNA poly-
merase of prokaryotes, with a binding constant in the range of 10–8 M (Floss and Yu,
2005). Fluoroquinolones have been used as bactericidal by inhibiting its DNA gyrase at
concentrations within achievable serum levels (Leysen et al., 1989; De Souza et al., 2006;
Kubendiran et al., 2006; Sriram et al., 2006; Shandil et al., 2007). Bedaquiline (TMC207)
inhibits the mycobacterial ATP synthase; this drug was recently approved as new drug
for MDR-TB. Aminoglycosides disrupt protein synthesis by targeting the bacterial 30S
ribosomal subunit. Due to the drug-resistance problem caused by the bacteria, it is
necessary to develop new drugs that should inhibit the specific targets; this might be
different from those currently used drugs. New drugs should inhibit the target pres-
ent in bacteria, not in the human host.
O
OCH3 H
HN N
H3CO OCH3
O CH3 S
O
O O
H3C O O O O H O
N
H3C O N NH N COOH
O HO H H
N O
CH3 HO H
O N H2N 67
66 H3CN NCH3 O
N O OH
O O
H2N O O
OH
HO OH
OH OH
HO OH
O
69
N O CO R OH
2
H H
O N N
O O N
H N
O O O H
O
O N 70
N H
RO2C
68
R2
O H
(R)
NH HN
HN (R) N H H
H (R)
(s)
(R) HO R1
O HO O
O
O (R) HN (R)
71. R1 = H, R2 = OH
HN 72. R1 = OH, R2 = OH
71 O (s) (s) NH
OH
O 73
O
HO
H 77
H
H
N
OH H NH H H AcO
O
H
H OH H
O HO
74 N
O
HO
OMe HN 78
NH HO Me
76 N
O H O
OAc S
O O
H N R
Me HO H
HO O
N H
O OAc
O H
H X O OH
75 79
80
N
22.3.3 Marine-derived active lead compound as TB drug discovery

Pseudopteroxazole is a marine-derived compound that was isolated from the Indian gor-
gonian coral, Pseudopterogorgia elisabethae. It has been used to test against M. tuberculosis
in vitro; pseudopteroxazole (63) was found to possess higher (97%) antimycobacterial
activity (Rodriguez, 1999). However, attempts at in vivo efficacy studies have not yet been
studied. If this compound is found to be efficient in vivo, then there is a possibility of
medicinal chemistry to design a novel drug for the treatment of TB. Massetolide A (64)
and viscosin (65) compound was isolated from Pseudomonas sp., which was symbiotically
associated with marine alga, used as a lead compound to test against M. tuberculosis, and
it was found to be effective in minimum level of dosage (Said et al., 2002). Caprazamycin
B (66) compounds were isolated from Streptomyces sp., which were tested in vitro against
MDR-TB. Majority of these compounds were isolated from marine species listed in
Table 22.1, and their structures were also listed (66–80). Though most of the works were
done through in vitro studies, there is no evidence for these isolated compounds to be
tested in vivo. Sometimes, in vitro testing results will not reappear during the experiment
conducted in vivo. This is due to metabolism: if compounds with potent in vitro activity
are rapidly and extensively metabolized into inactive products, this may lead the isolated
compound to a failed activity in vivo. Studies need to be conducted to determine whether
the natural compounds exert a bactericidal and bacteriostatic effect against M. tuberculosis.
Whole-cell assays and target-based assays are normally used in high-throughput screen-
ing (HTS). Target-based assay is more directional, but some natural products with good
inhibition of pure enzymes may also show a limited inhibitory effect on Mtb cells due to
their limited membrane permeability (Dhiman et al., 2005; Henriksson et al., 2007). In such
cases, proper modification of the natural compound will be proposed to overcome the
problem. In order to combine the advantages of the traditional whole-cell assays and the
target-inhibiting assays, target-based whole-cell assays were developed in recent years to
make HTS process more facile and efficient.
References
Altomare, C., Perrone, G., Zonno, M.C., Evidente, A., Pengue, R., Fanti, F., and Polonelli, L. 2000.
Biological characterization of fusapyrone and deoxyfusapyrone, two bioactive secondary
metabolites of Fusarium semitectum. J. Nat. Prod. 63(8): 1131–1135.
Amador, M.L., Jimeno, J., Paz-Ares, L., Cortes-Funes, H., and Hidalgo, M. 2003. Progress in the
development and acquisition of anticancer agents from marine sources. Ann. Oncol. 14(11):
1607–1615.
Andreas, G.N, Christian, J.H., Albert, Z., Ulrike, S., and Hjalmar, G.H. 1997. Molecular evidence for
tuberculosis in an ancient Egyptian mummy. The Lancet 350(9088): 1404.
Arai, M., Sobou, M., Vicheze, C., Baughn, A., Hashizume, H., Pruksakorn, P., Ishida, S., Matsumoto, M.,
and Jacobs, W.R. Jr, Kobayashi M. 2008. Halicyclamine A, a marine spongean alkaloid as a lead
for anti-tuberculosis agent. Bioorgan. Med. Chem. 16: 6732–6736.
Asolkar, R.N., Jensen, P.R., Kauffman, C.A., and Fenical, W. 2006. Daryamides A-C, weakly cytotoxic
polyketides from a marine-derived actinomycete of the genus Streptomyces strain CNQ-085.
J. Nat. Prod. 69(12): 1756–1759.
Asolkar, R.N., Kirkland, T.N., Jensen, P.R., and Fenical, W. 2010. Arenimycin, an antibiotic effective
against rifampin- and methicillin-resistant Staphylococcus aureus from the marine actinomy-
cete Salinispora arenicola. J. Antibiot. 63(1): 37–39.
Ayyazian, L.F. 1993. History of tuberculosis. In: Reichman, L.B. and Hershfield, E.S. (Eds.),
Tuberculosis. Dekker, New York.
Bai, X., Chen, Y., Chen, W., Lei, H., and Shi, G. 2011. Volatile constituents, inorganic elements and
primary screening of bioactivity of black coral cigarette holders. Marine Drugs 9: 863–878.
Barrero, A.F., Oltra, J.E., Herrador, M.M., Cabrera, E., Sanchez, J.F., Quílez, J.F., and Reyes, J.F. 1993.
Gibepyrones: α-pyrones from Gibberella fujikuroi. Tetrahedron 49(1): 141–150.
Basel, H.H. 1998. History of tuberculosis. Respiration 65: 5–15.
Belanger, A.E., Besra, G.S., Ford, M.E., Mikusova, K., Belisle, J.T., Brennan, P.J., and Inamine, J.M.
1996.The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in
cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.
Proc. Natl. Acad. Sci. 93: 11919–11924.
Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58(1): 1–26.
Bloom, B.R. and Murray, C.J. 1992. Tuberculosis: Commentary on re-emergent killer. Science 257:
1055–1064.
Chay, C.I.C., Cansino, R.G., Pinzón, C.I.E., Torres-Ochoa, R.O., and Martínez, R. 2014. Synthesis and
Anti-tuberculosis activity of the marine natural product caulerpin and its analogues. Mar.
Drugs 12(4): 1757–1772.
Chen, L., Sabo, J.P., Philip, E., Mao, Y., Norris, S.H., MacGregor, T.R., and Valdez, H. 2007. Steady-state
disposition of the nonpeptidic protease inhibitor tipranavir when coadministered with ritona-
vir. Antimicrob. Agents. Chemother. 51(7): 2436–2444.
De Oliveira, J.H.H.L., Grube, A., Kock, M., Berlinck, R.G., Macedo, M.L., Ferreira, A.G., and Hajdu, E.
2004. Ingenamine G and cyclostellettamines G–I, K, and L from the new brazilian species of
marine sponge Pachychalina sp. J. Nat. Prod. 67: 1685–1689.
De Souza, M.V., Vasconcelos, T.R., de Almeida, M.V., and Cardoso, S.H. 2006. Fluoroquinolones: An
important class of antibiotics against tuberculosis. Curr. Med. Chem. 13: 455–463.
Demain, A.L. and Sanchez, S. 2009. Microbial drug discovery: 80 years of progress. J. Antibiot. 62(1):
5–16.
Dhiman, R.K., Schaeffer, M.L., Bailey, A.M., Testa, C.A., Scherman, H., and Crick, D.C. 2005. 1-Deoxy-
D-xylulose 5-phosphate reductoisomerase (Ispc) from Mycobacterium tuberculosis: towards
understanding mycobacterial resistance to fosmidomycin. J. Bacteriol. 187: 8395–8402.
Dickinson, J.M. 1993. Microbial pyran-2-ones and dihydropyran-2-ones. Nat. Prod. Rep. 10(1): 71–98.
Ding, L., Maier, A., Fiebig, H.H., Lin, W.H., and Hertweck, C. 2011. A family of multicyclic indoloses-
quiterpenes from a bacterial endophyte. Organ. Biomol. Chem. 9(11): 4029–4031.
El Sayed, K.A., Bartyzel, P., Shen, X., Perry, T.L., Zjawiony, J.K., and Hamann, M.T. 2000. Marine natu-
ral products as antituberculosis agents. Tetrahedron 56: 949–953.
Fenical, W. 1993. Chemical studies of marine bacteria: Developing a new resource. Chem. Rev. 93(5):
1673–1683.
Floss, H.G. and Yu, T.W. 2005. Rifamycin-mode of action, resistance, and biosynthesis. Chem. Rev.
105: 621–632.
Fu, P., Liu, P., Qu, H., Wang, Y., Chen, D., Wang, H., and Zhu, W. 2011a. Alpha-pyrones and diketo-
piperazine derivatives from the marine-derived actinomycete Nocardiopsis dassonvillei HR10-5.
J. Nat. Prod. 74(10): 2219–2223.
Fu, P., Wang, S., Hong, K., Li, X., Liu, P., Wang, Y., and Zhu, W. 2011b. Cytotoxic bipyridines from
the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6. J. Nat. Prod. 74(8):
1751–1756.
Gärtner, A., Ohlendorf, B., Schulz, D., Zinecker, H., Wiese, J., and Imhoff, J.F. 2011. Levantilides A and
B, 20-membered macrolides from a Micromonospora strain isolated from the Mediterranean
deep sea sediment. Mar. Drugs 9(1): 98–108.
Gerwick, W.H., Moore, B.S. 2012. Lessons from the past and charting the future of marine natural
products drug discovery and chemical biology. Chemistry & Biology 19: 85–98.
Goldberger, M.J. 1988. Antituberculous agents. Med. Clin. North Am. 72: 661–668.
Grabley, S.T.R. (ed.). 1999. The impact of natural products on drug discovery. In: Drug Discovery from
Nature. Berlin Heidelberg: Springer-Verlag, pp. 1–37.
Gregory, M.R. 2015. Tuberculosis retrenched at Saranac Lake: A herald for contemporary hospitals.
News-Medical.net—An AZoNetwork Site.
Gupta, R. and Espinal, M. 2003. Stop TB Working Group on DOTSPlus for MDR-TB. A prioritized
research agenda for DOTS-Plus for multidrug-resistant tuberculosis (MDRTB). Int. J. Tuberc.
Lung Dis. 7: 410–414.
Hayakawa, Y., Shirasaki, S., Shiba, S., Kawasaki, T., Matsuo, Y., Adachi, K., and Shizuri, Y. 2007.
Piericidins C7 and C8, new cytotoxic antibiotics produced by a marine Streptomyces sp.
J. Antibiot. (Tokyo) 60(3): 196–200.
Helmke, E. and Weyland, H. 1984. Rhodococcus marinonascens sp. nov., an actinomycete from the sea.
Int. J. Syst. Bacteriol. 34(2): 127–138.
Henriksson, L.M., Unge, T., Carlsson, J., Aqvist, J., Mowbray, S.L., and Jones, T.A. 2007. Structures
of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new
insights into catalysis. J. Biol. Chem. 282: 19905–19916.
Hopewood, D.A. 2007. Therapeutic treasures from the deep. Nat. Chem. Biol. 3(8): 457–458.
Huang, H., Yao, Y., He, Z., Yang, T., Ma, J., Tian, X., and Ju, J. 2011. Antimalarial carboline and indol-
actam alkaloids from Marinactinospora thermotolerans, a deep sea isolate. J. Nat. Prod. 74(10):
2122–2127.
Igarashi, M., Nakagawa, N., Doi, N., Hattori, S., Naganawa H., and Hamada, M. 2003. Caprazamycin
B, a novel anti-tuberculosis antibiotic, from Streptomyces sp. J. Antibiot. 56: 580–583.
Iguchi, K., Saitoh, S., and Yamada, Y. 1989. Novel 19-oxygenated sterols from the Okinawan soft coral
Litophyton viridis. Chem. Pharm. Bull. 37: 2553–2554.
Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., and Omura, S. 2003.
Complete genome sequence and comparative analysis of the industrial microorganism
Streptomyces avermitilis. Nat. Biotechnol. 21(5): 526–531.
Iseman, M.D. 1993. Treatment of multidrug-resistant tuberculosis. N. Engl. J. Med. 329: 784–791.
Izumikawa, M., Khan, S.T., Takagi, M., and Shin-ya, K. 2010. Sponge-derived Streptomyces producing
isoprenoids via the mevalonate pathway. J. Nat. Prod. 73(2): 208–212.
Jadulco, R., Brauers, G., Edrada, R.A., Ebel, R., Wray, V., Sudarsono, S., and Proksch, P. 2002. New
metabolites from sponge-derived fungi Curvularia lunata and Cladosporium herbarum. J. Nat.
Prod. 65(5): 730–733.
Jensen, P.R., Dwight, R.Y.A.N., and Fenical, W. 1991. Distribution of actinomycetes in near-shore
tropical marine sediments. Appl. Environ. Microbiol. 57(4): 1102–1108.
Kamperdick, C., Van, N.H., and Van Sung, T. 2002. Constituents from Miliusa balansae (Annonaceae).
Phytochemistry 61(8): 991–994.
Kelecom, A. 2002. Secondary metabolites from marine microorganisms. An. Acad. Bras. Cienc. 74(1):
151–170.
Kobayashi, S., Tsuchiya, K., Kurokawa, T., Nakagawa, T., Shimada, N., and Iitaka, Y. 1994. Pironetin,
a novel plant growth regulator produced by Streptomyces sp. NK10958. II. Structural elucida-
tion. J. Antibiot. 47(6): 703–707.
Kondoh, M., Usui, T., Nishikiori, T., Mayumi, T., and Osada, H. 1999. Apoptosis induction via
microtubule disassembly by an antitumour compound, pironetin. Biochem. J. 340(Part 2):
411–416.
Kouchi, H., Nakamura, K., Fushimi, K., Sakaguchi, M., Miyazaki, M., Ohe, T., and Namba, M. 1999.
Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat
vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 264(3): 915–920.
Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K. 2011. The challenge of new drug
discovery for tuberculosis. Nature 469: 483–490.
Kubendiran, G., Paramasivan, C.N., Sulochana, S., and Mitchison, D.A. 2006. Moxifloxacin and gati-
floxacin in an acid model of persistent Mycobacterium tuberculosis. J. Chemother. 18: 617–623.
Kwon, H.C., Kauffman, C.A., Jensen, P.R., and Fenical, W. 2006. Marinomycins A−D, antitumor-
antibiotics of a new structure class from a marine actinomycete of the recently discovered
genus “Marinispora”. J. Am. Chem. Soc. 128(5): 1622–1632.
Lam, K.S. 2006. Discovery of novel metabolites from marine actinomycetes. Curr. Opin. Microbiol.
9(3): 245–251.
Leysen, D.C., Haemers, A., and Pattyn, S.R. 1989. Mycobacteria and the new quinolones. Antimicrob.
Agents Chemother. 33: 1–5.
Li, S., Tian, X., Niu, S., Zhang, W., Chen, Y., Zhang, H., and Zhang, C. 2011. Pseudonocardians A-C,
new diazaanthraquinone derivatives from a deep-sea actinomycete Pseudonocardia sp. SCSIO
01299. Mar. Drugs 9(8): 1428–1439.
Liu, Y. 2012. Renaissance of marine natural product drug discovery and development. J. Marine. Sci.
Res. Development 2: 2.
Maldonado, L.A., Fenical, W., Jensen, P.R., Kauffman, C.A., Mincer, T.J., Ward, A.C., and Goodfellow,
M. 2005. Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov., obligate marine acti-
nomycetes belonging to the family Micromonosporaceae. Int. J. Syst. Evol. Microbiol. 55(Part 5):
1759–1766.
Maskey, R.P., Li, F.C., Qin, S., Fiebig, H.H., and Laatsch, H. 2003. Chandrananimycins A approxi-
mately C: Production of novel anticancer antibiotics from a marine Actinomadura sp. isolate
M048 by variation of medium composition and growth conditions. J. Antibiot. 56(7): 622–629.
Mayer, A.M.S., Rodriguez, A.D., Berlinck, R.G.S., and Fusetani, N. 2011 Marine pharmacology in
2007–8: Marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory,
antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune
and nervous system, and other miscellaneous mechanisms of action. Comp. Biochem. Physiol.
C-Toxicol. Pharmacol. 153: 191–222.
McArthur, K.A., Mitchell, S.S., Tsueng, G., Rheingold, A., White, D.J., Grodberg, J., and Potts, B.C. 2008.
Lynamicins a−e, chlorinated bisindole pyrrole antibiotics from a novel marine Actinomycete.
J. Nat. Prod. 71(10): 1732–1737.
McGlacken, G.P. and Fairlamb, I.J.S. 2005. 2-Pyrone natural products and mimetics: Isolation, char-
acterisation and biological activity. Nat. Prod. Rep. 22(3): 369–385.
Medical Research Council. 1948. Streptomycin treatment of pulmonary tuberculosis. British Medical
Journal 2: 769 –782.
Meena, L.S., Rajni. 2010. Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv.
FEBS 277: 2416– 2427.
Mincer, T.J., Jensen, P.R., Kauffman, C.A., and Fenical, W. 2002. Widespread and persistent popula-
68(10): 5005–5011.
Motohashi, K., Takagi, M., and Shin-ya, K. 2010b. Tetrapeptides possessing a unique skeleton, JBIR-
34 and JBIR-35, isolated from a sponge-derived actinomycete, Streptomyces sp. Sp080513GE-23.
J. Nat. Prod. 73(2): 226–228.
Motohashi, K., Toda, T., Sue, M., Furihata, K., Shizuri, Y., Matsuo, Y., and Seto, H. 2010a. Isolation
and structure elucidation of tumescenamides A and B, two peptides produced by Streptomyces
tumescens YM23-260. J. Antibiot. 63(9): 549–552.
Muller, D., Krick, A., Keluraus, S. 2006. Brunsvicamides A–C: sponge related cyanobacterial pep-
tides with Mycobacterium tuberculosis protein tyrosine phosphatase inhibitory activity. J. Med.
Chem. 49: 4871–4878.
Murphy, B.T., Narender, T., Kauffman, C.A., Woolery, M., Jensen, P.R., and Fenical, W. 2010.
Saliniquinones A–F, new members of the highly cytotoxic anthraquinone-γ-pyrones from the
marine actinomycete Salinispora arenicola. Aust. J. Chem. 63(6): 929–934.
Nachtigall, J., Schneider, K., Bruntner, C., Bull, A.T., Goodfellow, M., Zinecker, H., and Fiedler,
H.P. 2011. Benzoxacystol, a benzoxazine-type enzyme inhibitor from the deep-sea strain
Streptomyces sp. NTK 935. J. Antibiot. (Tokyo) 64(6): 453–457.
Olano, C., Méndez, C., and Salas, J.A. 2009. Antitumor compounds from actinomycetes: From gene
clusters to new derivatives by combinatorial biosynthesis. Nat. Prod. Rep. 26(5): 628–660.
Orabi, K.Y., El Sayed, K.A., Hamann, M.T., Dunbar, D.C., Al Said, M.S., Higa, T., and Kelly, M. 2002.
Araguspongines K and L, new bioactive bis-1-oxaquinolizidine N-oxide alkaloids from Red
Sea specimens of Xestospongia exigua. J. Nat. Prod. 65(12): 1782–1785.
Pathom-Aree, W., Stach, J.E., Ward, A.C., Horikoshi, K., Bull, A.T., and Goodfellow, M. 2006. Diversity
of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench.
Extremophiles 10(3): 181–189.
Peng, J., Hu, J.F., Kazi, A.B., Li, Z., Avery, M., Peraud, O., Hill, R.T. et al. 2003. Manadomanzamines
A and B: A novel alkaloid ring system with potent activity against mycobacteria and HIV-1.
J. Am. Chem. Soc. 125(44): 13382–13386.
Petersen, F., Zähner, H., Metzger, J.W., Freund, S., and Hummel, R.P. 1993. Germicidin, an autoregu-
lative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551. J. Antibiot. 46(7):
1126–1138.
Pruksakorn, P., Arai, M., Kotoku, N., Vilcheze, C., Baughn, A.D., Moodley, P., Jacobs, W.R. Jr, and
Kobayashi, M. 2010. Trichoderins, novel aminolipopeptides from a marine sponge derived
Trichoderma sp., are active against dormant mycobacteria. Bioorg. Med. Chem. Lett. 20: 3658–3663.
Pyle, M.M. 1947. Relative numbers of resistant tubercle bacilli in sputa of patients before and during
treatment with streptomycin. Proc. Staff Meet. Mayo Clin. 22: 465–472.
Rodriguez, A.D., Ramirez, C. Rodriguez II, Gonzalez, E. 1999. Novel antimycobacterialbenzoxazole
alkaloids, from the west Indian sea whip Pseudopterogorgiaelisabethae. Org. Lett. 1: 527–530.
Rossi, M.H., Yoshida, M., and Maia, J.G.S. 1997. Neolignans, styrylpyrones and flavonoids from an
Aniba species. Phytochemistry 45(6): 1263–1269.
Said, O., Khalil, K., Fulder, S., Azaizeh, M. 2002. Ethnopharmacological survey of medicinal herbs in
Israel, the Golan Heights and the West Bank region. J. Ethnopharmarmacol. 83: 251–265.
Shandil, R.K., Jayaram, R., Kaur, P., Gaonkar, S., Suresh, B.L., Mahesh, B.N., Jayashree, R., Nandi, V.,
Bharath, S., and Balasubramanian, V. 2007. Moxifloxacin, ofloxacin, sparfloxacin, and ciproflox-
acin against Mycobacterium tuberculosis: Evaluation of in vitro and pharmacodynamic indices
that best predict in vivo efficacy. Antimicrob. Agents Chemother. 51: 576–582.
Sriram, D., Bal, T.R., Yogeeswari, P., Radha, D.R., and Nagaraja, V. 2006. Evaluation of antimycobac-
terial and DNA gyrase inhibition of fluoroquinolone derivatives. J. Gen. Appl. Microbiol. 52:
195–200.
Stackebrandt, E., Rainey, F.A., and Ward-Rainey, N.L. 1997. Proposal for a new hierarchic classifica-
tion system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47(2): 479–491.
Suwanborirux, K., Charupant, K., Amnuoypol, S., Pummangura, S., Kubo, A., and Saito, N. 2002.
Ecteinascidins 770 and 786 from the Thai tunicate Ecteinascidia thurstoni. J. Nat. Prod. 65(6):
935–937.
Suzuki, K., Kuwahara, A., Yoshida, H., Fujita, S., Nishikiori, T., and Nakagawa, T. 1997. NF00659A1,
A2, A3, B1 and B2, novel antitumor antibiotics produced by Aspergillus sp. NF 00659. I.
Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 50(4): 314–317.
Thaisrivongs, S., Romero, D.L., Tommasi, R.A., Janakiraman, M.N., Strohbach, J.W., Turner, S.R., and
Watenpaugh, K.D. 1996. Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-
hydroxy-2-pyrones as effective, nonpeptidic inhibitors. J. Med. Chem. 39(23): 4630–4642.
Timmins, G.S. and Deretic, V. 2006. Mechanisms of action of isoniazid. Mol. Microbiol. 62:
1220–1227.
Tringe, S.G., Von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, H.W., and Rubin, E.M.
2005. Comparative metagenomics of microbial communities. Science 308(5721): 554–557.
Tsuchiya, K., Kobayashi, S., Nishikiori, T., Nakagawa, T., and Tatsuta, K. 1997. NK10958P, a novel
plant growth regulator produced by Streptomyces sp. J. Antibiot. 50(3): 259–260.
Turner, S.R., Strohbach, J.W., Tommasi, R.A., Aristoff, P.A., Johnson, P.D., Skulnick, H.I., and
Thaisrivongs, S. 1998. Tipranavir (PNU-140690): A potent, orally bioavailable nonpeptidic HIV
protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class â. J. Med. Chem.
41(18): 3467–3476.
Wachter, G.A., Franzblau, S.G., Montenegro, G., Hoffmann, J.J., Maiese, W.M., and Timmermann,
B.N. 2001. Inhibition of Mycobacterium tuberculosis growth by saringosterol from Lessonia
nigrescens. J. Nat. Prod. 64:1463–1464.
Wang, C., Wang, J., Huang, Y., Chen, H., Li, Y., Zhong, L., Chen, Y. et al. 2013. Anti-mycobacterial
activity of marine fungus-derived 4-deoxybostrycin and nigrosporin. Molecules 18(2):
1728–1740.
Ward, A.C. and N. Bora 2006. Diversity and biogeography of marine actinobacteria. Curr. Opin.
Microbiol. 9(3): 279–286.
Wirth, T., Hildebrand, F., Allix-Béguec, C., Wolbeling, F., Kubica, T., Kremer, K., and Niemann, S.
2008. Origin, spread and demography of the Mycobacterium tuberculosis complex. PLoS Pathog.
4(9): e1000160.
Wonganuchitmeta, S., Yuenyongsawad, S., Keawpradub, N., and Plubrukarn, A. 2004. Antitubercular
sesterterpenes from the Thai sponge Brachiaster sp. J. Nat. Prod. 67(10): 1767–1770.
Wu, Z., Xie, L., Xia, G., Zhang, J., Nie, Y., Hu, J., and Zhang, R. 2005. A new tetrodotoxin-producing
actinomycete, Nocardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes.
Toxicon 45(7): 851–859.
Xie, Y.Y., Chen, R.X., Si, S.Y., Sun, C.H., Xu, H.Z. 2007. A new nucleosidyl-peptide antibiotic, sansan-
mycin. J. Antibiot. 60: 158–161.
Xie, Y.Y., Xu, H.Z., Si, S.Y., Sun, C.H., and Chen, R.X. 2008. Sansanmycins B and C, new components
of sansanmycins. J. Antibiot. 61: 237–240.
Xu, L.-Y., Quan, X.-S., Wang, C., Sheng, H.-F., Zhou, G.-X., Lin, B.-R., Jiang, R.-W., and Yao, X.-S. 2011.
Antimycins A19 and A20, two new antimycins produced by marine actinomycete Streptomyces
antibioticus H74-18. J. Antibiot. (Tokyo) 64(10): 661–665.
Yano, K., Yokoi, K., Sato, J., Oono, J., Kouda, T., Ogawa, Y., and Nakashima, T. 1986. Actinopyrones
A, B and C, new physiologically active substances. II. Physico-chemical properties and chemi-
cal structures. J. Antibiot. 39(1): 38–43.
Youmans, G.P., Williston, E.H., Feldman, W.H., and Hinshaw, H.C. (1946) Increase in resistance of
tubercle bacilli to streptomycin: a preliminary report. Proc. Staff Meet. Mayo Clin. 21: 126–127.
Zhang, W., Liu, Z., Li, S., Yang, T., Zhang, Q., Ma, L., and Zhang, C. 2012. Spiroindimicins A-D: New
bisindole alkaloids from a deep-sea-derived actinomycete. Org. Lett. 14(13): 3364–3367.
Zhang, Y., Wade, M.M., Scorpio, A., Zhang, H., and Sun, Z. 2003. Mode of action of pyrazinamide:
Disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic
acid. J. Antimicrob. Chemother. 52: 790–795.
chapter twenty-three
Antiprotozoal agents derived from

natural soil and aquatic actinobacteria
Fighting one microbe with another
Contents
23.1 Introduction......................................................................................................................... 457
23.2 Hurdles in protozoan eradication: vector removal and vaccine development
strategies.............................................................................................................................. 459
23.2.1 Hurdles in protozoan treatment: current strategies and drug repurposing...... 461
23.3 New sources of antiprotozoal drugs................................................................................ 465
23.3.1 Terrestrial and endophytic microbes................................................................... 465
23.3.2 Aquatic microbes.................................................................................................... 467
23.4 Conclusions and further directions................................................................................. 470
References...................................................................................................................................... 471
23.1 Introduction
Human protozoan infections are common in developing nations, where economic
resources for their treatment are Spartan at best, but are an issue around the world
(Andrews et al., 2014). A list of the most common protozoa, their transmission methods,
and their mortality is shown in Table 23.1. Protozoa are transmitted via two main meth-
ods: some participate in the life cycle of insects and are acquired through blood meals
(insect vector–borne diseases), and others are acquired through ingestion of contaminated
food or water (Fletcher et al., 2012; Andrews et al., 2014).
Insect vector–borne diseases such as malaria, sleeping sickness, and Chagas’ disease
are typically associated with tropical and subtropical climates due to the limitations in
the temperature survival range of the vector as well as the protozoan itself (Beck-Johnson
et al., 2013). Typical in this respect is the parasite–vector relationship between Plasmodium,
the agent of malaria, and Anopheles, the mosquito, which transmits it (Beck-Johnson et al.,
2013). Studies by Beck-Johnson et al. (2013) suggest that the baseline for persistence of the
Anopheles mosquito is 20°C–26°C, that is, the minimum temperature at which juvenile
insects can grow into adults to generate a significant population. The authors’ research
also predicted that in order for mosquitoes to be long lived, temperatures needed to be
in the 20°C–30°C range (Beck-Johnson et al., 2013). These observations correspond with
decreased longevity of Anopheles at temperatures above 32°C and in the East Africa high-
lands where temperatures are cool. Similar temperature range data (20°C–26°C) have been
seen for the vector of Trypanosoma brucei, the tsetse fly (Moore et al., 2011). In contrast, the
457
Table 23.1 Global impact of common human protozoal parasites

Protozoa Annual rates Spread by Annual deaths
Cryptosporidium 20% of diarrhea in Contaminated 30%–50% of deaths in
parvuma–c developing countriesb food and water children under 5 yearsc
Entamoeba 500 millionb Contaminated 100,000b
histolytica food and water
Giardia lamblia 20%–30% prevalence in Contaminated Unknown
(giardiasis) developing countries food and water
Leishmania speciesa 12 million Phlebotamine fly 52,000 (2010)
Plasmodiuma species 207 milliond Anopheles 627,000 (2012)
(malaria) mosquito
Toxoplasma gondiia 25%–30% of the world’s Zoonosis or May contribute to
(toxoplasmosis) population are infected mother to child epilepsye or
schizophreniaf
Trypanasoma bruceia 7000–8000 reportedg Tsetse fly 9,000 (2010)
(HAT)
T. cruzia (Chagas’) 8–10 milliona Reduviid bug 10,000–20,000 (2010)a,h
Abbreviations: HAT, Human African trypanosomiasis (sleeping sickness).
a Andrews et al. (2014).
b Fletcher et al. (2012).
c Snelling et al. (2007).
d World Health Organization (2013).
e Flegr et al., (2002).
f Torrey et al. (2007).
g Mumba et al. (2011).
h Kirchhoff (2011).
reduviid (triatomine) bug, the agent of Chagas’ disease, requires slightly higher tempera-
tures but similarly possesses a very narrow optimal range of 26°C–29°C (Lazzari, 1991).
In contrast, although many of the insect vector–associated conditions are primarily
seen in developing nations, there is strong evidence that gastrointestinal protozoa are
carried asymptomatically across the globe but to a lesser degree in developed versus
developing nations (Fletcher et al., 2012). A prime example of differential carriage is seen
in Cryptosporidium parvum, which is the most common causes of gastrointestinal illness
in the world (Fletcher et al., 2012). While C. parvum has a asymptomatic carriage rate
of 0.1% among people in developed countries (Fletcher et al., 2012), typically 10%–30%
of those in developing nations carry the microbe (Current and Garcia, 1991). The num-
ber of individuals with antibodies against C. parvum, that is, demonstrating previous
exposure (seroprevalence rate), ranges from 25% to 35% in industrialized countries, up
to 68%–88% in Russia (Egorov et al., 2004), and 95% in South America (Casemore et al.,
1997). Seroprevalence rates increase with age (Egorov et al., 2004) and are higher in those
working with animals, for example, dairy farmers (Lengerich et al., 1993), and in day-care
centers (Kuhls et al., 1994).
As with other infectious diseases, the approach to reducing the incidence of proto-
zoan infection has been via a classical two-pronged attack, namely prevention of infec-
tion and effective treatment. However, unlike other infectious diseases, protozoa have not
been as simple to combat by traditional methods and to understand why requires a fuller
understanding of the microbes themselves. In terms of prevention, the first issue is that
several of the successful pathogenic protozoa have exploited transmission by insect vec-
tors and require sophisticated eradication techniques in order to reduce infection rates
Chapter twenty-three: Antiprotozoal agents from natural soil and aquatic actinobacteria 459
(Alphey et al., 2010). Furthermore, prevention by vaccination has been stymied since pro-
tozoa, unlike bacteria and the majority of viruses, have the capacity to rapidly alter their
surface antigens and thus exist in many forms (Gurarie and McKenzie, 2006; Baral, 2010).
In terms of effective treatment of protozoa, the main issue is that since they are precursors
and very similar to animal cells, the therapeutics that kill them or prevent their reproduc-
tion typically also damage human tissues (Andrews et al., 2014).
Given the issues described earlier, it is apparent that much of the focus remains on the
development of novel antiprotozoal therapies, which display lower toxicity and greater
specificity against the parasites they are designed to eliminate. Such compounds would be
expected to be the products of moist soil and water bacteria, which share their ecosystems
and wish to avoid predation (Fenical and Jensen, 2006). Indeed, important in this respect
are likely to be Actinobacteria, known antimicrobial-producing organisms that are domi-
nant in most soil and aquatic ecosystems (Hogan, 2010). Actinobacteria have been shown to
be less likely to be eaten by protozoa that are bacterial predators (Fenical and Jensen, 2006),
and it is of interest that the parent compound of the antiprotozoal drug metronidazole was
originally isolated from the actinomycete Streptomyces eurocidus (Osato et al., 1955). To fully
understand the difficulties in controlling protozoal infections, the next two sections deal
with the current status of eradication and treatment strategies. The final sections then
explore the currently available literature on natural antiprotozoal agents from soil and
aquatic Actinobacteria as well as suggesting further directions for drug discovery for this
urgently needed field of infectious disease.
23.2 Hurdles in protozoan eradication: vector removal

and vaccine development strategies
For those protozoa that are acquired through contaminated food or water, there are a
number of practices that can help in limiting disease acquisition, including safe cook-
ing practices, hand washing, and boiling or chlorination of water (Fletcher et al., 2012).
In 2012, only 67% of the world’s population had access to potable water and that statistic
is not expected to improve by 2015 according to the World Health Organization (2012). In
addition, two of the three major protozoal causes of gastrointestinal infection, namely, C.
parvum and G. lamblia, produce small cysts resistant to the effects of chlorination and are
easily able to enter municipal water supplies (Carmena et al., 2007). Indeed, in a study by
Carmena and colleagues (2007), Cryptosporidium cysts were found in 15.4%–63.5% of vari-
ous raw surface water samples, 30.8% of treated water from small treatment facilities, and
26.8% of chlorinated tap water. Hardy Giardia cysts were found in 26.9%–92.3% of vari-
ous raw surface water samples, 19.2% of treated water from small treatment facilities, and
26.8% of chlorinated tap water (Carmena et al., 2007). Thus, contact of food with contami-
nated water used for irrigation or food preparation or contact with sewage can then spread
protozoa further into the human food chain (Fletcher et al., 2012).
To improve potable water, bios and filtration has been used as a low-cost method to
purify water in a number of developing countries, most notably Kenya (Tiwari et al., 2009).
Hazard Analysis Critical Control Point (HACCP) principles, which identify and address
areas where water contamination can occur, have been applied in some countries as a
means of providing potable water free of microbiological health hazards (Gunnarsdottir
and Gissurarson, 2008). Slow sand filtration, solar technology, and membrane technology
are effective methods for reducing pollution from rainwater to be used as a safe drinking
water supply (Helmreich and Horn, 2009).
For insect eradication, insecticides have had some success in controlling these agents,
especially triatomine bugs that spread Chagas’ disease (Schofield and Dias, 1999). The
Southern Cone Initiative, mounted in six South American countries (Argentina, Bolivia,
Brazil, Chile, Paraguay, and Uruguay) in 1991, sought to keep triatomine bugs out of the
dwellings of residents by using housing with less crevasses to eliminate hiding spaces and
spraying insecticide (Schofield and Dias, 1999). These measures, together with screening
all blood donors for infection, reduced Chagas’ disease rates by over 70% in these coun-
tries (Schofield and Dias, 1999).
An additional approach to insect vector reduction has been the sterile insect technique
(SIT) in which irradiated, sterile male insects are bred and released (Alphey et al., 2010).
Since the sterile males are unable to have offspring with wild females, they reduce the
insect population over a series of seasons (Alphey et al., 2010). This strategy has recently
proven very successful in reducing the tsetse fly population in Senegal by 99% (Dicko
et al., 2014). Mosquito control has also been assisted by using the SIT program, and on the
surface, this seems a more effective method than spraying of insecticides from crop planes
(Alphey et al., 2010).
There are, however, two downsides to the SIT program. The first is that it is expensive
to run and therefore not cost-effective for most developing nations (Ansari et al., 1977;
Dame et al., 1981). Production costs for sterile males of the Anopheles albimanus MACHO
mosquito strain in El Salvador in 1979 were estimated at US$156 per million (Dame et al.,
1981) and for Aedes aegypti and Culex pipiens fatigans mosquito pupae at US$58 and US$50,
respectively, per million in India in 1975 (Ansari et al., 1977). These numbers do not sound
a great deal but when translated into the millions of insects needed make the options
very costly. Secondly, for insects where juveniles are also damaging, such as the redu-
viid (triatomine) bugs that spread Chagas’ disease, this technique has been less effective
(Alphey et al., 2010). Thirdly, simply removing the insect vector may not be enough as
some experts believe that if this occurred, others would merely take their place as carriers
of disease (Fang, 2010). Finally, cross-breeding between insect species might also increase
the area in which they can survive (Lounibos et al., 2002). Indeed, a study by Lounibos
et al. (2002) demonstrated that Aedes aegypti mosquitoes could be fertilized by another spe-
cies, A. albopictus (Asian tiger), which can survive much cooler temperatures. If we add in
cross-breeding to the equation, these results suggest that vector control is only part of the
solution to protozoal disease.
A second strike that has spelt doom for many infections has been the use of vac-
cines, which have delivered numerous bacteria and viruses into the ash can of history.
Despite years of trying, however, vaccine development against protozoa still remains in
its infancy, with only two vaccines showing at least partial efficacy (Dumonteil et al., 2012;
Walsh, 2013). The first is a vaccine against childhood malaria that has shown a 25% effi-
cacy against infection in early clinical trials (Walsh, 2013). The second is a sophisticated,
recombinant vaccine that appears to slow the progression of early Chagas’ disease (caused
by Trypanosoma cruzi) in patients who have either indeterminate (those who have symp-
toms but have not produced antibodies) or determinate (those with both symptoms and
antibodies) disease (Dumonteil et al., 2012). A G. lamblia vaccine has been available for dogs
since 2000, but no effective human vaccine has yet followed (Olsen et al., 2000). Finally, it is
possible that a live attenuated vaccine may be made for L. donovani using gene knockout–
created versions of the pathogen (Dey et al., 2013). This vaccine, which involves mutation
of the p27 gene in the protozoan, has recently been shown to be effective in murine models
(Dey et al., 2013), but its efficacy in humans remains to be established. No other vaccines
for protozoa exist or are under development.
In the absence of successful preventative methods, research into treatment methods

has increased (Andrews et al., 2014). Currently used antiprotozoal pharmaceuticals include
those originally developed for the treatment of protozoa and those developed for other
conditions, including cancer treatment, have been successfully redirected for protozoal
therapy. The focus of the next section is to delve into these strategies further with a view to
underlying weaknesses that might be rectified by future drug development.
23.2.1 Hurdles in protozoan treatment: current strategies and drug repurposing

Current treatment methods and their limitations for the treatment of the eight main
global protozoal diseases are shown in Table 23.2. Issues with drug treatment of protozoa
can be grouped into several criteria, namely resistant strain emergence, toxicity of the
drugs, and contraindicated patients. Resistant strains are a particular problem for the
treatment of malaria (Andrews et al., 2014) as well as trypanosomal diseases (Kirchhoff,
2011; Odero, 2013) and are thought to result from the significant antigenic variation dem-
onstrated by these species (Gurarie and McKenzie, 2006; Chitanga et al., 2011). Indeed,
in the case of malaria, studies by Gurarie and McKenzie (2006) have suggested that in a
typical infection, the protozoa present will vary in growth profile, antigenic presentation,
and consequently drug sensitivity/resistance. Selective pressure provided by the drug
being administered will then sway the distribution in favor of the typically slower grow-
ing drug-resistant strains and eliminate the faster growing drug-sensitive ones (Gurarie
and McKenzie, 2006).
Table 23.2 Treatment strategies for major protozoal diseases

Protozoa Treatments Issues with treatment
C. parvum Nitazoxanide (3–14 days) a AIDS patients need a higher dosagea
E. histolytica Metronidazole (mild) paromomycin or Issues with differential penetration
iodoquinol (invasive) (7 days)a of drugs to reach tissue amebab
G. lamblia Metronidazole (3 days), tinidazole (single Treatment failures and relapses
dose), or albendazole (5 days)a occur probably indicative of
resistant strainsc
Leishmania Pentamidine/Amp B (IV; 28 days)d Variable efficacy and toxicitye
Plasmodium Gold standard are ACTs (3 days)f Resistant strains emergingd,g
Toxoplasma Pyrimethamine, combined with various Adverse toxic effects and concerns
gondii drugs (4–6 weeks)h about efficacyc
Trypanosoma Early = suramin (IV; 21 days) or pentamidine Pentamidine is very toxic,
brucei (HAT) (IV; 14–21 days). Late = melarsoprol melarsoprol-resistant strains now
(10 days) or eflornithine (4 weeks)i very common (~30% of species)i
T. cruzi Nifurtimox, benznidazole (years)j Toxicity, resistancej
Abbreviations: ACTs, Artemisinin combination therapies; Amp B, amphoteracin B; HAT, Human African trypano-
somiasis (sleeping sickness); IV, Intravenous.
a Fletcher et al. (2012).
b Espinosa et al. (2012).
c Busatti et al. (2009).
d Andrews et al. (2014).
e Croft and Coombs (2003).
f Perez-Jorge (2014).
g Ariey et al. (2014).
h Hökelek (2014).
i Odero (2013).
j Kirchhoff (2011).
Trypanosome resistance is an even more complex phenomenon that seems tied to

their immunological diversity (Baral, 2010). These protozoa possess a unique capacity to
alter their variant specific glycoproteins (VSGs) into procyclic acidic repetitive proteins
(PARPs, or procyclins) and then later back to VSG (Baral, 2010). Apart from hampering
vaccine development, this also means that trypanosomes develop drug resistance that is
not driven by selective pressure but instead by favorable, random mutations (Chitanga
et al., 2011).
Studies by Chitanga et al. (2011) examined the incidence of diminazene aceturate
(DA) resistance in a trypanosome strain related to human African trypanosomiasis (HAT)
namely T. congolese. DA is typically given to livestock to treat trypanosome infections,
and although natural populations of T. congolese also infect wildlife, these would not be
expected to demonstrate resistance to it due to lack of exposure (Chitanga et al., 2011).
Chitanga et al. (2011) were surprised to learn that 33 of the 34 strains (97.1%) they isolated
from wildlife demonstrated either full resistance (12/34; 35.2%) or partial resistance (21/34;
61.8%) to DA, suggesting that protozoan resistance was a random mutational event, that is,
not driven by selective pressure.
Apart from resistance, the second major issue with treating protozoal infections has
been the toxicity of the drugs being employed (Kirchhoff, 2011; Croft and Coombs, 2003;
Odero, 2013; Andrews et al., 2014). The treatment of late-stage infections with T. brucei is a
case in point, with melarsoprol, a derivative of arsenic, demonstrating severe side effects
including heart failure and death (Brun et al., 2011). Even in the “safer” forms of antiproto-
zoal medications currently on the market, there are significant contraindications for many
of them (Table 23.2). Treatment failures whether due to protozoan resistance (Andrews
et al., 2014) or adverse effects are also common in nifurtimox (Coura et al., 1997; Jackson
et al., 2010) and benznidazole treatments (Coura et al., 1997; Pinazo et al., 2010). Indeed, in
a recent study by Pinazo et al. (2010), performed in an area with good clinical care (Spain),
adverse events from benznidazole treatment of Chagas’ disease in adults were of the order
of 9% of patients studied. The most common issues were severe skin rash (urticaria) and
gastrointestinal distress (Pinazo et al., 2010). Nifurtimox produced even higher numbers
of adverse events, according to a study by Jackson et al. (2010). These authors observed a
97.5% rate of adverse events, which were often gastrointestinal distress (35.1%) or neuro-
logical (27.5%) (Jackson et al., 2010). Sudden critical reactions were also seen in 6.4% of the
patients, necessitating immediate withdrawal from the study (Jackson et al., 2010). Jackson
et al. (2010) also made a very worrying observation, namely that their “results call into ques-
tion the safety of large-scale use of nifurtimox among adult patients in settings with difficult access
to medical care, such as poor rural areas of South America” and that “Easily accessible medical care
and close clinical follow-up appear to be prerequisite to nifurtimox treatment.”
In treating protozoan infections, it has also become apparent that combination thera-
pies seem to be optimal to avoid the development of resistance in the organism (Burrows
et al., 2013; Andrews et al., 2014). This has been particularly important in the treatment of
diseases like malaria, where the original quinine and then chloroquine–primaquine com-
binations resulted in rapid development of resistance (Burrows et al., 2013). Nifurtimox–
eflornithine combination therapy (NCET) has also recently become a treatment option for
HAT, but sadly this is only effective against the gambiense form of the disease and not the
rapidly fatal rhodiense form (Priotto et al., 2007, 2009).
Given that new combinations and therapies needed to be developed, pharmaceuti-
cal companies have had to make some choices. Although new drug discovery is appeal-
ing, cost (around US$800 million) and time (one to two decades) of drug development
have been major obstacles and have limited development of significant numbers of novel
antimicrobials from nature. In contrast, the use of high-throughput targeted searches of

existing drug mechanisms has led to their being put to different uses (Andrews et al.,
2014), termed repurposing. Repurposing an existing product has many benefits for both
the developer and the consumer other than time to market, namely extant safety data and
information about side effects (Andrews et al., 2014). Since many protozoan infections are
not associated with significant revenue for companies investing in them, they have there-
fore become the preferred route to identify new antiprotozoal strategies.
At least nine protozoan drugs exist that were originally developed for use in other
areas, most notably chemotherapy and infectious disease treatment (Andrews et al., 2014).
Such repurposing can occur because the drug is found to have multiple efficacies, as
with doxycycline (Tan et al., 2011) and amphotericin B (Ostrovsky-Ziechner et al., 2003),
or because the drug was not effective as a chemotherapeutic agent, as with miltefosine
(Dorlo et al., 2012). Like other extant antimicrobial drugs, some repurposed drugs are of
natural origin including paromomycin (Wiwanitkit, 2012), semisynthetic like doxycycline
(Pickens and Tang, 2010; Tan et al., 2011), or synthetic like miltefosine (Croft and Engel,
2006) and sulfadiazine (Leport et al., 1988). A list of some commonly used protozoal treat-
ments and their origins is shown in Table 23.3.
Drug repurposing has been facilitated by an understanding of protozoan biology as
well as a definition of the molecular targets for the therapeutics themselves (Nwaka and
Hudson, 2006). Studies have shown that T. brucei and T. cruzi together with malaria and
leishmania express adenylate cyclase, phosphodiesterase (PDE), and kinase enzymes,
Table 23.3 Origin and usage of drugs repurposed for protozoal treatment regimens
Protozoan Therapeutic Origin Original uses
Leishmaniasis Miltefosinea Synthetica Breast cancera
Plasmodium Clindamycinb Natural; Streptomyces lincolnensisb Antibioticb
Doxycyclinec,d Semi-synthetic; oxytetracycline Antibioticc,d
parent molecule from S. rimosusc
Sulfamethoxazolef Synthetic; derived from dye Pe Antibiotice
Trimethoprimf Syntheticg. Antibiotice
T. gondii Spiramycinh Natural; S. ambofaciensi Antibiotici
T. brucei Amphotericin Bj Natural; S. nodosusk Antifungalj
Eflornithinel Syntheticm Antitumorn and hirsutismo
Paromomycinp Natural; S. rimosusp Antibioticp
Source: Modified from Andrews, K.T. et al., Int. J. Parasitol. Drugs Drug Resist., 4, 95, 2014.
a Croft and Engel (2006).
b Spízek and Rezanka (2004).
c Pickens and Tang (2010).
d Tan et al. (2011).
e Lesch (2007).
f Manyando et al. (2013).
g Eliopoulos and Huovinen (2001).
h Couvreur et al. (1988).
i Karray et al. (2007).
j Ostrovsky-Zeichner et al. (2003).
k Caffrey et al. (2001).
l Kennedy (2013).
m Metcalf et al. (1978).
n Gerner and Mayskens (2004).
o Wolf et al. (2007).
p Wiwanitkit (2012).
which have slightly differing structures to those of humans (Parsons et al., 2005). Drugs
antagonistic to these three target enzymes have been used with much success in tumor
therapy (Andrews et al., 2014), where they are effective against rapidly multiplying cells,
a situation also seen in malaria, trypanosome, and leishmania infections (Andrews et al.,
2014). To date, four classes of chemotherapeutic drugs have been explored for their ability
to suppress protozoa, namely histone deacetylase (HDAC) enzyme inhibitors (Kelly et al.,
2012; Subathdrage et al., 2012), PDE inhibitors (De Koning et al., 2012), carbonic anhydrase
inhibitors (Krungrkai et al., 2008), and tyrosine kinase inhibitors (Patel et al., 2013).
HDAC are a group of therapeutics that have received general acceptance for the treat-
ment of T cell lymphoma and have an effect on the proteins that regulate cell proliferation
and differentiation in mammalian cells (Dokmanovic et al., 2007). These agents are cur-
rently being tested as to their potential efficacy in the treatment of malaria (Subathdrage
et al., 2012) and HAT (Kelly et al., 2012) with promising in vitro results already being
reported at predicted human serum physiological levels (micromolar range). Recent stud-
ies by De Koning et al. (2012) demonstrated that the use of the PDE inhibitor tetrahydroph-
thalazinone, which they termed compound A (Cpd A), was able to significantly affect the
in vitro growth of T. brucei. Their results showed that T. brucei proliferation was inhibited
immediately and that protozoa died within 3 days (De Koning et al., 2012). De Koning et al.
(2012) also observed that Cpd A prevented cell division of the trypanosome, resulting in
multinucleated, multiflagellated cells that eventually lysed (De Koning et al., 2012).
Krungrai et al. (2008) tested a library of carbonic anhydrase inhibitors for efficacy
in inhibiting the growth of P. falciparum in cell cultures. They observed one compound
to be optimal in this respect, the sulfonamide derivative 4-(3,4-dichlorophenylureido)
thioureido-benzenesulfonamide, which was inhibitory when used at concentrations as
low as 0.18 µM (Krungrai et al., 2008). Finally, the anilinoquinazoline drugs lapatinib and
canertinib, which are potent synthetic tyrosine kinase inhibitors, have also been shown
to be effective against T. cruzi at micromolar levels (estimated dose 50 µM) through
blocking the parasite’s uptake of transferrin and effectively starving it (Patel et al., 2013).
Quinazoline-based drugs have at their heart a dicyclic benzene plus pyrimidine ring and
have had numerous uses, most notably antiviral and antibacterial (Wang and Gao, 2013).
This latter class of drugs is of interest since they are also structurally similar to the pow-
erful fluoroquinolone antibiotics, which include ciprofloxacin (Cipro) and levafloxacin
(Levaquin) (Wang and Gao, 2013).
Despite the advantages of repurposing extant drugs, this strategy is not, however,
without risks, and these mostly surround the usage of synthetic agents. The first is that
the rapidity with which protozoa develop resistance toward artificial compounds cannot
be predicted. The second concerns the reason for repurposing, that is, whether the drugs
have significant side effects and therefore require patient monitoring. This second issue
may make usage nonviable in developing countries, which is unfortunately where most
protozoan infections occur. Third, natural antimicrobials and their derivatives typically
possess better bioavailability and capacity to bind to cellular targets than their synthetic
counterparts, again lending support for new therapeutics to come from natural sources
(Wright, 2010). Thus, despite the considerable time and costs, it still behooves pharmaceuti-
cal companies to invest and investigate new sources for antiprotozoal drugs.
As with antibiotics, it appears that many of these will come from the common ter-
restrial and aquatic Actinobacteria species, with the dominant genus of interest being
Streptomyces (Raja and Prabakarana, 2011). Actively feeding and reproducing protozoa
have a need for water, although they can exist in cyst form in dry, desert soils (Darby
et al., 2006). Since they are common to all types of water environments and moist soils, it
is to be expected that Actinobacteria isolated from these locations will secrete antiprotozo-
als to avoid being grazed on (Fenical and Jensen, 2006). The next section deals with novel
antiprotozoal drugs that have been identified from naturally occurring microbes in these
environments.
23.3 New sources of antiprotozoal drugs

Bioprospecting for antibiotic agents began with the first discoveries of the soil-dwelling
Streptomyces species by Selman Waksman in 1952 (Waksman and Lechevalier, 1962) and
have continued apace ever since (Raja and Prabakarana, 2011). Actinobacteria are a substan-
tial and diverse group of microbes able to retain dominance in extreme habitats (Kieft, 1991;
Kurapova et al., 2012) as well as those with rich microenvironments (Piechoski et al., 2012).
They are the source of 80% of the world’s currently available antimicrobial drugs (Raja and
Prabakarana, 2011), and this usefulness has not even been fully explored yet. Given that
they are also the source of the majority of repurposed antiprotozoal drugs (Table 23.3),
many scientists are now investigating the capacity of terrestrial and aquatic Actinobacteria
to generate novel therapeutic agents. The next two sections explore the state-of-the-art
knowledge on novel antiprotozoal agents gleaned from these two types of environments.
23.3.1 Terrestrial and endophytic microbes

Several currently available antiprotozoal drugs have been derived from at least four
Streptomyces species, with S. lincolensis (Ryu et al., 2005) and S. rimosus (Duggar, 1948) being
isolated from cultivated, temperate soils in Lincoln, Nebraska and Columbia, Missouri,
respectively. The remaining two have come from diverse tropical soils including China
(S. ambofaciens; Pinnert-Sindico, 1954) and the Orinoco Basin of Venezuela (S. nodosus;
Donovick et al., 1954). Although these sound very disparate places, it is important to note
that each would be expected to boast a rich microbial ecosystem in which competition
for organic nutrients would be intense (Hawksworth and Colwell, 1992). Surviving such
locations would need a “competitive edge,” such as that provided by antimicrobials, and
dominant microbes would therefore be expected to possess this capability. It is not unsur-
prising that Streptomyces species constitute 40% of all soil bacteria (Boone et al., 2001), and
under dry and alkaline conditions, these dominate the terrestrial microbial population
(Vetsigian et al., 2011).
Although soil and water are primary Streptomyces habitats (Ward and Bora 2006;
Mokrane et al., 2013), they can also be found in hay (Horn et al., 2012) such as endophytes
associating with plants (Ezra et al., 2004; Castillo et al., 2006) and in the gastrointestinal
tracts of insects such as termites (Maheswarappa et al., 2013) and wasps (Oh et al., 2011).
Each of these locations has its own series of Actinobacteria and very different antimicrobial
abilities, and several have potential antiprotozoal activities as shown in Table 23.4.
At least two of the newly discovered antiprotozoal agents from terrestrial Streptomyces,
pyrocoll and prodigiosin, contain pyrolle rings (Dieter et al., 2003; Maheswarappa et al.,
2013). Pyrocoll’s pyrolle ring is attached to a diketopiperazine core (Dieter et al., 2003),
whereas prodigiosin possesses a tripyrrole structure (Maheswarappa et al., 2013), but for
both, the pyrolle ring structure is at the heart of their activities (Genes et al., 2011). The
importance of the pyrolle ring as an antimicrobial entity has been known for over a decade
(Bijev et al., 2000, 2002) and underlined by the more recent studies of Varshney et al.
(2014). Early studies by Bijev et al. (2000, 2002) synthesized a total of 12 novel pyrolle ring-
containing cephalosporin derivatives with antibacterial activity comparable to cefalexin
Table 23.4 Novel antiprotozoal agents from terrestrial Actinobacteria

Location/Soil Type Species Agent Protozoal target
Consett, County Durham Streptomyces AK409 Pyrocoll (pyrrole-2- Plasmodium and
United Kingdom carboxylic acid) Trypanosoma
(alkaline, black soil)a species
Yogyakarta, Indonesia Streptomyces Jogyamycin (analog of Plasmodium
(monsoon climate, a-WM-JG-16.2 pactamycin)c species
volcanic-mix soil)b
Manu region, Peru on Endophytic Streptomyces Coronomycin Plasmodium
Monstera plant speciesd (MSU-2110) (functionalized peptide) species
Northern Territory, Endophytic Streptomyces Munumbicins Plasmodium
Australia on Kennedia NRRL 3052 species
(snakevine) plantse
Soil termite gutf Unknown Streptomyces Prodigiosin (tripyrrole) T. cruzi (Chagas’
disease)g
Tamil Nadu, India Streptomyces VITVSK1 Quinone derivative Plasmodium
(saltpan)h species
a Dieter et al. (2003).
b Iwatsuki et al. (2012).
c Hanessian et al. (2013).
d Ezra et al. (2004).
e Castillo et al. (2006).
f Maheswarappa et al. (2013).
g Genes et al. (2011).
h Gopal et al. (2013).
(Bijev et al., 2000, 2002). This group also observed that those cephalosphorins in which
the beta-free position and ester groups remained accessible, that is, uncoupled, were typi-
cally the most antimicrobial (Bijev et al., 2000, 2002). Varshney et al. (2014) confirmed these
observations and additionally observed increased antimicrobial potency with substi-
tuted 2,5-dimethyl pyrrole derivatives and substituted 1,3-benzoxazine-4-one derivatives.
Indeed, a patent for the use of diaryl piperidyl pyrrole derivatives as antiprotozoal agents
was issued in 2006 (Biftu et al., 2006).
Interestingly, prodigiosin possesses all these qualities since it is a 4-methoxy-5-[(Z)-
(5-methyl-4-pentyl-2H-pyrrol-2-ylidene) methyl]-1H,1′H-2,2′-bipyrrole (Khanafari et al.,
2006). Furthermore, prodigiosin has been shown to be effective in killing not only bacteria
but also the protozoan T. cruzi (Genes et al., 2011). Genes et al. (2011) determined that the
death of T. cruzi occurred through the induction of apoptotic death of their mitochondria,
in a similar manner to that of cancer cells.
Although prodigiosin and the pyrolle-ringed derivatives that could be made from
it have potential, the question at this point may be why are some Actinobacteria mak-
ing these compounds? In the case of prodigiosin, this synthesis may be a success-
ful evolutionary strategy related to continued survival of the triatomine-associated
Streptomyces during T. cruzi infection (Genes et al., 2011). Early studies by Azumbuja
et al. (2004) demonstrated that ingestion of a blood meal containing T. cruzi caused an
increase in triatomine midgut bacteria populations 10,000 fold within a few hours. The
authors showed that this increase was specific to Serratia and Actinobacteria species and
was associated with a decline in viability in T. cruzi parasites, presumably through the
antiprotozoal actions of prodigiosin (Azambuja et al., 2004). Such Actinobacteria–insect
mutualism has been previously associated with the manufacture of dentigerumycin, a
selective antifungal in fungus-farming ants (Oh et al., 2011), and sceliphrolactam, from
Streptomyces living in wasp midguts (Oh et al., 2011).
In addition to mutualism with insects, some Streptomyces have established endophytic
relationships with plants, which may or may not be beneficial (Ezra et al., 2004; Castillo
et al., 2006). Since the plant–bacterial interaction is a specific one, this is clearly another
potential source of unique antimicrobials. At least two useful antimalarial agents have
been isolated to date, the munumbicins in 2002 (Castillo et al., 2006) and the coronamy-
cins in 2004 (Ezra et al., 2004). Both appear to be peptide antibiotics with strong efficacy
against the malaria parasite but have not been further developed since their discoveries
a decade ago.
Bioprospecting yields new compounds but may frequently also uncover modifica-
tions of known antimicrobials. This has certainly been the case for jogyamycin, which
appears to be a structural analog of pactamycin (Iwatsuki et al., 2012). Pactamycin was
first isolated from S. pactum in 1961 and appeared to have great potential as an anti-
microbial agent until its toxicity emerged (Hanessian et al., 2013). Structure–activity
analysis of pactamycin has recently led to the generation of successful synthetic and
semisynthetic antiprotozoal agents, which have revolved around modifications of the
6-methylsalicylic acid (6-MSA) ester moiety (Hanessian et al., 2013). Interestingly, the
natural product jogyamycin, which is a de-6-methylsalicylyl-7-deoxypactamycin con-
gener of pactamycin, is more potent than the parent molecule against trypanosomes
and possesses less toxicity (Iwatsuki et al., 2012; Hanessian et al., 2013). Finally, the
discovery of the quinone derivative being secreted by saltpan Streptomyces (Gopal et al.,
2013) may open up possibilities for this class of agents to be used in malarial treatment.
Although structurally separate from quinine and its derivatives, which have been tra-
ditionally used for malaria, quinones and their substitutions have been long known to
possess broad-spectrum antimicrobial activity (Valderrama et al., 1999). They have also
proven particularly useful in the treatment of cancer; for example, the semisynthetic
drug adriamycin (doxorubicin) is an anthracycline that contains a quinone ring at the
heart of its activities (Weiss, 1992). Given that many of the repurposed drugs being used
for antiprotozoal therapy seem to have originated as chemotherapeutics (Table 23.2),
quinones and their derivatives may also have a role to play in the treatment of both
types of conditions.
23.3.2 Aquatic microbes

The marine environment is the largest ecosystem on the planet and probably possesses
the greatest biodiversity in the world (Felczykowska et al., 2012). Actinobacteria species
abound in marine environments, comprising about 10% of the species, are harvested from
marine aggregates and sediments (Ward and Bora, 2006). Some of these species may be
unique to ocean environments (Jensen et al., 2005). For example, Salinispora (Maldonado
et al., 2005) and Marinispora (Jensen et al., 2005), which require seawater for growth, have
distinct marine chemotype signatures.
Although able to exist independently of vertebrate and invertebrate life, Actinobacteria
are often found in association with ocean corals (Lam, 2006). Coral-dwelling species
include members of the Streptomyces, Gordoniacae, and Actinokineospora genera and are
often unique to each type of cnidarian (Lam, 2006). As with terrestrial locations, those
Actinobacteria able to live in extreme environments, including those of the Arctic and
Antarctic oceans, in the deep oceans, or in seas with high levels of pollutants, are poorly
understood since their culture is problematic (Barone et al., 2014). Indeed, it has been
estimated that less than 0.1% of all microbes in the oceans today have been discovered so
far (Simon and Daniel, 2009). Given the issues with isolation and culture of these impor-
tant microbes, many investigators have turned to the use of metagenomics (Stach et al.,
2003; Mincer et al., 2005). Metagenomic methods, through which genes are identified via
high-throughput DNA sequencing technology and cloned into culturable vectors, may
well be one major method to allow the natural products they code for to be identified and
harvested (Tringe et al., 2005). In recent years, the discovery of several antiprotozoal agents
using both classical isolation and metagenomic techniques has been seen, and the charac-
teristics of those of note are shown in Table 23.5.
Some of the agents originating from marine Actinobacteria have already been discov-
ered and utilized. A prime example of this is echinomycin, which is also made by the
terrestrial species S. lansiliensis (Steinerová et al., 1987) and S. echinatus (Onnis et al., 2009).
Echinomycin was studied as a potential chemotherapeutic agent, when it was found to
inhibit hypoxia-inducible factors (HIFs), but clinical trials were halted in the 1980s due to
lack of efficacy (Onnis et al., 2009). The observations of Espinosa et al. (2012) that marine
Streptomyces-isolated echinomycin inhibited the multiplication of Entamoeba histolytica
suggest that we may again see a chemotherapeutic being repurposed as an antiprotozoal.
Similarly, tirandamycin, also originally identified from two terrestrial Streptomyces species
(Rahman et al., 2010), has now been shown to possess both antiamoebic (Espinosa et al.,
2012) and antinematodal activities (Yu et al., 2011), suggesting that this drug may also be
repurposed.
Others are novel antimalarial agents, including specific protease inhibitors (Karthik
et al., 2014), the polyketide mollemycin (Raju et al., 2014), and the trioxacarcin gutingimy-
cin (Manivasagan et al., 2014). Marine Actinobacteria have long been known to produce a
Table 23.5 Antiprotozoal agents from marine Actinobacteria

Protozoa
Location Species Agent(s) Target affected
Fishers Island Sound, Novel Streptomyces Echinomycin, DNA replication E. histolytica
United Statesa UR-F11 and URI-F39 tirandamycin RNA polymerase
Nicobar, Indian Unknown Unknown Protease Plasmodium
Oceanb Actinobacteria inhibition species
South Molle Island, Novel Streptomyces Mollemycin Unknown Plasmodium
Queenslandc sp. (CMB-M0244) (polyketide) species
Götingen, Swedend,e Streptomyces species Gutingimycin Unknown Plasmodium
B8652 (trioxacarcin) speciese
Sponge-Associated Actinobacteria
Mediterraneanf Unknown Valinomycin Ion transport Leishmania,
staurosporine T. brucei
Streptomyces Protein kinase C
Butenolide Proapoptoticg
Red seah Actinokineospora Actinosporins Unknown T. bruceih
a Espinosa et al. (2012).
b Karthik et al. (2014).
c Raju et al. (2014).
d Maskey et al. (2004).
e Manivasagan et al. (2014).
f Pimento-Elardo et al. (2010).
g Zhang et al. (2011).
h Abdelmohsen et al. (2014).
variety of important enzymes, including protease inhibitors (Karthik et al., 2014). Although
originally used in laboratory studies of enzyme kinetics, these are now being explored for
their potential as pharmacological agents (Drag and Salvesen, 2010). Antimalarial agents
functioning as highly specific protease inhibitors have also been recently recovered from
marine Actinobacteria (Karthik et al., 2014). These potential therapeutics appear to produce
no liver or spleen toxicity in a murine model (Swiss albino mice), again reinforcing their
potential as future antimalarial agents (Karthik et al., 2014).
Further, these as yet unknown Streptomyces species have the advantage of also being able
to synthesize gold nanoparticles, which exhibit significant inhibitory effects on Plasmodium
multiplication (Karthik et al., 2013). In a murine model, animals infected with a malarial
model strain (P. berghei) demonstrated delayed onset of parasitemia together with increased
survival 8 days postinfection (85% in treated compared with 50% in untreated mice) (Karthik
et al., 2013). Angiotensin-converting enzyme (ACE) and HIV-protease-inhibitor drugs have
already achieved widespread usage, and it would seem, therefore, that marine Actinobacteria
have still much untapped potential in this area.
The antimalarial compounds mollemycin and gutingimycin were only discovered
and/or ascribed this property recently and currently have no established mode of action
against the protozoan (Manivasagan et al., 2014; Raju et al., 2014). Mollemycin has been
classified as a polyketide, but since this is a large group of Streptomyces-derived agents
with diverse effects and targets (Gomes et al., 2013), the delineation does not narrow
down its mode of action. One might speculate that, given its effects, mollemycin is related
to type II polyketide chemotherapeutic drugs, which include doxorubicin (Gomes et al.,
2013). If this is the case, mollemycin exerts its effects by interfering with the enzyme topoi-
somerase II, thereby impairing DNA uncoiling and ultimately cell replication (Pommier
et al., 2010). Thus, mollemycin has the potential to become a chemopreventative or anti-
malarial drug or both.
Gutingimycin is a trioxacarcin, which is a class of compounds now being carefully
scrutinized for chemotherapeutic usage in various types of tumors (Magauer et al., 2013).
Given that trioxacarcins are also able to prevent cell proliferation, it is not therefore unex-
pected that gutingimycin can also prevent protozoan multiplication. With an invention
patent just launched for trioxocarcin A and its derivatives (Gruen-Wollny et al., 2014), it
remains to be seen whether the trioxacarcins and their future derivatives will in turn be
developed or repurposed for use as antimalarial drugs.
Several antiprotozoal compounds have been identified from Mediterranean coral-
associated Actinobacteria including valinomycin, staurosporine, and butenolide (Pimento-
Elardo et al., 2010). None of these agents are novel with staurosporine, for example, being
discovered in 1977 (Omura et al., 1977) and currently in use for the treatment of neuro-
blastoma (Mukthavaram et al., 2013). This discovery that several species of Actinobacteria
synthesizing the same antimicrobial is not unexpected; valinomycin, for example, is pro-
duced by at least 11 separate species of terrestrial and now aquatic Streptomyces species
(Matter et al., 2009). Instead, because Pimento-Elardo et al. (2010) screened for aquatic
microbes producing antitrypanosomal and leishmanial compounds, they may become
repurposed drugs.
In contrast, the actinosporins from Red Sea coral Actinobacteria possess an activity that
is novel, highly effective, and specific against T. brucei (Abdelmohsen et al., 2014). Finally,
studies by Sosovele et al. (2012) have also identified as yet unknown Actinobacteria species
from Dar Es Salaam, Tanzanian mangrove swamps capable of inhibiting the multiplica-
tion of Plasmodium falciparum in vitro. Whether these prove to be novel compounds or new
properties ascribed to extant therapeutics still remains to be clarified.
23.4 Conclusions and further directions

Protozoan infections are a significant cause of morbidity and mortality, particularly in
developing nations (Table 23.1). Unfortunately, at least three of these types of infections,
namely HAT, Chagas’ disease, and leishmaniasis, fall under the parameters of neglected
tropical diseases (NTDs), which have been largely ignored in terms of novel drug devel-
opment and discovery (Fevre et al., 2008). The reason for this is one of simple economics;
people in poorer countries cannot afford to pay for the drugs, and it has, therefore, not
been in the interest of pharmaceutical companies to develop them (Dimitri, 2012).
New initiatives in the area of protozoal drug development are primarily partnerships
that combine industry, research, and government agencies, which combine their abilities
to create and market cheap, effective, and available drugs (Bompart et al., 2011). This began
with the Sanofi-Aventis and Drugs for Neglected Diseases partnership in 2004 (Bompart
et al., 2011). Sanofi-Aventis was able to formulate a nonpatented, fixed-dose combination
of the antimalarial drugs artesunate and amodiaquine and made them relatively inex-
pensive, less than US$1 for an adult and 50 cents for a child’s full treatment (Bompart
et al., 2011). In addition, Sanofi-Aventis also partnered with local experts in the countries
where the antimalarial drugs were being administered in order to fully educate patients
and healthcare professionals about all forms of community malarial prevention (Bompart
et al., 2011). Since then, the combined efforts of pharmaceutical companies and the public
sector have resulted in several new treatments being developed for NTDs, including an
oral pill for HAT currently in late-stage clinical trials (Tran, 2014). Only in the year 2013,
pharmaceutical companies donated 1.4 billion NTD treatments to countries that desper-
ately need them, and each dollar invested is working to make US$10 of pharmaceuticals
(Tran, 2014).
These liaisons address the question of financial backing for antiprotozoal drug devel-
opment and how they will be appropriately administered, but still does not examine where
new, nonrepurposed drugs are going to come from. As can be seen from this chapter, there
are many viable candidates and, clearly, many more out there still waiting to be identified.
Metabolomics has been touted as an optimal screening method for antimicrobials since
it is highly cost-effective and provides rapid answers as to the potential utility of novel
agents (Creek and Barrett, 2014). Two types of studies are typically employed, targeted and
untargeted (Creek and Barrett, 2014). In targeted studies, the investigators are looking for
novel molecules that affect specific pathways or sites in the microbe (Creek and Barrett,
2014). For example, in malaria, there are a series of highly conserved and unique serine
proteases not found in other parasites or in humans and antagonists of these enzymes
have great potential as selective, specific drugs (Alam, 2014).
In reality, most studies are untargeted in nature, being primarily concerned with
determining that a potential agent can kill/prevent the multiplication of the protozoan
target (Creek and Barrett, 2014). Untargeted metabolomics may also uncover the true mode
of action of a drug as was seen in studies of the effective antiprotozoal drug eflornithine
(Vincent et al., 2012). Eflornithine, being a repurposed drug, has long been known to affect
human tumor cells by inhibiting ornithine decarboxylase (ODC) activity and subsequent
polyamine accumulation (Grishin et al., 1999). Metabolomic studies by Vincent et al. (2012)
confirmed its efficacy against ODC when they observed an increase in ornithine and
decrease in putrescine levels in T. brucei protozoa. Interestingly, they also observed that
eflornithine downregulated spermidine levels, and results indicate a targeted activity on
the polyamine pathway of the parasite. The polyamine pathway in trypanosomes is both
essential and unique, combining polyamines and glutathione to generate trypanothione,
which protects the protozoan against oxidative stress (Vincent et al., 2012). Thus, in addi-
tion to identifying the mode of action for eflornithine, these studies have also uncovered a
new target for future drug development, namely those targeting the trypanothione path-
way. Metabolomics will be assisted by understanding of the biochemical pathways within
protozoa, and this is expected to further unearth specific proteins as potential drug targets
(Creek and Barrett, 2014).
Once discovered, there is a second major issue to any drug development from natural
products, which is the ability to generate sufficient quantities. One strategy has been to use
metagenomics, through which natural organisms producing an antimicrobial compound
can be identified and matched with suitable candidate hosts to manufacture these agents
(Gomes et al., 2013). Unfortunately, Streptomyces species have been shown to be unlike any
other bacteria in that they possess a set of preferred codons that runs throughout their
genome (Baltz, 2006; Peirú et al., 2008). This has meant that for other Streptomyces antimi-
crobial products, novel strategies have to be employed for gene insertion and cloning; the
use of mutant species of Escherichia coli will allow for these genes to be inserted (Baltz,
2006; Peirú et al., 2008). Two types of mutation have been successful in this regard (Baltz,
2006; Peirú et al., 2008). The first is to modify the codons present in the Streptomyces gene
sequence to match those found most abundantly in E. coli, thus allowing the host machin-
ery to remain fully functional (Baltz, 2006). The second consists of changes throughout
the E. coli genome that will increase the occurrence of the rare codons seen in Streptomyces
(Peirú et al., 2008). To date, both have proven successful in generating genetically modified
host bacteria able to generate more Streptomyces antimicrobials than the parent organism
(Baltz, 2006; Peirú et al., 2008).
Given that the financial, production, and dissemination aspects of antiprotozoal dis-
covery have successful strategies in place, it may now be anticipated that new and repur-
posed drugs should be appearing over the next decade. Actinobacteria have long been the
primary sources of these agents, responsible for at least 10,000 bioactive compounds cur-
rently in use (Raja and Prabakarana, 2011). Their diversity and untapped potential, espe-
cially in unique and marine environments, should continue to provide novel and effective
antimicrobial agents to replace those of the past. The literature is full of partially or fully
characterized antiprotozoal agents being secreted from these ubiquitous soil and water
microbes, with some being described only recently (Tables 23.4 and 23.5). Whether in their
native form or as synthetic or semisynthetic versions, it is likely that these drugs represent
the future of antiprotozoal drugs able to combat the diseases that currently afflict millions
on our planet. The need for effective antiprotozoals is certainly there and is likely to be met
by agents made by Actinobacteria species, the successful mainstay sources of our antimi-
crobials for the past 62 years.
References
Abdelmohsen, U.R., Cheng, C., Viegelmann, C., Zhang, T., Grkovic, T., Ahmed, S., and Edrada-Ebel,
R. 2014. Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins
A and B from a marine sponge associated-Actinokineospora sp. EG49. Mar. Drugs, 12, 1220–1244.
Alam, A. 2014. Serine proteases of malaria parasite Plasmodium falciparum: Potential as antimalarial
drug targets. Interdiscip. Perspect. Infect. Dis., 2014, 453186.
Alphey, L., Benedict, M., Bellini, R., Clark, G.G., Dame, D.A., Service, M.W., and Dobson, S.L. 2010.
Sterile-insect methods for control of mosquito-borne diseases. Vector Borne Zoonotic Dis., 10(3),
295–311.
Andrews, K.T., Fisher, G., and Skinner-Adams, T.S. 2014. Drug repurposing and human parasitic
protozoan diseases. Int. J. Parasitol. Drugs Drug Resist., 4, 95–111.
Ansari, M.A., Singh, K.R., Brooks, G.D., Malhotra, P.R., and Vaidyanathan, V. 1977. The development
of procedures and techniques for mass rearing of Aedes aegypti. Ind. J. Med. Res., 65(Suppl.),
91–99.
Ariey, F., Witkowski, B., Amaratunga, C., Beghain, J., Langlois, A.C., Khim, N., and Ménard, D. 2014.
A molecular marker of artemisinin resistant Plasmodium falciparum malaria. Nature 505(7481),
50–55.
Azambuja, P., Feder, D., and Garcia, E.S. 2004. Isolation of Serratia marcescens in the midgut of
Rhodnius prolixus: Impact on the establishment of the parasite Trypanosoma cruzi in the vector.
Exp. Parasitol., 107(1–2), 89–96.
Baltz, R.H. 2006. Molecular engineering approaches to peptide, polyketide and other antibiotics.
Nat. Biotechnol., 24, 1533–1540.
Baral, T.N. 2010. Immunobiology of African trypanosomes; need for alternative interventions.
J. Biomed. Biotechnol., 2010, 389153.
Barone, R., De Santi, C., Palma Esposito, F., Tedesco, P., Galati, F., Visone, M., and De Pascale, D. 2014.
Marine metagenomics, a valuable tool for enzymes and bioactive compounds discovery. Front.
Mar. Sci., 1, 38.
Beck-Johnson, L.M., Nelson, W.A., Paaijmans, K.P., Read, A.F., Thomas, M.B., and Bjørnstad, O.N.
2013. The effect of temperature on anopheles mosquito population dynamics and the potential
for malaria transmission. PLoS One, 8(11), e79276.
Biftu, T., Feng, D.D., Liang, G.B., Ponpipom, M.M., Qian, X., Fisher, M.H., and Wyvratt, M.J. 2006.
Diaryl piperidyl pyrrole derivatives as anti-protozoal agents, EP 1278520 B1.
Bijev, A., Radev, I., and Borisova, Y. 2000. Synthesis and antibacterial activity of new cephalosporines
containing a pyrrole ring in the N-acyl chain. Pharmazie, 55(8), 568–571.
Bijev, A.T., Prodanova, P.P., and Nankov, A.N. 2002. Synthesis of new 1H-1-pyrrolylcarboxamides by
comparative N-aceylation. Comptes Rendus de l’Academie Bulgare des Sciences, 55, 9–49.
Bompart, F., Kiechel, J.R., Sebbag, R., and Peccoul, B. 2011. Innovative public-private partnerships to
maximize the delivery of anti-malarial medicines: Lessons learned from the ASAQ Winthrop
experience. Malar. J., 10, 143.
Boone, R., Castenholtz, R., and Garrity, G. 2001. Bergey’s Manual of Systematic Bacteriology. Springer-
Verlag, New York, vol. 1, pp. 163–164.
Brun, R., Don, R., Jacobs, R.T., Wang, M.Z., and Barrett, M.P. 2011. Development of novel drugs for
human African trypanosomiasis. Future Microbiol., 6(6), 677–691.
Burrows, J.N., van Huijsduijnen, R.H., Möhrle, J.J., Oeuvray, C., and Wells, T.N. 2013. Designing the
next generation of medicines for malaria control and eradication. Malar. J., 12, 187.
Busatti, H.G., Santos, J.F., and Gomes, M.A. 2009. The old and new therapeutic approaches to the
treatment of giardiasis: Where are we? Biologics, 3, 273–287.
Caffrey, P., Lynch, S., Flood, E., Finnan, S., and Oliynyk, M. 2001. Amphotericin biosynthesis in
Streptomyces nodosus: Deductions from analysis of polyketide synthase and late genes. Chem.
Biol., 8 (7), 713–723.
Carmena, D., Aguinagalde, X., Zigorraga, C., Fernández-Crespo, J.C., and Ocio, J.A. Presence of
Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain. J. Appl.
Microbiol., 102, 619–629.
Casemore, D.P., Wright, S.E., and Coop, R.L. 1997. Cryptosporidiosis—Human and animalepide-
miology. In Fayer, R. ed., Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL,
pp. 65–92.
Castillo, U.F., Strobel, G.A., Mullenberg, K., Condron, M.M., Teplow, D.B., Folgiano, V., and Jensen,
J. 2006. Munumbicins E-4 and E-5: Novel broad-spectrum antibiotics from Streptomyces NRRL
3052. FEMS Microbiol. Lett., 255(2), 296–300.
Chitanga, S., Marcotty, T., Namangala, B., Van den Bossche, P., Van Den Abbeele, J., and Delespaux,
V. 2011. High prevalence of drug resistance in animal trypanosomes without a history of drug
exposure. PLoS Negl. Trop. Dis., 5(12), e1454.
Choi, D.B. et al. 2005. Recovery and purification of lincomycin from the culture broth of Streptomyces
lincolnensis. J. Ind. Eng. Chem. 11(6), 932–937.
Couvreur, J., Desmonts, G., and Thulliez, P. 1988. Prophylaxis of congenital toxoplasmosis. Effects of
spiramycin on placental infection. J. Antimicrob. Chemother., 22(Suppl. B), 193–200.
Croft, S.L. and Coombs, G.H. 2003. Leishmaniasis—Current chemotherapy and recent advances in
the search for novel drugs. Trends Parasitol., 19(11), 502–508.
Croft, S.L. and Engel, J. 2006. Miltefosine—Discovery of the antileishmanial activity of phospholipid
derivatives. Trans. R Soc. Trop. Med. Hyg., 100(Suppl. 1), S4–S8.
Coura, J.R., De Abreu, L.L., Willcox, H.P., and Petana, W. 1997. Comparative controlled study on the
use of benznidazole, nifurtimox and placebo, in the chronic form of Chagas’ disease, in a field
area with interrupted transmission. I. Preliminary evaluation. Rev. Soc. Bras. Med. Trop., 30(2),
139–144.
Creek, D.J. and Barrett, M.P. 2014. Determination of anti-protozoal drug mechanisms by metabolo-
mics approaches. Parasitology, 141(1), 83–92.
Current, W.L. and Garcia, L.S. 1991. Cryptosporidiosis. Clin. Microbiol. Rev., 4(3), 225–258.
Dame, D., Lowe, R., and Williamson D. 1981. Assessment of released sterile Anopheles albimanus
and Glossina morsitans morsitans. In: Kyoto, R.P., Kitzmiller, J., Kanda, T., (eds.). Cytogenetics and
Genetics of Vectors. Proc XVI Internat. Cong. Entomol. New York: Elsevier Science, pp. 231–248.
Darby, B.J., Housman, D.C., Zaki, A.M., Shamout, Y., Adl, S.M., Belnap, J., and Neher, D.A. 2006.
Effects of altered temperature and precipitation on desert protozoa associated with biological
soil crusts. J. Eukaryot. Microbiol., 53(6), 507–514.
De Koning, H.P., Gould, M.K., Sterk, G.J., Tenor, H., Kunz, S., Luginbuehl, E., and Seebeck, T. 2012.
Pharmacological validation of Trypanosoma brucei phosphodiesterases as novel drug targets.
J. Infect. Dis., 206(2), 229–237.
Dey, R., Dagur, P.K., Selvapandiyan, A., McCoy, J.P., Salotra, P., Duncan, R., and Nakhasi, H.L. 2013.
Live attenuated Leishmania donovani p27 gene knockout parasites are nonpathogenic and elicit
long term effective immunity in BALB/c mice. J. Immunol., 190(5), 2138–2149.
Dicko, A.H., Lancelot, R., Seck, M.T., Guerrini, L., Sall, B., Lo, M., and Bouyer, J. 2014. Using species
distribution models to optimize vector control in the framework of the tsetse eradication cam-
paign in Senegal. Proc. Natl. Acad. Sci., 111(28), 10149.
Dieter, A., Hamm, A., Fiedler, H. P., Goodfellow, M., Muller, W. E., Brun, R., and Bringmann, G. 2003.
Pyrocoll, an antibiotic, antiparasitic and antitumor compound produced by a novel alkaliphi-
lic Streptomyces strain. J. Antibiot., 56(7), 639–646.
Dimitri, N. 2012. R and D investments for neglected diseases can be sensitive to the economic goal
of pharmaceutical companies. Drug Discov. Today, 17, 818–823, 2012.
Dokmanovic, M., Clarke, C., and Marks, P.A. 2007. Histone deacetylase inhibitors: Overview and
perspectives. Mol. Cancer Res., 5, 981–989.
Donovick, R., Gold, W., Pagano, J.F., and Stout, H.A. 1954. Amphotericins A and B, antifungal
antibiotics produced by a streptomycete. I. In vitro studies. Antibiot. Ann., 3, 579–586.
Dorlo, T.P., Balasegaram, M., Beijnen, J.H., and de Vries, P.J. 2012. Miltefosine: A review of its phar-
macology and therapeutic efficacy in the treatment of leishmaniasis. J. Antimicrob. Chemother.,
67(11), 2576–2597.
Drag, M. and Salvesen, G.S. 2010. Emerging principles in protease-based drug discovery. Nat. Rev.
Drug Discov., 9, 690–701.
Duggar, B.M. 1948. Aureomycin: A product of the continuing search for new antibiotics. Ann. N. Y.
Acad. Sci., 51, 177–181.
Dumonteil, E., Bottazzi, M.E., Zhan, B., Heffernan, M.J., Jones, K., Valenzuela, J.G., and Hotez,
P.J. 2012. Accelerating the development of a therapeutic vaccine for human Chagas disease:
Rationale and prospects. Expert Rev. Vaccines, 11(3), 1044–1055.
Egorov, A., Frost, F., Muller, T., Naumova, E., Tereschenko, A., and Ford, T. 2004. Serological evidence
of Cryptosporidium infections in a Russian city and evaluation of risk factors for infections.
Ann. Epidemiol., 14, 129–136.
Eliopoulos, G.M. and Huovinen, P. 2001. Resistance to trimethoprim-ulfamethoxazole. Clin. Infect.
Dis., 32(11), 1608–1614.
Espinosa, A., Socha, A.M., Ryke, E., and Rowley, D.C. 2012. Antiamoebic properties of the actinomy-
cete metabolites echinomycin A and tirandamycin A. Parasitol. Res., 111(6), 2473–2477.
Ezra, D., Castillo, U.F., Strobel, G.A., Hess, W.M., Porter, H., Jensen, J.B., and Yaver, D. 2004.
Coronamycins, peptide antibiotics produced by a verticillate Streptomyces sp. (MSU-2110)
endophytic on Monstera sp. Micobiology, 150, 785–793.
Fang, J. 2010. A world without mosquitoes. Nature, 466, 432–434.

Felczykowska, A., Bloch, S.K., Nejman-Falenczyk, B., and Baranska, S. 2012. Metagenomic approach
in the investigation of new bioactive compounds in the marine environment. Acta Biochim. Pol.,
59, 501–505, 2012.
Fenical, W. and Jensen, P. R. 2006. Developing a new resource for drug discovery: Marine actinomy-
cete bacteria. Nat. Chem. Biol., 2,666–673.
Fevre, E.M., Wissmann, B.V., Welburn, S.C., and Lutumba, P. 2008. The burden of human African
trypanosomiasis. PLoS Neglect. Trop. Dis., 2(12), e333.
Flegr, J., Havlícek, J., Kodym, P., Malý, M., and Smahel, Z. 2002. Increased incidence of traffic acci-
dents in subjects with latent toxoplasmosis: A retrospective case-control study. BMC Infect.
Dis., 2, 11.
Fletcher, S.M., Stark, D., Harkness, J., and Ellis J. 2012. Enteric protozoa in the developed world: A
public health perspective. Clin. Microbiol. Rev., 25(3), 420–449.
Genes, C., Baquero, E., Echeverri, F., Maya, J.D., and Triana, O. 2011. Mitochondrial dysfunction in
Trypanosoma cruzi: The role of Serratia marcescens prodigiosin in the alternative treatment of
Chagas disease. Parasit. Vectors, 4(1), 66.
Gerner, E.W. and Meyskens F.L. 2004. Polyamines and cancer: Old molecules, new understanding.
Nat. Rev. Cancer, 4(10), 781–792.
Gomes, E.S., Schuch, V., and Lemos, E.G.D.M. 2013. Biotechnology of polyketides: New breath of life
for the novel antibiotic genetic pathways discovery through metagenomics. Braz. J. Microbiol.,
44(4), 1007–1034.
Gopal, J.V., Subashini, E., and Kannabiran, K. 2013. Extraction of quinone derivative from Streptomyces
sp. VITVSK1 isolated from Cheyyur saltpan, Tamilnadu, India. J. Kor. Soc. Appl. Biol. Chem.,
56(4), 361–367.
Grishin, N.V., Osterman, A.L., Brooks, H.B., Phillips, M.A., and Goldsmith, E.J. 1999. X-ray structure
of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in
complex with α-difluoromethylornithine. Biochemistry, 38, 15174–15184.
Gruen-Wollny, I., Hansske, F., Helmke, E., Kayser, O., Laatsch, H., and Maskey, R.P. 2014. Trioxacarcins
and their use against infections. Patent WO 2005080549 A2.
Gunnarsdottir, M.J. and Gissurarson L.R. 2008. HACCP and water safety plans in Icelandic water
supply: Preliminary evaluation of experience. J. Water Health, 6, 377–382.
Gurarie D. and McKenzie, F.E. 2006. Dynamics of immune response and drug resistance in malaria
infection. Malar. J., 5, 86.
Hanessian, S., Vakiti, R.R., Chattopadhyay, A.K., Dorich, S., and Lavallée, C. 2013. Probing functional
diversity in pactamycin toward antibiotic, antitumor, and anti-protozoal activity. Bioorgan.
Med. Chem., 21(7), 1775–1786.
Hawksworth, D.L. and Colwell, R.R. 1992. Biodiversity amongst microorganisms and its relevance.
Biodivers. Conserv., 1, 221–345.
Helmreich, B. and Horn, H. 2009. Opportunities in rainwater harvesting. Desalination, 248, 118–124.
Hogan, C.M. 2010. Bacteria. In Draggan, S. and Cleveland, C.J., eds., Encyclopedia of Earth. National
Council for Science and the Environment, Washington, DC.
Hökelek, M. 2014. Toxoplasmosis medication. Updated September 8.
Horn, S., Vaaje-Kolstad, G., Westereng, B., and Eijsink, V. 2012. Novel enzymes for the degradation
of cellulose. Biotechnol. Biofuels, 5(45), 1–12.
Iwatsuki, M., Nishihara-Tsukashima, A., Ishiyama, A., Namatame, M., Watanabe, Y., Handasah, S.,
and Ōmura, S. 2012. Jogyamycin, a new anti-protozoal aminocyclopentitol antibiotic, produced
by Streptomyces sp. a-WM-JG-16.2. J. Antibiot., 65, 169–171.
Jackson, Y., Alirol, E., Getaz, L., Wolff, H., Combescure, C., and Chappuis, F. 2010. Tolerance and
safety of nifurtimox in patients with chronic Chagas disease. Clin. Infect. Dis., 51(10), e69–e75.
Jensen, P.R., Mincer, T.J., Williams, P.G., and Fenical, W. 2005. Marine actinomycete diversity and
natural product discovery. Antonie Van Leeuwenhoek, 87, 43–48.
Karray, F., Darbon, E., Oestreicher, N., Dominguez, H., Tuphile, K., Gagnat, J., and Pernodet, J.L.
2007. Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in
Streptomyces ambofaciens. Microbiology, 153(12), 4111–4122.
Karthik, L., Kumar, G., Keswani, T., Bhattacharyya, A., Chandar, S.S., and Rao, K.B. 2014. Protease
inhibitors from marine actinobacteria as a potential source for antimalarial compound. PLoS
One, 9(3), e90972.
Karthik, L., Kumar, G., Keswani, T., Bhattacharyya, A., Reddy, B.P., and Rao, K.B. 2013. Marine acti-
nobacterial mediated gold nanoparticles synthesis and their antimalarial activity. Nanomed.
Nanotechnol., Biol. Med., 9(7), 951–960.
Kelly, J.M., Taylor, M.C., Horn, D., Loza, E., Kalvinsh, I., and Björkling, F. 2012. Inhibitors of human
histone deacetylase with potent activity against the African trypanosome Trypanosoma brucei.
Bioorg. Med. Chem. Lett., 22(5), 1886–1890.
Kennedy, P.G. 2013. Clinical features, diagnosis, and treatment of human African trypanosomiasis
(sleeping sickness). Lancet Neurol., 12(2), 186–194.
Khanafari, A., Assadi, M.M., and Fakar, F.A. 2006. Review of prodigiosin, pigmentation is Serratia
marssescens. J. Biol. Sci., 1, 1–13.
Kieft, T.L. 1991. Soil microbiology in reclamation of arid and semi-arid lands. In Skujins, J., ed.,
Semiarid Lands and Deserts: Soil Resource and Reclamation. Marcel Dekker, New York, pp. 209–256.
Kirchhoff, L.V. 2011. Chagas disease (American trypanosomiasis) diagnosis and treatment.
Updated June 3, 2011. Accessed September 22, 2014, http://emedicine.medscape.com/article/
214581-overview#a0101.
Krungkrai, J., Krungkrai, S.R., and Supuran, C.T. 2008. Carbonic anhydrase inhibitors: Inhibition of
Plasmodium falciparum carbonic anhydrase with aromatic/heterocyclic sulfonamides-in vitro
and in vivo studies. Bioorg. Med. Chem. Lett., 18(20), 5466–5471.
Kuhls, T.L., Mosier, D.A., Crawford, D.L., and Griffis, J. 1994. Seroprevalence of cryptosporidial anti-
bodies during infancy, childhood, and adolescence. Clin. Infect. Dis., 18(5), 731–735.
Kurapova, A.I., Zenova, G.M., Sudnitsyn, I.I., Kizilova, A.K., Manucharova, N.A., Norovsuren, Z.,
and Zvyagintsev, D.G. 2012. Thermotolerant and thermophilic actinomycetes from soils of
Mongolia desert steppe zone. Microbiology, 81, 105–116.
Lam, K.S. 2006. Discovery of novel metabolites from marine actinomycetes. Curr. Opin. Microbiol.,
2, 9(3), 245–251.
Lazzari, C.R. 1991. Temperature preference in Triatoma infestans (Hemiptera: Reduviidae). Bull.
Entomol. Res., 81, 273–276.
Lengerich, E.J., Addiss, D.G., Marx, J.J., Ungar, B.L., and Juranek, D.D. 1993. Increased exposure to
cryptosporidia among dairy farmers in Wisconsin. J. Infect. Dis., 167(5), 1252–1255.
Leport, C., Raffi, F., Matheron, S., Katlama, C., Regnier, B., Saimot, A.G., and Vilde, J.L. 1988. Treatment
of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in
35 patients with the acquired immunodeficiency syndrome. Efficacy of long-term continuous
therapy. Am. J. Med., 84(1), 94–100.
Lesch, J.E. 2007. The first miracle drugs: how the sulfa drugs transformed medicine. Chapter 3:
Prontosil. p. 51. Oxford University Press.
Lounibos, L.P., Suárez, S., Menéndez, Z., Nishimura, N., Escher, R.L., O’Connell, S.M., and Rey, J.R.
2002. Does temperature affect the outcome of larval competition between Aedes aegypti and
Aedes albopictus? J. Vector Ecol., 27(1), 86–95.
Magauer, T., Smaltz, D.J., and Myers, A.G. 2013. Component-based syntheses of trioxacarcin A,
DC-45-A1 and structural analogues. Nat. Chem., 5(10), 886–893.
Maheswarappa, G., Kavitha, D., Vijayarani, K., and Kumanan, K. 2013. Prodigiosin as anticancer
drug produced from bacteria of termite gut. Ind. J. Basic Appl. Med. Res., 3(1), 257–266.
Maldonado, L.A., Fenical, W., Jensen, P.R., Kauffman, C.A., Mincer, T.J., Ward, A.C., and Goodfellow,
M. 2005. Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov., obligate marine
actinomycetes belonging to the family Micromonosporaceae. Int. J. Syst. Evol.Microbiol., 55,
1759–1766, 2005.
Manivasagan, P., Venkatesan, J., Sivakumar, K., and Kim, S.K. 2014. Pharmaceutically active second-
ary metabolites of marine actinobacteria. Microbiol. Res., 169(4), 262–278.
Manyando, C., Njunju, E.M., D’Alessandro, U., and Van Geertruyden, J.P. 2003. Safety and efficacy
of co-trimoxazole for treatment and prevention of Plasmodium falciparum malaria: A systematic
review. PLoS One, 8(2), e56916.
Maskey, R.P., Sevvana, M., Usón, I., Helmke, E., and Laatsch, H. 2004. Gutingimycin: A highly com-
plex metabolite from a marine streptomycete. Angew. Chem. Int. Ed., 43(10), 1281–1283.
Matter, A.M., Hoot, S.B., Anderson, P.D., Neves, S.S., and Cheng, Y.Q. 2009. Valinomycin biosynthetic
gene cluster in Streptomyces: Conservation, ecology and evolution. PLoS One, 4(9), e7194.
Metcalf, B.W., Bey, P., Danzin, C., Jung, M. J., Casara, P., and Vevert, J.P. 1978. Catalytic irreversible
inhibition of mammalian ornithine decarboxylase (EC.4.1.1.17) by substrate and product ana-
logs. J. Am. Chem. Soc., 100, 2551–2553.
Mincer, T.J., Fenical, W., and Jensen P.R. 2005. Culture-dependent and culture-independent diver-
sity within the obligate marine actinomycete genus Salinispora. Appl. Environ. Microbiol., 71(11),
7019–7028.
Mokrane, S., Bouras, N., Sabaou, N., and Mathieu, F. 2013. Actinomycetes from saline and non-saline
soils of Saharan palm groves: Taxonomy, ecology and antagonistic properties. Afr. J. Microbiol.
Res., 7(20), 2167–2178.
Moore, S., Shrestha, S., Tomlinson, K.W., and Vuong, H. 2011. Predicting the effect of climate change
on African trypanosomiasis: Integrating epidemiology with parasite and vector biology. J. R.
Soc. Interf., 9, 817–830.
Mukthavaram, R., Jiang, P., Saklecha, R., Simberg, D., Bharati, I.S., Nomura, N., and Kesari, S.2013.
High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved
in vivo toxicity and tumor response profile. Int. J. Nanomed. (Dovepress), 8(1), 3991–4006.
Mumba, D., Bohorquez, E., Messina, J., Kande, V., Taylor, S.M., Tshefu, A.K., and Meshnick, S.R. 2011.
Prevalence of human African trypanosomiasis in the Democratic Republic of the Congo. PLoS
Negl. Trop. Dis., 5(8), e1246.
Nwaka, S. and Hudson, A. 2006. Innovative lead discovery strategies for tropical diseases. Nat. Rev.
Drug Discov., 5(11), 941–955.
Odero, R.O. 2013. African trypanasomiasis medication. Updated December 13, 2013. Accessed
September 23, 2014, http://emedicine.medscape.com/article/228613-medication.
Oh, D.C., Poulsen, M., Currie, C.R., and Clardy, J. 2011. Sceliphrolactam, a polyene macrocyclic lac-
tam from a wasp-associated Streptomyces sp. Org. Lett., 13(4), 752–755.
Olsen, M.E., Ceri, H., and Morck, D.W. 2000. Giardia vaccination. Parasitol. Today, 16(5), 213–217.
Omura, S., Iwai, Y., Hirano, A., Nakagawa, A., Awaya, J., Tsuchiya, H., and Asuma, R. 1977. A new
alkaloid AM-2282 of Streptomyces origin taxonomy, fermentation, isolation and preliminary
characterization. J. Antibiot., 30(4), 275–282.
Onnis, B., Rapisarda, A., and Melillo, G. 2009. Development of HIF-inhibitors for cancer therapy.
J. Cell. Mol. Med., 13, 2780–2786.
Osato, T., Ueda, M., Fukuyama, S., Yagishita, K., Okami, Y., and Umezawa, H. 1955. Production of
tertiomycin (a new antibiotic substance), azomycin and eurocidin by S. eurocidus. J. Antibiot.,
8, 105–109.
Ostrosky-Zeichner, L., Marr, K.A., Rex, J.H., Cohen, S.H. 2003. Amphotericin B: time for a new ‘‘gold
standard’’. Clin. Infect. Dis. 37(3), 415–425.
Parsons, M., Worthey, E.A., Ward, P.M., and Mottram, J.C. 2005. Comparative analysis of the kinomes
of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma
cruzi. BMC Genomics, 6, 127.
Patel, G., Karver, C.E., Behera, R., Guyett, P.J., Sullenberger, C., Edwards, P., and Pollastri, M.P. 2013.
Kinase scaffold repurposing for neglected disease drug discovery: Discovery of an efficacious,
lapatinib-derived lead compound for trypanosomiasis. J. Med. Chem., 56(10), 3820–3832.
Peirú, S., Rodríguez, E., Menzella, H.G., Carney, J.R., and Gramajo, H. 2008. Metabolically engineered
Escherichia coli for efficient production of glycosylated natural products. Microb. Biotechnol., 1,
476–486, 2008.
Perez-Jorge, E.V. 2014. Malaria medication. Updated March 14, 2014. Accessed September 23, 2014,
http://emedicine.medscape.com/article/221134-medication.
Pickens, L.B. and Tang, Y. 2010. Oxytetracycline biosynthesis. J. Biol. Chem., 285, 27509–27515.
Piechoski, M.P., Cundell, D.R., Bower, A.H., and Porter, J.R. 2012. Bioassay and antibiotic activity
of jamaican actinomyces isolates. J. Young Investigators. October 2012. Accessed January 13,
2014. http://www.jyi.org/issue/bioassay-and-antibiotic-activity-of-jamaican-actinomycetes-
isolates/.
Pimentel-Elardo, S.M., Kozytska, S., Bugni, T.S., Ireland, C.M., Moll, H., and Hentschel, U. 2010.
Anti-parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges.
Mar. Drugs., 8(2), 373–380.
Pinazo, M.J., Muñoz, J., Posada, E., López-Chejade, P., Gállego, M., Ayala, E., and Gascon, J. 2010.
Tolerance of benznidazole in treatment of Chagas’ disease in adults. Antimicrob. Agents
Chemother., 54(11), 4896–4899.
Pinnert-Sindico, S. 1954. Une nouvelle espe’ ce de Streptomyces productrice d’antibiotiques:
Streptomyces ambofaciens n. sp. caracte’ res culturaux. Ann. Inst. Pasteur (Paris), 87, 702–707.
Pommier, Y., Leo, E., Zhang, H., and Marchand, C. 2010. DNA topoisomerases and their poisoning
by anticancer and antibacterial drugs. Chem. Biol., 17(5), 421–433.
Priotto, G., Kasparian, S., Mutombo, W., Ngouama, D., Ghorashian, S., Arnold, U., and Kande, V.
2009. Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma
brucei gambiense trypanosomiasis: A multicentre, randomised, phase III, non-inferiority trial.
Lancet, 374(9683), 56–64.
Priotto, G., Kasparian, S., Ngouama, D., Ghorashian, S., Arnold, U., Ghabri, S., and Karunakara,
U. 2007. Nifurtimox-eflornithine combination therapy for second-stage Trypanosoma brucei
gambiense sleeping sickness: A randomized clinical trial in Congo. Clin. Infect. Dis., 45(11),
1435–1442.
Rahman, H., Austin, B., Mitchell, W.J., Morris, P.C., Jamieson, D.J., Adams, D.R., and Schweizer, M.
2010. Novel anti-infective compounds from marine bacteria. Marine Drugs, 8(3), 498–518.
Raja, A. and Prabakarana, P. 2011. Actinomycetes and drug-an overview. Am. J. Drug Discov. Dev.,
1, 75–84.
Raju, R., Khalil, Z.G., Piggott, A.M., Blumenthal, A., Gardiner, D.L., Skinner-Adams, T.S., and Capon,
R.J. 2014. Mollemycin A: An antimalarial and antibacterial glyco-hexadepsipeptide-polyketide
from an Australian marine-derived Streptomyces sp. (CMB-M0244). Org. Lett., 16(6), 1716–1719.
Ryu, S.R., Choi, O.Y., Yin, P., and Kwun, K. H. 2005. Recovery and purification of lincomycin from the
culture broth of Streptomyces lincolnensis. J. Ind. Eng. Chem., 11(6), 932–937.
Schofield, C.J. and Dias, J.C.P. 1999. The southern cone initiative against Chagas disease. Adv.
Parasitol., 42, 1–27.
Simon, C. and Daniel, R. 2009. Achievements and new knowledge unraveled by metagenomic
approaches. Appl. Microbiol. Biotechnol., 85(2), 265–276.
Snelling, W.J., Xiao, L., Ortega-Pierres, G., Lowery, C.J., Moore, J.E., Rao, J.R., and Dooley, J.S. 2007.
Cryptosporidiosis in developing countries. The Journal of Infection in Developing Countries, 1(03),
242–256.
Sosovele, M.E., Bergmann, B., Lyimo, T.J., Hosea, K.M., and Mueller, B.I. 2012. Antimalarial activity
of marine Actinomycetes isolated from Dar es Salaam mangrove sediments. Int. Res. Biol. Sci.,
2(4), 177–181.
Spížek, J. and Řezanka, T. 2004. Lincomycin, cultivation of producing strains and biosynthesis. Appl.
Microbiol. Biotechnol., 63(5), 510–519.
Stach, J.E., Maldonado, L.A., Ward, A.C., Goodfellow, M., and Bull, A.T. 2003. New primers for the
class Actinobacteria: Application to marine and terrestrial environments. Environ. Microbiol.,
5(10), 828–841.
Steinerova, N., Lipavská, H., Stajner, K., Čáslavská, J., Blumauerová, M., Cudlín, J., and Vank, Z. 1987.
Production of quinomycin A in Streptomyces lasaliensis. Folia Microbiol., 32(1), 1–5.
Sumanadasa, S.D., Goodman, C.D., Lucke, A.J., Skinner-Adams, T., Sahama, I., Haque, A., and
Andrews, K.T. 2012. Antimalarial activity of the anticancer histone deacetylase inhibitor
SB939. Antimicrob. Agents Chemother., 56(7), 3849–3856.
Tan, K.R., Magill, A.J., Parise, M.E., and Arguin, P.M. 2011. Doxycycline for malaria chemoprophy-
laxis and treatment: Report from the CDC expert meeting on malaria chemoprophylaxis. Am.
J. Trop. Med. Hyg., 84(4), 517–531.
Tiwari, S.S.K., Schmidt, W.P., Darby, J., Kariuki, Z.G., and Jenkins, M.W. 2009. Intermittent slow sand
filtration for preventing diarrhoea among children in Kenyan households using unimproved
water sources: Randomized controlled trial. Trop. Med. Int. Health, 14(11), 1374–1382.
Torrey, E.F., Bartko, J.J., Lun, Z.R., and Yolken, R.H. 2007. Antibodies to Toxoplasma gondii in patients
with schizophrenia: A meta-analysis. Schizophr. Bull., 33(3), 729–736.
Tran, M.T. 2014. Global partners are taking the “Neglect” out of “Neglected Tropical Diseases”.
Uniting to Combat Neglected Tropical Diseases. April 1, 2014, Press Release. Accessed
October 8, 2014, http://unitingtocombatntds.org/news/global-partners-are-taking-neglect-
out-neglected-tropical-diseases.
Tringe, S.G., Von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, H.W., and Rubin, E.M.
2005. Comparative metagenomics of microbial communities. Science, 308(5721), 554–557.
Valderrama, J., Fournet, A., Valderrama, C., Bastias, S., Astudillo, C., Rojas de Arias, A., and Yaluff, G.
1999. Synthesis and in vitro antiprotozoal activity of thiophene ring-containing quinones.
Chemical and Pharmaceutical Bulletin, 47(9), 1221–1226.
Varshney, H., Ahmad, A., Rauf, A., Husain, F.M., and Ahmad, I. 2014. Synthesis and antimicrobial
evaluation of fatty chain substituted 2,5-dimethyl pyrrole and 1,3-benzoxazin-4-one deriva-
tives. J. Saudi Chem. Soc. May 9, 2014. Accessed October 6, 2014, http://www.sciencedirect.com/
science/article/pii/S1319610314000647.
Vetsigian, K., Jajoo, R., and Kishony, R. 2011. Structure and evolution of Streptomyces interaction net-
works in soil and in silico. PLoS Biol., 9(10), e1001184.
Vincent, I.M., Creek, D.J., Burgess, K., Woods, D.J., Burchmore, R.J., and Barrett, M.P. 2012. Untargeted
metabolomics reveals a lack of synergy between nifurtimox and eflornithine against
Trypanosoma brucei. PLoS Negl. Trop. Dis., 6(5), e1618.
Waksman, S.A. and Lechevalier, H.A. 1962. The Actinomycetes, Volume III: Antibiotics of Actinomycetes.
Baltimore, The Williams and Wilkins Company.
Walsh, F. Malaria vaccine trial shows promise. BBC News Health, October 10, 2013.
Wang, D. and Gao, F. 2013. Quinazoline derivatives: Synthesis and bioactivities. Chem. Cent. J., 7, 95.
Ward, A.C. and Bora, N. 2006. Diversity and biogeography of marine actinobacteria. Curr. Opin.
Microbiol., 9(3), 279–286.
Weiss, R.B. 1992. The anthracyclines: Will we ever find a better doxorubicin? Semin. Oncol., 19(6),
670–686.
Wiwanitkit, V. 2012. Interest in paromomycin for the treatment of visceral leishmaniasis (kala-azar).
Therap. Clin. Risk Manage., 8, 323.
Wolf, J.E., Shander, D., Huber, F., Jackson, J., Lin, C.S., Mathes, B.M., and Schrode, K. 2007. Randomized,
double‐blind clinical evaluation of the efficacy and safety of topical eflornithine HCl 13.9%
cream in the treatment of women with facial hair. Int. J. Dermatol., 46(1), 94–98.
World Health Organization Report. 2012. Global costs and benefits of drinking-water supply and
sanitation interventions to reach the MDG target and universal coverage. Accessed September
22, 2014.
World Health Organization. 2013. Malaria report. Accessed September 22, 2014. http://www.who.
int/malaria/publications/world_malaria_report_2013/wmr2013_no_profiles.pdf.
Wright, G.D. 2010. Q and A: Antibiotic resistance: Where does it come from and what can we do
about it? BMC Biol., 8(1), 123.
Yu, Z., Vodanovic-Jankovic, S., Ledeboer, N., Huang, S.X., Rajski, S.R., Kron, M., and Shen, B. 2011.
Tirandamycins from Streptomyces sp. 17944 inhibiting the parasite Brugia malayi asparagine
tRNA synthetase. Org. Lett., 13(8), 2034–2037.
Zhang, Y.F., Xiao, K., Chandramouli, K.H., Xu, Y., Pan, K., Wang, W.X., and Qian, P.Y. 2011. Acute tox-
icity of the antifouling compound butenolide in non-target organisms. PloS One, 6(8), e23803.
chapter twenty-four
Bioactive compounds from actinomycetes

and their antiviral properties
Present trends and future prospectives
Avilala Janardhan, Arthala Praveen Kumar, and Golla Narasimha
Contents
24.1 Introduction......................................................................................................................... 479
24.2 Antiviral activity.................................................................................................................480
24.3 Screening methods............................................................................................................. 481
24.4 Viral entry inhibition assay............................................................................................... 482
24.5 Viral replication inhibition assay..................................................................................... 482
24.6 Anticancer activity..............................................................................................................483
References...................................................................................................................................... 483
24.1 Introduction
The search for bioactive compounds in nature is a multistep procedure that begins with
the selection of suitable sources and then the biological, chemical, or physical interac-
tions of metabolites with test systems that are then qualitatively or quantitatively evalu-
ated (Omura, 1992). This is called screening. Natural products are the organic molecules
derived from primary or secondary metabolism of living organisms such as microorgan-
isms. Among them, 50%–60% are produced by plants (alkaloids, flavonoids, terpenoids,
steroids, and carbohydrates), and 5% have a microbial origin (Berdy, 2005). The natural
products from plants (Han et al., 2007; Huang et al., 2008; Huo et al., 2008), fungi (Krohn
et al., 2001; Lin et al., 2002; Wu et al., 2004; Gao et al., 2007), bacteria (Lin et al., 2008), and
actinomycetes (Xie et al., 2006; Tang et al., 2007) are the most anti-infectious, anticancer,
antibacterial, antiviral, anti-inflammatory, antimalarial, and antidiabetic drugs on the
market today. The increasing role in the production of natural products such as antibiot-
ics and other drugs for treatment of serious diseases has been dramatic, but the develop-
ment of resistance in pathogens and tumor cells has become a major problem and requires
much research effort to screen it. The basic premises of a screening program are as fol-
lows: (1) drugs operate in a dose–response manner and produce toxicity in higher doses;
(2) each class of drug has a characteristic dose–response profile; (3) for the majority of
drugs, route of administration produces only a quantitative change in action; (4) absolute
potency is not of major importance in therapeutics; and (5) it is possible to predict use-
fulness and toxicity of a new compound by utilizing a dose–response spectra library of
various prototype drugs. The criteria of a good screening program are that it should be
simple, economical, reliable, able to pick up new unexpected or unique activity, unbiased,
479
and comprehensive (Irwin, 1962; Lucas and Lewis, 1944; Taylor et al., 1952; Laurence and
Bacharach, 1964; Turner, 1965; Mantegazza and Piccinni, 1966; Turner and Hoborn, 1971;
Dhawan and Srimal, 1984, 1992; Kamboj and Dhawan, 1989).
Viruses cause many important diseases in humans, with viral-induced emerging and
reemerging infectious diseases representing a major health threat to the public. In addi-
tion, viruses can also infect livestock and marine species, causing huge losses of many
vertebrate food species. Effective control of viral infection and disease has remained an
unachieved goal, due to the virus’ intracellular replicative nature and readily mutating
genome, as well as the limited availability of antiviral drugs and measures. In relation
to infectious diseases, the exploration of the marine environment represents a promising
strategy in the search for active compounds, whereas there is a need for new medicines,
due to the appearance of resistance to available treatments in many microorganisms, spe-
cifically concerning antiviral activities. Among the microorganisms, the actinomycetes
are the gram-positive bacteria belonging to the order Actinomycetales, which play a sig-
nificant role in the production of new metabolites (Goodfellow et al., 1988; Demain, 1995).
Especially, the Streptomyces and Micromonospora strains have proven to produce novel anti-
biotics (Omura et al., 2001; Watve et al., 2001; Bentlley et al., 2002). The screening of micro-
bial natural products leads to the discovery of novel chemicals for the development of new
therapeutic agents (Bull et al., 2000). So, it is necessary to continue the screening for new
metabolites and evaluate the potential of less-known and new bacterial strains so that the
new and improved compounds for future use against drug-resistant bacteria or for chemi-
cal modification purposes may be developed (Kurtboke, 2005).
24.2 Antiviral activity

Some compounds have been used for testing antiviral activity in our laboratory. Marine
antiviral agents (MAVAs) (Fujioka and Loh, 1996) can be used for the biological c ontrol
of human enteropathogenic virus contamination and disease transmission in sewage-
polluted waters, as chemotherapy for viral diseases of humans and lower animals, as
well as the biological control of viral diseases of marine animals. The seeding of MAVAs
under natural conditions, or when marine mammals are kept in captivity for various uses,
could control viral disease transmission within these select populations. It is clear that
the marine environment will play a vital role in the future development and trials of anti-
infective drugs. The purpose of this study was to establish an in vitro model to screen
marine extracts for antiviral activity and to evaluate some marine extracts for their antivi-
ral potential, with a long-term goal of discovering new marine compounds to be used as
potential antiviral drug candidates.
Viruses cause many diseases in animals; effective control of viral diseases and infec-
tions has remained an unachieved goal due to virus intracellular replicative nature and
readily mutating genome. Due to the limited availability of antiviral drugs, the use of natu-
ral products as drugs was well established. Different studies were conducted to determine
the effectiveness of the natural products. Ager (1984) had done experiments on 25 isolates
of actinomycetes, which were given as feed for cultured shrimp and tested for their abil-
ity to reduce the white spot syndrome virus (WSSV) infection in shrimp. Among these 25
isolates, 6 isolates have shown to be most potential. The pentalactones are extracted from
the fermentation broth of Streptomyces sp. M-2719 has been reported to be active against
several DNA viruses (Kumar et al., 2006). Researchers have reported that guanine-7-N-
oxide produced by Streptococcus sp. was found to inhibit in vitro replication of fish her-
pes virus, rhabdovirus, and infectious pancreatic necrovirus (Nakagawa et al., 1985).
Chapter twenty-four: Bioactive compounds from actinomycetes and their antiviral properties 481
The antibiotic SF 2487 from Actinomadura sp. was found to exhibit antiviral activity against

influenza virus in vitro (Hasobe et al., 1985). A Streptomyces sp. isolated from Brazilian tropi-
cal forest soil possessed antiviral activity against herpes simplex virus type 1 (HSV-1) on
HEP-2 cells (Hatsu et al., 1990). An antibiotic enriching from Streptomyces lavendulae showed
inhibition of influenza A and influenza B virus in vitro (Sacrament et al., 2004). Current
antiviral drugs comprise of over 40 compounds that have been officially approved for clini-
cal use. Among these drugs, half of them were used to treat HIV infections (Bhakuni et al.,
1990; Schaeffer and Krylov, 2000; Tziveleka et al., 2003; Mayer and Hamann, 2005). MAVAs
were used for biological control of human enteropathogenic virus contamination and dis-
ease transmission in sewage-polluted water (Fujioka and Loh, 1996).
MAVAs represent a significantly unique natural marine resource whose multipotential
uses include the following applications: (1) One is the biological control of human entero-
pathogenic virus contamination and disease transmission in sewage-polluted waters. This
application would be particularly important to communities that utilize the coastal waters
for recreational activities and for food industries (e.g., fish, shellfish), as well as to those
regions of the country, such as Hawaii, where the loss of these marine resources would
have a devastating effect on the lifestyle and economy of the people. (2) The other one is
the chemotherapy of viral diseases of humans and lower animals. To be of practical use,
it is imperative that MAVAs are isolated from pure cultures, identified, and characterized.
Their spectrum and mechanism of antiviral activity should also be clearly established.
Their active principle and moieties should be identified and chemically characterized in
order to facilitate application of biotechnological methods for increased yields and cost-
effective production.
Currently, it appears that there have been only a few compounds derived from marine
actinobacteria with antiviral activity. Benzastatin C (56), a 3-chloro-tetrahydroquinolone
alkaloid obtained from Streptomyces nitrosporeus, showed antiviral activity in a dose-
dependent manner with EC50 values of 1.92, 0.53, and 1.99 g/mL against HSV-1, HSV-2,
and vesicular stomatitis virus, respectively (Lee et al., 2007). Kumar et al. (2006) reported
the antiviral property of a marine Streptomyces against WSSV in penaeid shrimp. WSSV
infection can cause cumulative mortality up to 100% within 3–10 days, thereby causing
considerable economic loss to the shrimp farmers.
24.3 Screening methods

It is suggested that the potential antiviral agents must be screened in a living cell or ani-
mal host. Testing for antiviral activity is usually performed in cell culture or embryonated
chicken eggs and animal models. In vitro antiviral testing using cell cultures involves the
virus of interest and a primary or permanent cell line that can support its multiplication.
The cells are infected with the virus, or already viral-infected cell lines are exposed to the
extracted compound. If the compound has antiviral activity, the multiplication of the virus
will be inhibited, which will be evident from the morphology of the cell monolayer. It is
important to assess the toxic effect of the test substance on cells at each dilution. This can
be done by examining the uninfected cell monolayers exposed to the extracted compound
only. From the observed ED50 and LD50 of the compound, its therapeutic index is calcu-
lated. Several viral targets are studied to estimate the antiviral effect of compound in a cell
culture system. Some of these are viral DNA polymerase activity, ribonucleotide diphos-
phate reductase, mRNA polyadenylation and RNA-dependent RNA polymerase, termi-
nal deoxynucleotidyl transferase, thymidine kinase, uracil-DNA glycolase, d-UTPase,
and reverse transcriptase. Testing for antiviral activity in chicken eggs is very simple.
Here, prophylactic and therapeutic assays may be carried with different test substances
since a wide choice of routes and timing of application of both virus and antiviral agents is
possible. There are three main routes by which the bioactive compound could be admin-
istered into embryonated eggs: allantoic cavity inoculation, amniotic cavity inoculation,
and chorioallantoic membrane inoculation. The virus and the compound may be given
through different routes; it depends on the type of virus and the compound. The test sub-
stance can be given before, along with, or after virus infection. Testing in animal models
has relatively the maximum predictive value among the various methods employed for
detecting antiviral activity. Testing in these model systems can identify both antiviral
activity and antiviral agents. The ideal animal model should have three features: (1) use
of a human virus with minimal alteration by adaptation; (2) use of the natural route of
infection and size of inoculum as in humans; and (3) similarity of infection, pathogenesis,
host response, drug metabolism, and drug toxicity. Animal models exist for both local
and systemic virus infections. Antiviral activity of a test substance can also be assessed
by titrating the virus in blood and other target organs. The details of these models are
described (Bhakuni et al., 1990).
24.4 Viral entry inhibition assay

Cells at exponential growth phase were harvested and seeded into multiwell plates at
densities that would allow the formation of an approximately 90% cell monolayer over-
night. Marine extracts were diluted with serum-free medium to twice the effective safe
concentrations, as determined by the cytotoxicity tests. A 250 μL solution of each extract
at twice the maximum nontoxic concentration (e.g., 200 μg/mL for those found to be non-
toxic at 100 μg/mL) was mixed with an equal volume of the virus dilution. Positive con-
trols were made by mixing 250 μL of virus dilution with 250 μL of serum-free medium
with 0.2% DMSO, in order to yield a final DMSO concentration of 0.1%. The 500 μL virus/
extract mixtures were preincubated for 1 h, along with controls, and then assayed for viral
infectivity using the optimized plaque assay protocols. Antiviral effect of each extract was
categorized as having no meaningful inhibition (<20%), slight inhibition (≥20%), moderate
inhibition (≥50%), or high inhibition (≥80%).
24.5 Viral replication inhibition assay

Test cells were seeded into TC 12.5 cm2 flasks at a density that would allow the forma-
tion of an approximately 90% monolayer the next day. Marine extracts were diluted with
a medium containing 5% serum to their safe and effective concentrations. The medium
was completely aspirated from the flasks, and then the cell monolayer was briefly washed
with Dulbecco`s Phosphate Buffered Saline (DPBS) buffer, before infection with test virus
at a multiplicity of infection of 0.1. Following a 1 h viral adsorption, all medium in the
flask was removed and the flasks were washed twice with DPBS; infected cultures were
incubated with 2.5 mL/flask of diluted extract. Two flasks were tested per extract, and
these cultures were allowed to incubate for 3 days. Pictures were taken every 12 h using
an inverted microscope equipped with a camera, starting at time zero, in order to track the
progression of viral-induced CPE. To track viral progression, 200 μL samples of medium
were taken from each flask, every 12 h, and stored at −20°C until the end of the experiment.
The viral titers of these samples were later determined by standard plaque assay, as previ-
ously described. Test extracts shown to produce a visually noticeable reduction in CPE, as
well as a reduction in viral titer, were considered for further characterization.
24.6 Anticancer activity

Cancer still remains one of the most serious human health problems, and breast cancer
is the second most universal cause of cancer death in women (Ravikumar et al., 2010).
Therapeutic methods for cancer treatment are surgery, radiotherapy, immunotherapy, and
chemotherapy (Gillet et al., 2007), and these techniques are individually useful in particu-
lar situations, and when combined, they offer a more efficient treatment for tumor. Many of
the antitumor compounds from marine drugs are derived from marine actinobacteria, and
these metabolites play an important role in identification of pharmaceutical compounds
(Ravikumar et al., 2012). Currently, it appears that there have been only a few studies focus-
ing on finding bioactive compounds derived from marine actinobacteria to be used as
anticancer agents as well as agents against infectious organisms. Pure active compounds
extracted from the marine actinobacterium Salinispora tropica have shown inhibitory effects
in many malignant cell types (Prudhomme et al., 2008). In particular, salinosporamide A
is a novel rare bicyclic beta-lactone gamma-lactam isolated from an obligate marine acti-
nobacterium, S. tropica (Feling et al., 2003; Jensen et al., 2007). Salinosporamide A is an
orally active proteasome inhibitor that induces apoptosis in multiple myeloma cells with
mechanisms distinct from the commercial proteasome inhibitor anticancer drug bortezo-
mib (Chauhan et al., 2005). The first anticancer chemical compound for cancer treatment is
produced from obligate marine actinnobacterium (Fenical et al., 2009). Prudhomme et al.
(2008) tested salinosporamide A for its utility as an anticancer and antimalarial drug. It was
shown to have inhibitory activity against parasite development in vitro (Plasmodium falci-
parum) and in vivo (Plasmodium yoelii). The exact mode by which salinosporamide A inhibits
Plasmodium erythrocytic development is unknown; however, it is likely due to the inhibition
of the proteasome complex. It is interesting to note that chloroquine-resistant strains are
still sensitive to salinosporamide A. Targeting the proteasome system has a huge thera-
peutic implication as it can restrain growth and survival of most cell types (Prudhomme
et al., 2008). These attributes, taken with the fact that it is already in phase I clinical trials
as an antitumor agent, make it an excellent candidate for alternative therapies, such as anti-
bacterial, antiparasitic, antifungal, or antiviral treatments. Caprolactones are new antibiot-
ics isolated from Streptomyces sp. showing moderate phytotoxicity and promising activity
against cancer cells with concomitant low general cytotoxicity (Stritzke et al., 2004).
References
Ager, Jr., A.L. 1984. Rodent malaria models. In: Peters, W., Ed., Antimalarial Drugs, Springer, Berlin,
Germany, pp. 225–264.
Bentley, S.D., Chater, K.F., Cerdeno-Tarragfa, A.M., Challis, G.L., Thompson, N.R., James, K.D.,
Harris, D.E., Quail, M.A., Kieser, H., and Harper, D. 2002. Complete genome sequence of the
model actinomycete Streptomyces coelicolor A3(2). Nature, 417, 141–147.
Berdy, J. 2005. Bioactive microbial metabolites, a personal view. J. Antibiot., 58, 1–26.
Bhakuni, D.S., Goel, A.K., Jain, S., Mehrotra, B.N., and Srimal, R.C. 1990. Screening of Indian plants
for biological activity: Part XIV. Ind. J. Exp. Biol., 28, 619.
Bull, A.T., Ward, A.C., and Goodfellow, M. 2000. Search and discovery strategies for biotechnology:
The paradigm shift. Microbiol. Mol. Biol. Rev., 64, 573–606.
Chauhan, D., Catley, L., Li, G., Podar, K., Hideshima, T., Velankar, M., and Anderson, K.C. 2005. A
novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with
mechanisms distinct from Bortezomib. Cancer Cell, 8(5), 407–419.
Demain, A.L. 1995. Why do microorganisms produce antimicrobials? In: Hunter, P.A., Darby, G.K.
and Russell, N.J., Eds., Fifty Years of Antimicrobials: Prospective and Future Trends—Symposium 53,
Society of General Microbiology, Cambridge University Press, Cambridge, pp. 205–228.
Dhawan, B.N. and Srimal, R.C. 1984. In the use of pharmacological techniques or the evaluation of
natural products, UNESCO-CDRI, Worshop on the Pharmacological Techniques Used for Evaluation
of Natural Products, Lucknow, India, 159pp
Dhawan, B.N. and Srimal, R.C. 1992. In The Use of Pharmacological Techniques for the Study of Natural
Products, UNESCO-CDRI, Lucknow, India.
Feling, R.H., Buchanan, G.O., Mincer, T.J., Kauffman, C.A., Jensen, P.R., and Fenical, W. 2003.
Salinosporamide A: A highly cytotoxic proteasome inhibitor from a novel microbial source, a
marine bacterium of the new genus Salinospora. Angew. Chem. Int. Ed., 42(3), 355–357.
Fenical, W., Jensen, P.R., Palladino, M.A., Lam, K.S., Lloyd, G.K., and Potts, B.C. 2009. Discovery and
development of the anticancer agent salinosporamide A (NPI-0052). Bioorg. Med. Chem., 17(6),
2175–2180.
Fujioka, R.S., Loh, P.C. Characterization of the microbiological quality of water in Mamala Bay.
Project MB-7 In Mamala Bay, Final Report. 1996;1.
Gao, H., Hong, K., Zhang, X., Liu, H.W., Wang, N.L., Zhang, L., and Yao, X.S. 2007. Polyhydroxylated
sterols and new sterol fatty esters from the mangrove fungus Aspergillus awamori exhibiting
potent cytotoxic activity. Helv. Chim. Acta, 90, 1165–1178.
Gillet, J.-P., Efferth, T., and Remacle, J. 2007. Chemotherapy-induced resistance by ATP binding cas-
sette transporter genes. Biochim. Biophys. Acta Rev. Cancer, 1775(2), 237–262.
Goodfellow, M., Williams, S.T., and Mordarski, M. 1988. Actinomycetes in Biotechnology, vol. 12,
pp. 73–74. Academic Press, London, U.K.
Han, L., Huang, X.S., Sattler, I., Fu, H.Z., Grabley, S., and Lin, W.H. 2007. Two new constituents from
mangrove Bruguiera gymnorrhiza. J. Asian Nat. Prod. Res., 9, 327–331.
Hasobe, M., Saneyoshi, M., and Isono, K. 1985. Antiviral activity and its mechanism of guanine 7-N
oxide on DNA and RNA viruses derived from Salmonid. J. Antibiot., 38, 1581–1587.
Hatsu, M., Sasaki, T., Miyadoh, S., Watabe, H., Takeuchi, Y., Kodama, Y., Orikasa, Y. et al. 1990. SF2487,
a new polyether antibiotic produced by Actinomadura. J. Antibiot., 43(3), 259–266.
Huang, H., Lv, J., Hu, Y., Fang, Z., Zhang, K., and Bao, S. 2008. Micromonospora rifamycinica sp. nov., a
novel actinomycete from mangrove sediment. Int. J. Syst. Evol. Microbiol., 58, 17–20.
Huo, C., Liang, H., Tu, G., Zhao, Y., and Lin, W. 2008. A new 5, 11-epoxymegastigmane glucoside
from Acanthus ilicifolius. Nat. Prod. Res., 22, 896–900.
Irwin, S. 1962. Drug screening and evaluative procedures current approaches do not provide the
information needed for properly predicting drug effects in man. Science, 136(3511), 123–128.
Jensen, P.R., Williams, P.G., Oh, D.C., Zeigler, L., and Fenical, W. 2007. Species-specific second-
ary metabolite production in marine actinomycetes of the genus Salinispora. Appl. Environ.
Microbiol., 73(4), 1146–1152.
Kamboj, V.P. and Dhawan, B.N. 1989. Contraceptive Research Today and Tomorrow. Indian Council of
Medical Research, New Delhi, India, pp. 115–125.
Krohn, K., Steingröver, K., and Zsila, F. 2001. Five unique compounds: Xyloketals from the mangrove
fungus Xylaria sp. from the South China Sea coast. J. Org. Chem., 66, 6252–6256.
Kumar, S.S., Philip, R., and Achuthankutty, C. 2006. Antiviral property of marine actinomycetes
against white spot syndrome virus in penaeid shrimps. Curr. Sci., 91(6), 807–811.
Kurtboke, D.I. 2005. Actinophages as indicators of actinomycete taxa in marine environments.
Antonie Van Leeuwenhoek, 1, 19–28.
Laurence, D.R. and Bacharach, A.L. 1964. Evaluation of Drug Activities: Pharmacometrics. Academic
Press, London, U.K., vol. 1, pp. 135–166.
Lee, J.-G., Yoo, I.-D., and Kim, W.-G. 2007. Differential antiviral activity of benzastatin C and its
dechlorinated derivative from Streptomyces nitrosporeus. Biol. Pharm. Bull., 30(4), 795–797.
Lin, Y.C., Wu, X.Y., Deng, Z.J., Wang, J. Zhou, S.N., Vrijmoed, L.L.P., and Jones, E.B.G. 2002. The
metabolites of the mangrove fungus Verruculina enalia No. 2606 from a salt lake in the Bahamas.
Phytochemistry, 59, 469–471.
Lin, Z., Zhu, T., Fang, Y., and Gu, Q. 2008. 1H and 13C NMR assignments of two new indolic enamide
diastereomers from a mangrove endophytic fungus Aspergillus sp. Magn. Reson. Chem., 46,
1212–1216.
Lucas, E.H. and Lewis, R.W. 1944. Antibacterial substances in organs of higher plants. Science, 100,
597–599.
Mantegazza, P. and Piccini, F. (eds.) 1966. Methods in drug evaluation. Proceedings of the International
Symposium, Milan 1965. North-Holland, Amsterdam, the Netherlands, 1966, pp. 548–573.
Mayer, A.M. and Hamann, M.T. 2005. Marine pharmacology in 2001–2002: Marine compounds with
anthelmintic, antibacterial, anticoagulant, antidiabetic, antifungal, anti-inflammatory, anti-
malarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the
cardiovascular, immune and nervous systems and other miscellaneous mechanisms of action.
Comp. Biochem. Physiol. Part C, 140 (3–4), 265–286.
Nakagawa, A., Tomoda, H., Hao, V. M., Iwai, Y., and Omura, S. 1985. Antiviral activities of pentale-
nolactones. J. Antibiot., 8, 1114–1115.
Omura, S. 1992. The Search for Bioactive Compounds from Microorganisms, 1st edn. Springer, Berlin,
Germany.
Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M., Takahashi, Y. et al. 2001.
Genome sequence of an industrial microorganism Streptomyces avermitilis: Deducing the abil-
ity of producing secondary metabolites. Proc. Natl. Acad. Sci., 98, 12215–12220.
Prudhomme, J., McDaniel, E., Ponts, N., Bertani, S., Fenical, W., and Jensen, P. 2008. Marine actino-
mycetes: A new source of compounds against the human malaria parasite. PLoS ONE, 3(6),
e2335.
Ravikumar, S., Gnanadesigan, M., Saravanan, A., Monisha, N., Brindha, V., and Muthumari, S. 2012.
Antagonistic properties of seagrass associated Streptomyces sp., RAUACT-1: A . Asian Pac. J.
Trop. Med., 5(11), 887–890.
Ravikumar, S., Gnanadesigan, M., Thajuddin, N., Chakkaravarthi, V., and Banerjee, B. 2010.
Anticancer property of sponge associated actinomycetes along Palk Strait. J. Pharm. Res., 3(10),
2415–2417.
Sacrament, O.D.R., Coelho, R.R.R., Wigg, M.D., Linhares, L.F.T.D., Santos, M.G.M.D., Semedo,
D.S.L., and DaSilva, A.J.R. 2004. Antimicrobial and antiviral activities of an actinomycete
(Streptomyces sp.) isolated from a Brazilian tropical forest soil. World J. Microbiol. Biotechnol.,
20(3), 225–229.
Schaeffer, D.J. and Krylov, V.S. 2000. Anti-HIV activity of extracts and compounds from algae and
cyanobacteria. Ecotoxicol. Environ. Saf., 45, 208–227.
Stritzke, K., Schulz, S., Laatsch, H., Helmke, E., and Beil, W. 2004. Novel caprolactones from a marine
Streptomycete. J. Nat. Prod., 67(3), 395–401.
Tang, J.S., Gao, H., Hong, K., Yu, Y., Jiang, M.M., Lin, H.P., Ye, W.C., and Yao, X.S. 2007. Complete
assignments of 1H and 13C NMR spectral data of nine surfactin isomers. Magn. Reson. Chem.,
45, 792–796.
Taylor, A., McKenna, G.F., and Burlage, H.M. 1952. Cancerchemotherapy experiments with plant
extracts. Tox. Rep. Biol. Med., 10, 1062.
Turner, R.A. 1965. Analgesics. In Screening Methods in Pharmacology. Academic Press, New York,
pp. 113–116.
Turner, R.A. and Hoborn, P. 1971. In Screening Methods in Pharmacology, Academic Press, New York,
2nd edition.
Tziveleka, L.A., Vagias, C., and Roussis, V. 2003. Natural products with anti-HIV activity from
marine organisms. Curr. Top. Med. Chem., 3, 1512–1535.
Watve, M.G., Tickoo, R., Jog, M.M., and Bhole, B.D. 2001. How many antibiotics are produced by the
genus Streptomyces. Arch. Microbiol., 176, 386–390.
Wu, J., Xiao, Q., Huang, J., Xiao, Z., Qi, S., Li, Q., Zhang, S., and Xyloccensin, O. 2004. Unique
8,9,30-phragmalin ortho esters from Xylocarpus granatum. Org. Lett., 6, 1841–1844.
Xie, X.C., Mei, W.L., Zhao, Y.X., Hong, K., and Dai, H.F. 2006. A new degraded sesquiterpene from
marine actinomycete Streptomyces sp. 0616208. Chinese Chem. Lett., 17, 1463–1465.
chapter twenty-five
Novel antidermatophytic drug

candidates from nature
Didem Deliorman Orhan and Nilüfer Orhan
Contents
25.1 Introduction......................................................................................................................... 487
25.2 Superficial fungal infections............................................................................................. 488
25.2.1 Candidiasis.............................................................................................................. 488
25.2.2 Malassezia infections............................................................................................... 488
25.2.3 Dermatophyte infections....................................................................................... 488
25.3 Treatment of dermatophyte infections............................................................................ 490
25.4 Studies on antidermatophytic activity of natural products and plant extracts......... 491
25.4.1 Plant extracts........................................................................................................... 492
25.4.2 Secondary metabolites........................................................................................... 492
25.4.2.1 Essential oils and terpenes..................................................................... 492
25.4.2.2 Phenolic compounds...............................................................................504
25.4.2.3 Saponins....................................................................................................505
25.4.2.4 Fatty acids................................................................................................. 506
25.4.2.5 Other compounds.................................................................................... 506
25.5 Conclusion........................................................................................................................... 507
References...................................................................................................................................... 507
25.1 Introduction
Historically, natural products have been used since ancient times traditionally for treat-
ment of many diseases. Chemistry and bioactivity studies ongoing for ages enabled scien-
tists to discover thousands of active molecules from a diverse range of chemical structures
from plants, fungi, lichens, and microorganisms. Additionally, a huge number of natural
compounds have become models for semisynthetic or synthetic compounds and are used
by the modern pharmaceutical industry. Hence, nature is a rich source for new drug can-
didates to cure various diseases.
Over 300 million people suffer from many fungal diseases, including superficial,
systemic, cutaneous, subcutaneous, and systemic mycoses, every year (Hean et al., 2011;
Global Action Fund for Fungal Infections, 2014). Among all, superficial mycoses are the
most frequent fungal diseases throughout the world (Avelar Pires et al., 2014).
487
Currently, several synthetic antifungal drugs used in the treatment of superficial

mycoses exhibit toxic side effects, including headaches, skin hypersensitivity, hepatic tox-
icity, and gastrointestinal disturbances (Bang et al., 2000). Therefore, many researchers
have been focusing on the discovery of safe and effective antidermatophytic drugs from
natural resources. For this purpose, the screening of the plants used in traditional medi-
cine is an approach to drug discovery.
25.2 Superficial fungal infections

Superficial fungal pathogens usually infect the outer layer of keratinized tissues such as
skin, hair, and nails and cause dermatophytosis, candidiasis, and Malassezia infections.
25.2.1 Candidiasis
Candidiasis is a fungal infection caused by the genus Candida, which could be found in
vagina, mouth, oropharynx, and gastrointestinal tract in many people. Candida albicans is
the first most common cause of 70%–80% of Candida infections. There are many types of
these infections such as mucosal candidiasis, candidemia, systemic candidiasis, vulvovag-
inal candidiasis, invasive candidiasis, cutaneous candidiasis, and oropharyngeal candi-
diasis. Today, amphotericin B, triazoles, echinocandins, and flucytosine are the treatment
options used for Candida infections (Ho and Cheng, 2010).
25.2.2 Malassezia infections

Malassezia-like lipophilic yeasts (M. furfur, M. sympodialis, and M. pachydermatis),
which are frequently found on human skin as commensals, cause Malassezia infec-
tions. M. f urfur is especially responsible for several skin diseases, including seborrheic
dermatitis, folliculitis, confluent and reticulated papillomatosis, and pityriasis versi-
color. First-line treatment of such infections is commonly with topical agents (imidazole
antifungals). Oral antifungal agents such as ketoconazole, itraconazole, and griseoful-
vin are preferred in the treatment of systemic and serious Malassezia infections. Prior
to use these oral antifungal drugs, liver function and blood tests of patients should be
performed (Ho and Cheng, 2010).
25.2.3 Dermatophyte infections

Fungi known as dermatophytes are from Microsporum, Trichophyton, and Epidermophyton
genera. These organisms are capable of causing fungal infections in keratinized tissues
(skin, hair, and nails) of both human and animals (Kishore et al., 1996). Systemic or top-
ical antifungal drugs could be preferred in the treatment of dermatophyte i nfections.
First-line therapy of dermatophyte infections is topical agents as in the treatment of
Malassezia infections. Dermatophytes are colonized in stratum corneum layer of the
epidermis and cause inflammatory changes in this layer. Topical antifungal drugs dif-
fuse into skin and inhibit the development and growth of fungus. Therefore, these
drugs should be capable of binding to stratum corneum cells. The vast majority of
patients with dermatophyte infections do not respond to topical drug therapy. In those
cases, systemic antifungal drugs or combination therapy should be chosen.
Chapter twenty-five: Novel antidermatophytic drug candidates from nature 489
According to clinical appearances, dermatophytosis is categorized into the following

nine major groups:
1.
Tinea barbae: Tinea barbae is one of the most common dermatophyte infections
especially among males. It is a rare infection involving the bearded areas of the
face and neck that is closely similar to tinea capitis. The transmission of the disease
occurs via direct contact with an infected animal. Typical clinical appearances of
this infection are severe pustular eruption, deep inflammatory plaques, or nonin-
flammatory superficial patches with invasion of the hair shaft (Sabota et al., 1996;
Baran et al., 2004).
2.
Tinea capitis: Tinea capitis is a dermatophyte infection of eyebrows, eyelashes,
scalp hair follicles, and the surrounding skin that is most common in children.
Typical symptoms are pustules, large inflammatory swellings (kerion), patchy
and scaly alopecia, broken-off hairs, and swollen lymph nodes. This infection is
transmitted from personal belongings (combs, bedding, towel, etc.) of children
with tinea capitis to other healthy children. Also, house pets, such as kittens
and puppies, can spread tinea capitis (Higgins et al., 2000; Rebollo et al., 2008;
Moriarty et al., 2012).
3.
Tinea corporis: Tinea corporis infection occurs in all body parts except for scalp, beard
area, feet, groin, and palms. Common clinical appearances are one or more round
or oval erythematous scaly skin (ringworm-like lesions). People may be contami-
nated with dermatophytes causing tinea corporis from clothing, combs, pool sur-
faces, shower floors, and walls. Tinea corporis has been implicated in several other
skin disorders such as nummular eczema, psoriasis, annular erythemas, and pityria-
sis rosea. It mainly affects children but can occur in people of all ages (Jacyk, 2004;
Karakoca et al., 2010).
4.
Tinea cruris (groin): It is an acute to chronic infection of the groin area, genitals, pubic
area, perineum, and perianal skin. Clinical features are a pruritic erythematous
rash with an active scaly palpable edge within pustules or vesicles. This infection
usually occurs predominantly in adult men. In diabetic patients, obese individuals,
and excessive sweating people, the risk of developing tinea cruris is high (Ho and
Cheng, 2010).
5.
Tinea favosa (favus): It is a chronic dermatophyte infection of the scalp and gla-
brous skin, generally caused by Trichophyton schoenleinii. This infection passes
from human to human and is most common in Africa and Eurasia. Tinea favosa,
also known as favus, is characterized by the presence of scutula (yellowish,
cup-shaped crusts) and severe alopecia (favosa) (Khaled et al., 2007; Anane and
Chtourou, 2013).
6.
Tinea imbricata: It is a kind of tinea corporis that is caused by Trichophyton concentri-
cum. Contamination is usually by direct contact between family members sharing
household items. This infection is characterized by various scaly papulosquamous
plaques arranged in concentric rings and appears on skin and scalp. Men and women
are affected equally by tinea imbricata infection (Satter, 2009).
7.
Tinea manuum: Tinea manuum, fungal infection of the hand, is commonly caused
by Trichophyton rubrum but also associated with Trichophyton tonsurans and
also known as “the two-feet-one-hand syndrome.” This infection often begins
to develop on the hands after the onset of tinea pedis. Scaly lesions on palmar
surface, crops of tiny blisters especially on the sides of the fingers and palm, itch-
ing, burning, and ringlike appearance of the infection on the skin are typically
observed (Ho and Cheng, 2010).
8.
Tinea pedis: Tinea pedis is also known as athlete’s foot and one of the most com-
mon types of dermatophyte infections. The strains (Epidermophyton floccosum,
Microsporum gypseum, Trichophyton mentagrophytes, T. rubrum, T. tonsurans) of derma-
tophyte causing the infection thrive in moist environments. Therefore, athlete’s foot
is generally picked up from swimming pools, showers, or locker rooms. Commonly
affected areas include the feet, especially the soles and toe webs. Symptoms and
signs of this infection are itchiness, redness, fine silvery white flakes on slightly
erythematous skin, and sometimes blistering or cracking of skin (Weitzman
and Summerbell, 1995; Ayatollahi Mousavi et al., 2009; American Orthopaedic
Foot & Ancle Society, 2013).
9.
Tinea unguium: Tinea unguium caused by E. floccosum, M. gypseum, T. mentagrophytes,
T. rubrum, and T. tonsurans is also known as onychomycosis (Ayatollahi Mousavi
et al., 2009). Infection can occur in nail tissues of hands or feet. It is usually picked up
in damp areas such as public gyms, showers, or swimming pools and can be spread
from human to human. It is characterized by thickening of the nail, discoloration,
and destructive and flaky lesions in nails. Symptoms are similar to many condi-
tions such as those of psoriasis, onychogryphosis, and lichen planus (Weitzman and
Summerbell, 1995). Thus, diagnosis of the disease should be confirmed by laboratory
examinations before the treatment.
25.3 Treatment of dermatophyte infections

Drugs used in the treatment of dermatophyte infections can be classified into six c ategories
(Del Palacio et al., 2000; Gupta and Cooper 2008; Dias et al., 2013). Clinical diagnosis and
treatment options of dermatophyte infections and dermatophyte species also are given in
Table 25.1.
1.
Azoles
a. Imidazoles: clotrimazole (cream, spray, lotion and solutions, vaginal tablet), eber-
conazole (cream), econazole (water miscible cream and lotion), ketoconazole
(cream, shampoo, tablet), luliconazole (cream), miconazole (oral gel, powder,
ointment, cream, solution, spray, lotion), oxiconazole (cream and lotion), sertacon-
azole (cream), sulconazole (cream and solution), tioconazole (cream, nail solution)
b. Triazoles: itraconazole (capsule, liquid preparations), fluconazole (capsule, tablet,
powder and intravenous infusion)
2.
Allylamines: terbinafine (tablet, solution, and cream), naftifine (cream)
3.
Benzylamines: butenafine (cream)
4.
Hydroxypyridone: ciclopirox (cream, gel, lotion, solution, and nail lacquer)
5.
Morpholine derivatives: amorolfine (cream and nail lacquer)
6.
Other: spiro-benzo[b]furan derivative griseofulvin (tablet, spray, and cream)
Clinical diagnosis and treatment options of dermatophyte infections and dermato-

phyte species (Odom, 1993; Del Palacio et al., 2000; Asticcioli et al., 2008; Gupta and
Cooper, 2008, Ayatollahi Mousavi et al., 2009; Moodahadu-Bangera et al., 2012; Dicle
and Özkesici, 2013).
Table 25.1 Treatment options for dermatophytes

Clinical diagnosis Species of dermatophytes Treatment options
Tinea barbae Mg, Tm, Tve Itraconazole, terbinafine
Tinea capitis Ma, Mc, Md, Mf, Mfu, Fluconazole, griseofulvin, itraconazole, terbinafine
Mg, Mn, Tm, Tmi, Tsc,
Tso, Tt, Tv, Tve, Ty
Tinea corporis Ef, Mc, Mg, Tm, Tr, Tt, Amorolfine, butenafine, clotrimazole, eberconazole,
Tv, Tve econazole, fluconazole, griseofulvin, itraconazole,
ketoconazole, miconazole, naftifine, oxiconazole,
sertaconazole, sulconazole, terbinafine
Tinea cruris Ef, Mg, Tr Butenafine, clotrimazole, eberconazole, econazole,
fluconazole, griseofulvin, itraconazole,
ketoconazole, miconazole, naftifine, oxiconazole,
sertaconazole, sulconazole, terbinafine
Tinea favosa ( favus) Tsc Griseofulvin
Tinea imbricata Tc Terbinafine
Tinea manuum Tr, Tt Itraconazole, terbinafine
Tinea pedis Ef, Mg, Tm, Tr, Tt Amorolfine, clotrimazole, econazole, fluconazole,
griseofulvin, itraconazole, ketaconazole,
miconazole, naftifine, oxiconazole, sertaconazole,
sulconazole terbinafine
Tinea unguium Ef, Mg, Tm, Tr, Tt Amorolfine, fluconazole, griseofulvin itraconazole,
ketaconazole, terbinafine, tioconazole
Ef, Epidermophyton floccosum; Ma, Microsporum audouinii; Mc, Microsporum canis; Md, Microsporum distortum; Mf,
Microsporum ferrugineum; Mfu, Microsporum fulvum; Mg, Microsporum gypseum; Mn, Microsporum nanum;
Tc, Trichophyton concentricum; Tm, Trichophyton mentagrophytes; Tmi, Trichophyton megninii; Tr, Trichophyton
rubrum; Tsc, Trichophyton schoenleinii; Tso, Trichophyton soudanense; Tt, Trichophyton tonsurans; Tv, Trichophyton
violaceum; Tve, Trichophyton verrucosum; Ty, Trichophyton yaoudei.
25.4 Studies on antidermatophytic activity

of natural products and plant extracts
In today’s world, the incidence of antifungal infections in humans is increasing day
by day. Although there are a number of antifungal preparations, their effectiveness is
limited because of many reasons such as side effects, toxicity, arise of resistant strains,
and lack of oral and parenteral preparations due to solubility problems. Thus, there is
a great desire for the discovery of new antifungal and antidermatophytic drug candi-
dates that are soluble, broad spectrum, and having new mechanism of actions (Vila
et al., 2013). Natural products are the most important choices in antidermatophytic
activity researches. Many different techniques are used for the determination of anti-
fungal activity of natural products, and these methods are described in detail in numer-
ous reviews (Rios et al., 1998, Jacob and Walker, 2005; Cos et al., 2006; Engelmeier and
Hadacek, 2006; Das et al., 2010). Mainly extracts, essential oils, and different secondary
metabolite groups of medicinal plants and also isolated compounds were studied for
their antifungal activity.
25.4.1 Plant extracts

According to an extensive literature survey, it is known that there is an extremely high
number of studies on antidermatophytic activities of medicinal plants. Thus, the summary
of these studies is organized in Table 25.2 to be clear and easily understood. Only plants
having antidermatophytic activity are mentioned in the table; therefore, inactive plants
are not included. Latin names of 142 different plants, their families, parts of the plants that
have antidermatophytic activity, type of the extract, affected dermatophyte type, and data
of the references are given.
25.4.2 Secondary metabolites

Many secondary plant metabolites having different chemical structures are reported to
have antidermatophytic activities. However, most of the antidermatophytic plants, second-
ary metabolites, or pure compounds have only been tested in in vitro experimental models.
Thus, their effects, adverse effects, and toxic effects in humans still remain unknown.
Despite these facts, natural compounds might be the new precursors to design highly
effective drug candidates. A series of molecules from several chemical groups such as
terpenes, flavonoids, fatty acids, polyphenols, and alkaloids have been described to have
antidermatophytic activities (Dorman and Deans, 2000; Abad et al., 2007; Arif et al., 2009;
Lang and Buchbauer, 2012). In this part, examples of active compounds belonging to dif-
ferent secondary metabolite groups are summarized.
25.4.2.1 Essential oils and terpenes

Essential oils have shown several biological activities such as antimicrobial, antipara-
sitical, insecticidal, anti-inflammatory, wound healing, and antioxidant. Many studies
have reported the antifungal activity of essential oils against dermatophytes. In this
connection, the antifungal essential oils from the Lamiaceae and Asteraceae families
are very popular.
The essential oil obtained by water distillation from the aerial parts of Thymus pulegi-
oides (Lamiaceae) growing from Portugal was investigated against Microsporum canis FF1,
M. gypseum FF3, T. rubrum FF5, T. mentagrophytes FF7, and E. floccosum FF9. For T. rubrum,
essential oil at subinhibitory concentration (0.08 µg/mL) decreased ergosterol content
by around 70%. Ergosterol plays a fundamental role in maintaining the integrity and
function of the yeast cell membrane. In this report, antifungal activity of T. pulegioides
essential oil has been ascribed to the presence of high contents of carvacrol and thymol
(Pinto et al., 2006).
Effect of an essential oil mixture consisting of Thymus serpyllum, Origanum vulgare,
and Rosmarinus officinalis (Lamiaceae) in Prunus dulcis oil on dermatophyte infection in
sheep caused by T. mentagrophytes was evaluated. For this purpose, the mixture and its
main components (thymol, carvacrol, 1,8-cineole, α-pinene, p-cymene, and γ-terpinene)
were tested against fungal clinical isolate. As a result, it was thought that the essential oil
mixture would be effective for limiting fungal growth (Mugnaini et al., 2013).
Antifungal activity of the essential oil obtained by water distillation from flow-
ers of Matricaria recutita (Asteraceae) was tested on dermatophytes such as M. canis
PFCC 50691, M. gypseum PFCC 50701, T. rubrum PFCC 51431, T. tonsurans PFCC88-
1352, and T. mentagrophytes PFCC 50541 using microbioassay technique. The growth
of all tested dermatophytes was inhibited to varying degrees by the essential oil.
Gas c hromatography–mass spectroscopy (GC-MS) analysis exhibited that the main
Table 25.2 Active plant extracts on dermatophytes
Plant name Family Plant part Active extract Fungi Reference
Acacia erioloba Edgew. Fabaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Acalypha indica L. Euphorbiaceae Leaf Hexane, ethanol, methanol Ef, Tm, Tr, Ponnusamy et al. (2010)
Tt
Acalypha manniana Müll. Euphorbiaceae Leaf Ethyl acetate, methanol Mg, Te, Tm Noumedem et al. (2013)
Arg.
Acanthospermum australe Asteraceae Aerial parts Dichloromethane, Mg, Tm Portillo et al. (2001)
Kuntze methanol, water
Acanthospermum hispidum Asteraceae Aerial parts Dichloromethane Mg, Tm Portillo et al. (2001)
DC.
Acokanthera schimperi Apocynaceae Leaf Methanol Tm Tadeg et al. (2005)
(A. DC.) Schweinf.
Aegle marmelos (L.) Correa Rutaceae Leaf Ethanol, methanol, water Ef, Mc,Tm Balakumar et al. (2011)
Allium ascalonicum Liliaceae Bulb Water Mg, Tm, Tr Amin and Kapadnis (2005)
Allium cepa L. Liliaceae Bulb Water Ef, Mc, Mg, Amin and Kapadnis (2005),
Tm, Tr, Ts Shams-Ghahfarokhi et al. (2004,
2006)
Allium sativum L. Liliaceae Bulb Water Ef, Mc, Mg, Amin and Kapadnis (2005),
Tm, Tr Shams-Ghahfarokhi et al. (2006)
Aloe arborescens Mill. Xanthorrhoeaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Alpinia galanga (L.) Willd. Zingiberaceae Rhizome Chloroform, ethanol Mg, Tl Khattak et al. (2005), Phongpaichit
et al. (2005)
Chapter twenty-five: Novel antidermatophytic drug candidates from nature
Anagallis arvensis L. Primulaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Anchusa strigosa (Soland.) Boraginaceae Aerial parts Water Tv Ali-Shtayeh and Abu Ghdeib (1999)
Andira inermis H. B. & K. Fabaceae Bark Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
Andira surinamensis Fabaceae Bark Dichloromethane, Mg, Tm Freixa et al. (1998)
(Boudt.) Splitz methanol
Annona cherimola Mill. Annonaceae Seed Methanol Tm, Tr, García et al. (2003)
493
(Continued)
Table 25.2 (Continued) Active plant extracts on dermatophytes
494

Anogeissus leiocarpus (DC.) Combretaceae Bark, leaf, Chloroform, ethanol, ethyl Ma, Mg, Batawila et al. (2005), Mann et al.
Guill. & Perr. (L.) root acetate, methanol, water Mn, Tm, Tr (2008)
Aristea ecklonii Baker. Iridaceae Leaf, root Dichloromethane/ Tm Mabona et al. (2013)
methanol
Asclepia curassavica L. Asclepiadaceae Leaf Hexane, methanol Tr García et al. (2003)
Asphodelus microcarpus Liliaceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Salzm. et Viv.
Asphodelus luteus L. Liliaceae Aerial parts Water Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Azadirachta indica (Neem) Meliaceae Seed 5% DMSO Mn, Tm, Tr Natarajan et al. (2003)
Baccharis articulata Pers. Asteraceae Aerial parts Dichloromethane Mg, Tm Portillo et al. (2001)
Bixa orellana L. Bixaceae Leaf Dichloromethane, Mg, Tm, Tr Freixa et al. (1998), García et al.
methanol (2003)
Blepharocalyx tweediei Myrtaceae Leaf, seed Dichloromethane, Mg, Tm Freixa et al. (1998)
(Hook. et Arn.) Berg. methanol
Boesenbergia pandurata Zingiberaceae Rhizome Chloroform Mg Phongpaichit et al. (2005)
(Robx.) Schltr.
Calycophyllum multiflorum Rubiaceae Bark Dichloromethane, water Mg, Tm Portillo et al. (2001)
Griseb.
Capparis spinosa L. Capparidaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Cassia alata L. Fabaceae Leaf Hexane, ethanol, methanol Ef, Tm, Tr, Ponnusamy et al. (2010)
Ts, Tt
Cassia fistula L. Fabaceae Flower Chloroform, hexane, ethyl Ef, Tm, Tr, Duraipandiyan and Ignacimuthu
acetate, methanol, water Ts, Ss et al. (2007)
Cassia tora L. Fabaceae Leaf Chloroform, ethanol Ef, Tm Rath and Mohanty (2013)
Chenopodium ambrosioides Chenopodiaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
Bert. ex Steud. methanol
Cicca acida Merr. Euphorbiaceae Methanol Mg, Tm Chauhan et al. (2012)
Cichorium intybus L. Asteraceae Root Acetone Tt Mares et al. (2005)
Clematis cirrhosa L. Ranunculaceae Aerial parts Water Tv Ali-Shtayeh and Abu Ghdeib (1999)
(Continued)
Clerodendrum inerme (L.) Verbenaceae Leaf, stem Ethyl acetate, hexane Ef, Tm, Tr, Anitha and Kannan (2006)
Gaertn. Tt
Clerodendrum phlomidis L.f. Verbenaceae Leaf, stem Ethyl acetate, hexane Ef, Tm, Tr, Anitha and Kannan (2006)
Tt
Combretum fragrans Combretaceae Leaf Ethanol Mn, Tm, Tr Batawila et al. (2005)
F. Hoffm.
Croton gacilipes Baill. Euphorbiaceae Leaf Dichloromethane Mg, Tm Portillo et al. (2001)
Croton urucurana Baill. Euphorbiaceae Leaf Dichloromethane, water Mg, Tm Portillo et al. (2001)
Croton zehntneri Pax & K. Euphorbiaceae Leaf Dichloromethane Mg, Tm Freixa et al. (1998)
Hoffm.
Curcuma longa L. Zingiberaceae Rhizome Ethanol Ef, Tl, Tm Khattak et al. (2005), Rath and
Mohanty (2013)
Dicoma anomala Sond. Asteraceae Tuber Dichloromethane/ Tm Mabona et al. (2013)
methanol
Diospyros mespiliformis Ebenaceae Leaf Dichloromethane/ Mc, Tm Mabona et al. (2013)
Hochst. ex A.D.C. methanol
Dodonaea angustifolia L.f. Sapindaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Elephantorrhiza elephantina Fabaceae Rhizome, Dichloromethane/ Mc Mabona et al. (2013)
(Burch.) Skeels root methanol
Erythrina christi-galli L. Fabaceae Bark Dichloromethane Mg, Tm Portillo et al. (2001)
Eucalyptus camaldulensis Myrtaceae Leaf Methanol Ef, Tm, Tsc Falahati et al. (2005)
Dehnh.
Eupatorium Asteraceae Leaf Hexane, methanol Tm,Tr García et al. (2003)

aschenborniarum Schauer
Eupatorium buniifolium Asteraceae Aerial parts Methanol Mg, Tr, Tm Muschietti et al. (2005)
Hook & Arn.
Ficus natalensis Hochst. Moraceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
(Continued)
495
496

Ficus sur Forssk. Moraceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Gallesia integrifolia Phytolaccaceae Bark Dichloromethane, Mg, Tm Freixa et al. (1998)
(Spreng.) Harms methanol
Galphimia glauca Cav. Malpighiaceae Aerial parts Hexane, methanol Tm,Tr García et al. (2003)
Geophila repens (L.) I.M. Rubiaceae Whole Dichloromethane, Mg, Tm Portillo et al. (2001)
Johnston parts methanol, water
Gentianella nitida Griseb. Gentianaceae Whole Methanol Mg, Tm Rojas et al. (2004)
parts
Gunnera perpensa L. Gunneraceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Harpephyllum caffrum Anacardiaceae Bark Dichloromethane/ Tm Mabona et al. (2013)
Bernh. ex Krauss methanol
Hedyosmum anisodorum C. Chloranthaceae Leaf Dichloromethane Mg, Tm Freixa et al. (1998)
A. Todzia
Heterotheca inuloides Cass. Asteraceae Flower Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
Hura crepitans L. Euphorbiaceae Bark Dichloromethane, Tm Freixa et al. (1998)
methanol
Hymenaea martiana Hayne Fabaceae Bark, Trunk Butanol, ethanol, Mc, Tm, Tr Machado de Souza et al. (2009)
methanol
Hypericum ternum A. Sit. Hypericaceae Aerial parts Chloroform, petroleum Ef, Mc, Mg, Fenner et al. (2005)
Hil. ether Tm, Tr
Inula viscosa (L.) Aiton Asteraceae Aerial parts Water Mc, Mg, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Ixora coccinia L. Rubiaceae Methanol, water Mg, Tm Chauhan et al. (2012)
Jacaranda mimosifolia D. Bignoniaceae Aerial Parts Methanol Mc Muschietti et al. (2005)
Don
Juglans regia L. Juglandaceae Aerial parts Water Mc, Mg, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Lannea discolor Engl. Anacardiaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
(Continued)
Lantana rugosa Thunb. Verbenaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Lawsonia inermis L. Lythraceae Leaf Hexane, ethanol, methanol Ef, Tm, Tr, Ponnusamy et al. (2010)
Ts, Tt
Lippia adoensis Hochst. ex Verbenaceae Leaf Chloroform, methanol, Tm Tadeg et al. (2005)
Walp. petroleum ether
Lippia integrifolia (Griseb.) Verbenaceae Aerial Parts Methanol Mc, Mg, Ef, Muschietti et al. (2005)
Hieron. Tr, Tm
Lithrea molleoides (Vell.) Anacardiaceae Aerial Parts Methanol Mc, Mg, Ef, Muschietti et al. (2005)
Engl. Tr, Tm
Lysiloma acapulcensis Fabaceae Bark Methanol Tm,Tr García et al. (2003)
(Kunth) Benth.
Malva parviflora L. Malvaceae Leaf, root Dichloromethane/ Tm Mabona et al. (2013), Tadeg et al.
methanol, methanol (2005)
Mansoa alliaceae (Lam.) A. G. Bignoniaceae Leaf Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
Maytenus ilicifolia Mart. ex Celastraceae Stem Dichloromethane, Mg, Tm Portillo et al. (2001)
Reiss methanol
Melianthus comosus Vahl. Melianthaceae Leaf Dichloromethane/ Mc, Tm Mabona et al. (2013)
methanol, methanol
Melianthus major L. Melianthaceae Leaf Dichloromethane/ Mc, Tm Mabona et al. (2013)
methanol
Mentha longifolia Huds, Lamiaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
methanol
Micromeria nervosa (Desf.) Lamiaceae Aerial parts Water Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Benth
Ocimum gratissimum L. Lamiaceae Leaves Hexane Mc, Mg, Tm, Silva et al. (2005)
Tr
Ocimum micranthum Willd. Lamiaceae Aerial parts Dichloromethane Mg, Tm Freixa et al. (1998)
(Continued)
497
498

Olinia rochetiana A. Juss. Oliniaceae Leaf Acetone, chloroform, Tm Tadeg et al. (2005)
methanol, petroleum
ether
Parietaria diffusa Mert. & Urticaceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib (1999)
W.D.J. Koch
Paronychia argentea Lam. Caryophyllaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Peltophorum pterocarpum Fabaceae Methanol Mg, Tm Chauhan et al. (2012)
(DC.) K. Heyne
Persea laevigata H. B. Et K. Lauraceae Bark Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
Persicaria glabra Mill. Polygonaceae Methanol Mg, Tm Chauhan et al. (2012)
Phagnalon rupestre (L.) DC. Asteraceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Phyllanthus amarus Schum. Euphorbiaceae Aerial parts Chloroform Mg Agrawal et al. (2004)
& Thonn.
Piper betle L. Piperaceae Leaf Ethanol Mc, Mg, Tm Trakranrungsie et al. (2008)
Piper elongatum C. DC. Piperaceae Leaf Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
Piper fulvescens C. DC. Piperaceae Leaf Dichloromethane Mg, Tm Freixa et al. (1998)
Piper solmsianum C. DC. Piperaceae Leaf Dichloromethane, hexane, Ef, Mc, Mg, De Campos et al. (2005)
var. solmsianum methanol Tm, Tr
Pistacia lentiscus L. Anacardiaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Pistia stratiotes L. Araceae Leaf Methanol Ef, Mn, Mg, Premkumar and Shyamsundar
Tm, Tr (2005)
Pittosporum viridiflorum Pittosporaceae Leaf Dichloromethane/ Tm Mabona et al. (2013)
Sims. methanol
Plumbago europaea L. Plumbaginaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Polygonum hydropiperoides Polygonaceae Leaf Dichloromethane Mg, Tm Freixa et al. (1998)
Michx.
Potalia amara Aubl. Loganiaceae Leaf, stem Dichloromethane, Mg, Tm Freixa et al. (1998)
methanol
(Continued)
Pterocaulon alopecuroides Asteraceae Aerial parts Dichloromethane, hexane, Mg, Tr, Tm Stein et al. (2005)
(Lam.) D.C. methanol
Pterocaulon balansae Asteraceae Aerial parts Dichloromethane, hexane Mg, Tr, Tm Stein et al. (2005)
Chodat.
Pterocaulon polystachyum Asteraceae Aerial parts Hexane, methanol Mg, Tr, Tm Stein et al. (2005)
D.C.
Pterospermum suberifolium Sterculariaceae Methanol Mg, Tm Chauhan et al. (2012)
(L.) Lam.
Punica granatum L. Lythraceae Fruit rind Methanol Ef, Tm, Tr, Ponnusamy et al. (2010)
Ts, Tt
Retama raetam (Forssk.) Papilionaceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib
Webb (1999)
Ruscus aquleatus L. Liliaceae Aerial parts Water Mc, Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Ruta chalepensis L. Rutaceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Salvia fruticosa Mill. Lamiaceae Aerial parts Water Mc, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Sebastiania brasiliensis Euphorbiaceae Aerial parts Methanol Mc, Mg, Ef, Muschietti et al. (2005)
Spreng. Tr, Tm
Sebastiania commersoniana Euphorbiaceae Aerial parts Methanol Mc, Mg, Ef, Muschietti et al. (2005)
(Baill.) L. B. Sm. & B.J. Tr, Tm
Downs
Sedum oxypetalum HBK. Crassulaceae Leaf Hexane, methanol Tm,Tr García et al. (2003)
Senecio angulifolius DC. Asteraceae Leaf Tm García et al. (2003)
Senecio grisebachii Baker Asteraceae Flower Dichloromethane, Mg, Tm Portillo et al. (2001)
methanol, water
Senecio inaequidens DC. Asteraceae Aerial parts Chloroform, hexane, Mg, Tt Loizzo et al. (2004)
methanol
Senecio vulgaris L. Asteraceae Aerial parts Chloroform, hexane, Mg, Tt Loizzo et al. (2004)
methanol
Solanum nigrum L. Solanaceae Aerial parts Water Tv Ali-Shtayeh and Abu Ghdeib (1999)
Spilanthes cavla DC. Asteraceae Root Petroleum ether, water Tm Rai et al. (2004)
499
(Continued)
500

Tabebuia avellanedae Griseb. Bignoniaceae Bark Dichloromethane, Mg, Tm Portillo et al. (2001)
methanol, water
Terminalia avicennioides Combretaceae Root Ethanol, ethyl acetate, Ma, Tr Mann et al. (2008)
Guill. & Perr. Methanol, water
Terminalia brachystemma Combretaceae Leaf Acetone, dichloromethane, Mc Masoko et al. (2005)
Welw. ex Hiern hexane, methanol
Terminalia elliptica Willd. Combretaceae Methanol, water Mg, Tm Chauhan et al. (2012)
Terminalia gazensis Bak.f. Combretaceae Leaf Acetone, dichloromethane, Mc Masoko et al. (2005)
hexane, methanol
Terminalia glaucescens Combretaceae Leaf, root Ethanol Mg, Mn, Batawila et al. (2005)
Planh. ex Benth. Tm, Tr
Terminalia laxiflora Engl. Combretaceae Leaf, root Ethanol Mn, Tm, Tr Batawila et al. (2005)
et Diels
Terminalia macroptera Combretaceae Bark, leaf, Ethanol Mn, Tm, Tr Batawila et al. (2005)
Guill. et Perr. root
Terminalia mollis Laws. Combretaceae Leaf Acetone, dichloromethane, Mc Masoko et al. (2005)
hexane, methanol
Terminalia prunioides M.A. Combretaceae Leaf Acetone, dichloromethane, Mc Masoko et al. (2005)
Lawson hexane, methanol
Terminalia sabesica Engl. & Combretaceae Leaf Acetone, dichloromethane, Mc Masoko et al. (2005)
Diels hexane, methanol
Terminalia sericea Burch Combretaceae Leaf, root Acetone, dichloromethane, Mc, Tm Masoko et al. (2005), Mabona et al.
ex DC. dichloromethane/ (2013)
methanol, hexane,
methanol
(Continued)
Terminalia trifolia (Griseb.) Combretaceae Aerial parts Methanol Mg, Tr, Tm Muschietti et al. (2005)
Lillo
Thespesia populnea (L.) Sol. Malvaceae Leaf Chloroform, hexane, Ef, Tm, Tr, Ponnusamy et al. (2010)
ethanol, methanol Ts, Tt
Thevetia nerrifolia Juss. Apocynaceae Leaf Methanol Mg, Tm, Tr, Singh and Vidyasagar (2014)
Tt
Trachyspermum ammi Apiaceae Methanol Mg, Tm Chauhan et al. (2012)
Sprague
Vernonia tweedieana Baker Asteraceae Root Dichloromethane Mg, Tm Portillo et al. (2001)
Warburgia salutaris Canellaceae Bark, leaf Dichloromethane/ Tm Mabona et al. (2013)
(G.Bertol.) Chiov. methanol
Wrightia tinctoria R. Br. Apocynaceae Leaf, seed Chloroform, hexane, Ef, Tm, Tr, Ponnusamy et al. (2010)
ethanol, methanol Ts, Tt
Xanthosoma sagittifolium L. Araceae Leaf, stalk, Water Tr Schmourlo et al. (2005)
Scott. root
Xylosma longifolium Clos. Flacourtiaceae Bark, leaf Chloroform, methanol, Mc, Mg, Tr Devi et al. (2013)
petroleum ether
Zizyphus spina-christi (L.) Rhamnaceae Aerial parts Water Tm, Tv Ali-Shtayeh and Abu Ghdeib (1999)
Desf.
Ef, Epidermophyton floccosum; Ma, Microsporum audouinii; Mc, Microsporum canis; Mg, Microsporum gypseum; Mn, Microsporum nanum; Te, Trichophyton equinum;
Tl, Trichophyton longifusus; Tm, Trichophyton mentagrophytes; Tr, Trichophyton rubrum; Ts, Trichophyton simii; Tsc, Trichophyton schoenleinii; Tt, Trichophyton tonsurans;
Tv, Trichophyton violaceum; Ss, Scopulariopsis sp.
501
compounds of the essential oil were chamazulene (61.3%), isopropyl hexadecanoate

(12.7%), trans-trans-farnesol (6.9%), and E-β-farnesol (5.2%) (Jamalian et al., 2012).
Varying polarity extracts of the roots of Vernonanthura tweedieana (Asteraceae) used
for the treatment of skin diseases in Paraguay were screened in order to evaluate their
antifungal activity against M. gypseum and T. mentagrophytes. One active sesquiterpene,
identified as 6-cinnamoyloxy-1-hydroxyeudesm-4-en-3-one, against T. mentagrophytes was
isolated from the antifungal dichloromethane extract through bioassay-guided fraction-
ation procedures (Portillo et al., 2005).
Berry essential oil of Juniperus communis sp. alpina and leaf and berry essential oils
of Juniperus turbinata and Juniperus oxycedrus sp. oxycedrus (Cupressaceae) were tested
against T. rubrum FF5, T. mentagrophytes FF7, M. canis FF1, and M. gypseum FF3 using
a macrodilution method. In this study, J. oxycedrus sp. oxycedrus leaf oil was found to
be effective against all dermatophyte strains tested. One of the main components of
the essential oil is δ-3-carene, which has potent antidermatophyte activity (Cavaleiro
et al., 2006).
Melaleuca alternifolia (Thymelaeaceae) essential oil, also known as tea tree oil or
melaleuca oil, has been used externally as a traditional medicine for various condi-
tions including acne, athlete’s foot, nail fungus, wounds, oral candidiasis, cold sores,
and skin lesions. Antifungal activity of purchased tea tree oil samples in Australia was
investigated against seven dermatophyte strains. Tea tree oil was found to have both
its inhibitory and fungicidal effects using in vitro susceptibility and time-kill assays
(Hammer et al., 2002).
Antidermatophyte activity of Lonicera japonica (Caprifoliaceae) leaf essential oil was
studied against three strains of M. canis, three strains of T. rubrum, and two strains of
T. mentagrophytes. The essential oil (1000 ppm) exhibited moderate antifungal activity
(55.1%–70.3%) against all dermatophyte strains tested except for T. mentagrophytes KCTC
6085 and also displayed inhibitory effect on spore germination of all tested microorgan-
isms. The findings showed that L. japonica leaf essential oil could be a therapeutic alterna-
tive for dermatophyte infections (Rahman et al., 2014).
Fruit essential oil of Pimpinella anisum (Apiaceae) is utilized as expectorant, carmina-
tive, flavoring, and spice. Effect of the essential oil was evaluated in vitro on four clinical
isolates of dermatophytes (T. rubrum, T. mentagrophytes, M. canis, and M. gypseum). Anise
oil displayed potent inhibitory activity against tested fungi with minimum inhibitory con-
centration (MIC) lower than 0.78% (v/v) (Kosalec et al., 2005).
Natural coniferous resin (Norway spruce resin) obtained from Picea abies (Pinaceae)
has been known to have strong antifungal activity. Sipponen et al. (2012) examined the
clinical efficacy of Norway spruce resin for topical treatment of onychomycosis. Thirty-
seven patients with onychomycosis caused by T. rubrum and T. mentagrophytes used topical
resin lacquer therapy for 9 months. The 14 patients who completed treatment considered
the topical resin treatment to be effective.
The activity of natural essence of bergamot (Citrus bergamia, Rutaceae) was tested on
92 clinical isolates of dermatophytes (Trichophyton, Epidermophyton, and Microsporum spe-
cies) using in vitro susceptibility assays. Bergamot oil exhibited activity against all tested
fungi with MICs ranging from 0.156% to 2.5%. Therefore, this natural essence could be
recommended as potential source for the topical treatment of dermatophyte infections
(Sanguinetti et al., 2007).
The leaf essential oils of Ageratum houstonianum, Chenopodium ambrosioides, Citrus med-
ica, Corymbia citriodora, Hyptis suaveolens, Melaleuca leucadendron, Murraya koenigii, Ocimum
sanctum, Solidago canadensis, and Tagetes erecta were investigated for their fungitoxicity
against Microsporum audouinii and T. mentagrophytes. Only C. ambrosioides oil displayed anti-
mycotic activity against all dermatophyte strains tested. Chenopodium ointment prepared
with petroleum jelly was applied to guinea pigs with ringworm infection for 15 days. The
infection was completely treated at the end of the treatment. Chenopodium oil contains
mainly ascaridole, and this compound was thought to be responsible for the antimycotic
activity of the oil (Kishore et al., 1996).
Beikert et al. (2012) examined the efficacy of 6% coriander oil (Apiaceae) in unguen-
tum leniens in the treatment of tinea pedis patients. This cream exhibited a considerable
improvement in the clinical signs of tinea pedis. Also, this medication was well tolerated
by patients.
Antidermatophyte activity of several Iranian medicinal plant essential oils (Artemisia
sieberi, Cuminum cyminum, Foeniculum vulgare, Heracleum persicum, Mentha spicata, Nigella
sativa, Rosmarinus officinalis, Zataria multiflora, and Ziziphora clinopodioides) was studied
against T. mentagrophytes, T. rubrum, E. floccosum, M. gypseum, and M. canis using the broth
microdilution technique. The highest activity was exerted by A. sieberi, having a lower
MIC against dermatophytes than other plant essential oils (Khosravi et al., 2013).
The essential oil obtained from rhizomes of Homalomena aromatica (Araliaceae),
which is used in the treatment of skin infections and joint pains in India, was assayed
for antifungal activity against T. rubrum, T. mentagrophytes, Microsporum fulvum, and
M. gypseum. The rhizome oil significantly inhibited the growth of all tested strains of
dermatophytes. The composition of active essential oil was analyzed by GC–MS, and lin-
alool, terpene-4-ol, δ-cadinene, and T-muurolol were determined as main components
(Policegoudra et al., 2012).
The essential oils obtained by hydrodistillation from the rhizomes of nine
Zingiberaceae species (Zingiber officinale, Zingiber zerumbet, Curcuma aeruginosa, Curcuma
mangga, Curcuma xanthorrhiza, Kaempferia galanga, Alpinia galanga, and Boesenbergia
pandurata) were evaluated for their antifungal activities against five dermatophytes
(T. mentagrophytes, T. rubrum, M. canis, Microsporum nanum, and E. floccosum). Among the
samples tested, only B. pandurata essential oil exhibited the inhibitory activity on the
growth of all fungi. Camphor, geraniol, 1,8-cineole, methyl cinnamate, and camphene
were identified as main components in the essential oil. Previous studies reported that
the essential oils containing camphor have antifungal activity against some dermato-
phyte strains such as T. mentagrophytes (Jantan et al., 2003).
Five volatile column fractions of Cupressus lusitanica (Cupressaceae) leaf hexane extract
were screened for their antidermatophytic activity using the agar dilution method against
five reference dermatophyte strains (M. audouinii, Microsporum langeronii, M. canis, T. rubrum,
and T. tonsurans). The chemical composition of the column fractions with the highest activity
was analyzed by GC–MS, and the main components were identified as α-pinene, epi-bicy-
closesquiphellandrene, pimaric acid, kaurenoic acid, and 8-β-hydroxysandracopimarane
(Kuiate et al., 2006).
Triterpenoids (friedelin, β-amyrin acetate, betulinic acid, and lupeol) isolated from
the EtOAc extract of the stem bark of Syzygium jambos (Myrtaceae) used in traditional
medicine for the treatment of toothache, mouth sores, cough, and as a wound dress-
ing in Cameroon were investigated for their antifungal activity against M. audouinii,
T. mentagrophytes, and Trichophyton soudanense. Betulinic acid and friedelolactone have
been found to exhibit strong antidermatophytic activity against T. soudanense and
T. mentagrophytes (Kuiate et al., 2007).
Maytenin and pristimerin isolated from the bark of the roots of Maytenus ilicifolia
(Celastraceae) are quinonemethide triterpenoid compounds, and their antifungal activity
was tested against T. rubrum and T. mentagrophytes. Maytenin showed more strong antide-
rmatophytic activity than pristimerin on T. rubrum clinical isolate (Gullo et al., 2012).
25.4.2.2 Phenolic compounds

In recent years, many studies were conducted on the antidermatophytic activity of pheno-
lic compounds isolated from various plant species. These phenolic compounds are mainly
flavonoids, coumarins, anthraquinones, tannins, phenolic acids, and their derivatives.
Flavonoids constitute one of the largest groups of phenolics that are widely distrib-
uted in the plant kingdom. Antifungal compounds from the bark of the Peruvian plant
Swartzia polyphylla DC have been isolated as biochanin A and dihydrobiochanin A. These
two isoflavones have been found to be active against T. mentagrophytes and M. gypseum
(Rojas et al., 2006). Eight flavonoids have been isolated from the stem bark of Erythrina
burtii. The flavanones sigmoidin B 4′-methylether, the pterocarpans calopocarpin and
neorautenol, and the isoflavanone bidwillon A have been identified as the active com-
ponents against T. mentagrophytes and M. gypseum (Yenesew et al., 2005). Additionally,
biochanin A has been isolated from the bark of Andira surinamensis (Boudt.) Splitz and
has evidenced antifungal activity especially against yeasts and dermatophytes (Lock de
Ugaz et al., 1991).
Antidermatophytic activity of different extracts of Psoralea corylifolia seeds has been
examined on T. rubrum, T. mentagrophytes, E. floccosum, and M. gypseum by the disk dif-
fusion method. A 4′-metoxyflavone was isolated as the active compound of the active
methanol extract. The activity of compound has been tested by tube dilution method
showing MICs of 62.5 μg/mL for T. and T. rubrum and 125 μg/mL for other dermatophytes
(Rajendra Prasad et al., 2004).
Apigenin has been isolated as one of the antifungal principles of Terminalia chebula
stem extracts by Singh et al. (2014). After that, in vivo antifungal effect of apigenin
oilments (2.5 and 5 mg/g) was tested on mice, which were experimentally induced with
T. mentagrophytes. High doses of apigenin oilment led to complete recovery from the infec-
tion on the 12th day of the experiment. In this study, apigenin gave encouraging results in
the topical treatment of dermatophytosis in mice.
Antimicrobial and antifungal activities of 26 Eucalyptus species have been investigated,
and E. globulus, E. maculata, and E. viminalis were found to have strong antifungal activity
against T. mentagrophytes. After isolation and structure elucidation processes, three flavo-
noid compounds were identified as 2′,6′-dihydroxy-3′-methyl-4′-methoxy-dihydrochalcone,
eucalyptin, and 8-desmethyl-eucalyptin. All these flavonoids commonly exhibited signifi-
cant inhibitory activity against T. mentagrophytes with MIC ranging from 1 to 31 mg/L
(Takahashi et al., 2004).
According to ethnopharmacological use of Zuccagnia punctata, two chalcones
(2′,4′-dihydroxy-3′-methoxychalcone and 2′,4′-dihydroxychalcone) were isolated as the
active compounds after bioactivity-guided fractionation. Both chalcones have shown very
strong activities against several clinical strains of the dermatophytes T. mentagrophytes and
T. rubrum with MIC values in the range of 1.9–15.6 μg/mL and minimum fungucidal con-
centration (MFC) values between 1.9 and 7.8 μg/mL (Svetaz et al., 2007).
Asafoetida is a resinous mixture with a smell similar to garlic, which is obtained
by drying the exudates of different Ferula species. Asafoetida collected from vari-
ous Ferula species were tested for their antifungal activity, and F. foetida was found
to be the most active one. Nine prenylated coumarins were isolated from this sam-
ple and were tested against M. gypseum and Trichophyton interdigitale. Four of the
compounds exhibited strong antifungal activity against the dermatophytes with
5,8-dihydroxyumbelliprenin being most active with an MIC of 10 mM, the positive
control miconazole having an MIC of 0.5 mM (Houghton et al., 2006a).
Antifungal activity of dichloromethane extract from leaves of Piper fulvescens was
examined by using an agar overlay bioautographic method. Activity-guided fraction-
ation of the extract has led to the isolation of three antifungal neolignans named as cono-
carpan, eupomatenoid 5, and eupomatenoid 6. Conocarpan showed the widest activity,
whereas eupomatenoid 6 was the most active against dermatophytes M. gypseum and
T. mentagrophytes (Freixa et al., 2001). In another study, in vitro antidermatophytic activity
of extracts from leaves of another Piper species (P. regnellii) was investigated by microdi-
lution methods. The hydroalcoholic extract of leaves presented a strong activity against
T. mentagrophytes, T. rubrum, M. canis, and M. gypseum with MICs of 15.62, 15.62, 15.62, and
62.5 μg/mL, respectively. After bioactivity-guided fractionation studies, two neolignans
(eupomatenoid 3 and eupomatenoid 5) were isolated from the column fractions of chloro-
form subextract. The pure compounds showed strong activity on T. rubrum with MICs of
50 and 6.2 μg/mL, respectively (Koroishi et al., 2008).
Methanolic extract of Magnolia obovata stem bark has been found to have antifungal
activity against T. mentagrophytes, and further studies have revealed that two neolignan
compounds (magnolol, honokiol) were responsible for the antidermatophytic activity of
the stem bark extract. These two neolignans have shown significant inhibitory activi-
ties against M. gypseum, T. mentagrophytes, and E. floccosum with MICs between 25 and
100 μg/mL (Bang et al., 2000).
Lopes et al. (2012) have studied antifungal activities of purified phlorotannin extracts
from three brown seaweeds (Cystoseira nodicaulis, Cystoseira usneoides, and Fucus spiralis).
The purified phlorotannin extracts possessed both fungistatic and fungicidal activities
against yeast and dermatophytes. E. floccosum and T. rubrum were the most susceptible
species among dermatophytes.
Antimicrobial effect of phytoalexin resveratrol on dermatophytes and bacterial
pathogens of the skin was investigated by Chan (2002). Antifungal activity was tested on
T. mentagrophytes, T. tonsurans, T. rubrum, E. floccosum, and M. gypseum. The growth of der-
matophytes was inhibited at 25–50 μg/mL of resveratrol.
The methanol extract and subextracts of the aerial parts of Geophila repens were tested
against many dermatophytes. The butanol subextract was found to be the most active one
against Cryptococcus neoformans, M. gypseum, and T. mentagrophytes. After fractionation by
successive column chromatography, an ester of caffeic acid and maleic anhydride identi-
fied as maleic anhydride caffeate was determined as the active principle (Vila et al., 2013).
Zacchino et al. (1999) investigated in vitro antidermatophytic effect of 34 arylpro-
panoids and related compounds isolated from different plants against E. floccosum,
M. canis, M. gypseum, T. mentagrophytes, and T. rubrum by agar dilution method. Alpha-
halopropiophenones displayed a broad spectrum of activities against dermatophytes with
MICs between 0.5 and >50 µg/mL. Additionally, keto, alcohol, and alpha-haloketo propyl
derivatives of naphthalene and phenanthrene possessed high antidermatophytic activ-
ity. Also, phenanthryl derivatives were found to be more active (MICs: 3–20 µg/mL) than
naphthyl derivatives (MICs 3–50 µg/mL).
25.4.2.3 Saponins
Triterpene and steroidal saponins have also been isolated as antidermatophytic
constituents of some medicinal plants. These studies concern mainly species of the
Solanaceae family. A spirostanol saponin and three saponins have been isolated
from the leaves of Solanum hispidum. All the isolated compounds have revealed
antimycotic activity. The most active compound was a spirostanol derivative, with
IC50 values of 25 μg/mL against both T. mentagrophytes and T. rubrum (Gonzales
et al., 2004). Alvarez et al. (2001) have isolated a steroidal saponin named SC-1 from
the leaves of Solanum chrysotrichum. Its structure has been characterized as 3-O-{β-
quinovopyranosyl(1 → 6)-β-glucopyranosyl(1 → 6)-β-glucopyranosyl}chlorogenin, and
it has possessed straight fungitoxic activity against the dermatophyte T. mentagrophytes
(MIC = 40 μg/mL; nystatin MIC = 10 μg/mL). Additionally, five spirostan saponins and
two sterol glycosides have been isolated from the leaves of the same plant having anti-
dermatophytic activity. The most active compound is one of the spirostan derivatives
and had low MIC values (12.5 µg/mL) against T. mentagrophytes and T. rubrum (Zamilpa
et al., 2002).
Saponins isolated from roots and aerial parts of Medicago sativa, Medicago murex,
Medicago arabica, and Medicago hybrida were reported to be active against three dermato-
phytic fungi M. gypseum, T. interdigitale, and T. tonsurans (MIC < 0.09 mM). T. tonsurans was
the most sensitive dermatophyte. Activities of glycosides were higher than the activities
of aglycones and monodesmosidic glycosides of medicagenic acid were the most active
compounds (Houghton et al., 2006b).
25.4.2.4 Fatty acids

According to a recent study, fatty acids have antifungal activity mainly due to their capac-
ity to disrupt cell membranes. They are able to interfere with cell membrane structure,
displacing phospholipids and increasing its permeability. Undecylenic acid is a good
example of a semisynthetic antifungal compound mainly used in the treatment of super-
ficial mycoses. It is prepared from ricinoleic acid obtained from the seed oil of Ricinus
communis (Vila et al., 2013).
A number of fatty acids were isolated from the bark of Calycophyllum spruceanum
var. multiflorum by bioguided fractionation, and their structure has been elucidated as
6-hexadecinoic acid, 6-heptadecinoic acid, 6-octadecinoic acid, 6-nonadecinoic acid and
6-eicosinoic acid, palmitic acid, heptadecanoic acid, and stearic acid. The mixture of these
fatty acids was found to be active against the dermatophytes M. gypseum and T. mentagro-
phytes with MIC and MFC values of 0.25 μg/mL, lower than those of the reference drugs
nystatin and amphotericin B (Vila et al., 2013).
25.4.2.5 Other compounds

Two piperidine alkaloids and named as haloxylines A and B have been isolated from
Haloxylon salicornicum. Both alkaloids showed moderate to potent antifungal activities
against Trichophyton longifusus and M. canis (Ferheen et al., 2005).
In vitro antifungal activity of hexane, methanol, ethanol, and chloroform extracts
of some Indian plants has been studied by Ponnusamy et al. (2010). Active chloroform
extract of Wrightia tinctoria leaves has been fractionated, and the indole compound indiru-
bin was isolated as the active principle. It exhibited activity against dermatophytes such
as E. floccosum (MIC = 6.25 μg/mL), T. rubrum and T. tonsurans (MIC = 25 μg/mL), and
Trichophyton simii (MIC = 50 μg/mL).
After antifungal assay–directed fractionation of methanolic extract of Eleutherine
americana bulbs, a naphthoquinone derivative was isolated from the n-hexane-soluble
fraction. The compound was determined as eleutherin, and it was found to have antider-
matophytic activity against T. mentagrophytes in agar diffusion assay (Kusuma et al., 2010).
A study of the antimicrobial compounds from Moneses uniflora resulted in the isolation
of naphthoquinone derivatives named 8-chlorochimaphilin, together with chimaphilin
and 3-hydroxychimaphilin having antifungal activity against M. gypseum with MIC val-
ues 12.5 and 25 μg/mL (Saxena et al., 1996).
The leaves of Allamanda cathartica have been used to isolate an iridoid called plumi-
eride. It has been tested against dermatophytes E. floccosum and M. gypseum by a modi-
fied paper disk technique. Plumieride possessed significant fungitoxicity by inhibiting the
growth of both test dermatophytes completely (Tiwari et al., 2002).
Antidermatophytic activity of ether extract of Nigella sativa and its active principle thy-
moquinone have been tested against different dermatophyte species (E. floccosum, M. canis,
T. rubrum, T. mentagrophytes, and T. interdigitale) by agar diffusion method. The MICs of
ether extract of the plant and thymoquinone have been between 10 and 40 and 0.125 and
0.25 mg/mL, respectively. The MICs of thymoquinone have been less for E. floccosum and
M. canis than for the Trichophyton species (Aljabre et al., 2005).
Antidermatophytic activities of crude steroidal glycoside extract and two spirosta-
nol glucosides (yuccaloeside B and C) isolated from Yucca gloriosa were tested by using
agar dilution method on T. rubrum, T. mentagrophytes, T. soudanense, M. canis, M. gypseum,
and E. floccosum. The MICs of yuccaloeside B and C were found to be between 0.78 and
12.5 μg/mL (Favel et al., 2005).
25.5 Conclusion
Although the incidence of dermatophytic infections in humans is increasing day by day,
there are only a few therapeutic options for treatment because of side effects and toxicity of
medicines, drug resistance, etc. Beside synthetic drugs, traditional medicines and natural
products are widely used for fungal infections all over the world. Today, nearly 1000 plants
have been reported for their antifungal activities on different strains; however, only 100–200
of them have possessed antidermatophytic activity. Antidermatophytic activity screening
studies on plant extracts are almost common, but even so, only a few scientists have resumed
their studies to isolate and determine the active principles.
Additionally, the in vivo antidermatophytic activity studies of the in vitro active com-
pounds have to be tested on animal and humans to find their effectiveness, toxicity, and
dose–response relationship.
In this chapter, the studies on essential oils, terpenoids, phenolic compounds, sapo-
nins, other natural compounds, and plant extracts with antidermatophytic activity were
reviewed. Further studies are needed to enlighten structure–activity relationship of these
secondary metabolites and also to determine their active and safe doses on humans.
References
Abad, M.J., Ansuategui, M., and Bermejo, P. 2007. Active antifungal substances from natural sources.
Arkivoc 7:116–145.
Agrawal, A., Srivastava, S., Srivastava, J.N., and Srivasava, M.M. 2004. Evaluation of inhibitory effect
of the plant Phyllanthus amarus against dermatophytic fungi Microsporum gypseum. Biomed.
Environ. Sci. 17:359–365.
Ali-Shtayeh, M.S. and Abu Ghdeib, S.I. 1999. Antifungal activity of plant extracts against dermato-
phytes. Mycoses 42:665–672.
Aljabre, S.H.M., Randhawa, M.A., Akhtar, N., Alakloby, O.M., Alqurashi, A.M., and Aldossary, A.
2005. Antidermatophytic activity of ether extract of Nigella sativa and its active principle, thy-
moquinone. J. Ethnopharmacol. 101:116–119.
Alvarez, L., Perez, M.C., Villarreal, M.L., and Navarro, V. 2001. SC-1, An antimycotic spirostan sapo-
nin from Solanum chrysotrichum. Planta Med. 67:372–374.
American Orthopaedic Foot & Ankle Society. 2015. Athlete’s foot.

Amin, M. and Kapadnis, B.P. 2005. Heat stable antimicrobial activity of Allium ascalonicum against
bacteria and fungi. Indian J. Exp. Biol. 43:751–754.
Anane, S. and Chtourou, O. 2013. Tinea capitis favosa misdiagnosed as tinea amiantacea. Med.
Mycol. Case Rep. 2:29–31.
Anitha, R. and Kannan, P. 2006. Antifungal activity of Clerodendrum inerme (L.) and Clerodendrum
phlomidis (L.). Turk. J. Biol. 30:139–142.
Arif, T., Bhosale, J.D., Kumar, N., Mandal, T.K., Bendre, R.S., Lavekar, G.S., and Dabur, R. 2009.
Natural products-antifungal agents derived from plants. J. Asian Nat. Prod. Res. 11:621–637.
Asticcioli, S., Di Silverio, A., Sacco, L., Fusi, I., Vincenti, L., and Romero, E. 2008. Dermatophyte infec-
tions in patients attending a tertiary care hospital in northern Italy. New Microbiol. 31:543–548.
Avelar Pires, C.A., Monteiro Lobato, A., Oliveira Carneiro, F.R., Satos da Cruz, N.F., Oliveira de
Sousa, P., and Darwich Mendes, A.M. 2014. Clinical, epidemiological, and therapeutic profile
of dermatophytosis. Ann. Bras. Dermatol. 89:259–274.
Ayatollahi Mousavi, S.A., Salari Sardoui, S., and Shamsadini, S. 2009. A first case of tinea imbricate
from Iran. Jundishapur J. Microbiol. 2:71–74.
Balakumar, S., Rajan, S., Thirunalasundari, T., and Jeeva, S. 2011. Antifungal activity of Aegle marme-
los (L.) Correa (Rutaceae) leaf extract on dermatophytes. Asian Pac. J. Trop. Biomed. 1(4):309–312.
Bang, K.H., Kim, Y.K., Min, B.S., Na, M.K., Rhee, Y.H., Lee, J.P., and Bae, K.H. 2000. Antifungal activ-
ity of magnolol and hinokiol. Arch. Pharm. Res. 23:46–49.
Baran, W., Szepietowski, J.C., and Schwartz, R.A. 2004. Tinea barbae. Acta Dermatoven APA 13:91–94.
Batawila, K., Kokou, K., Koumaglo, K., Gbeassor, M., De Foucault, B., Bouchet, P., and Akpagana,
K. 2005. Antifungal activities of five Combretaceae used in Togolese traditional medicine.
Fitoterapia 76:264–268.
Beikert, F.C., Anastasiadou, Z., Fritzen, B., Frank, U., and Augustin, M. 2013. Topical treatment of
tinea pedis using 6% coriander oil in unguentum leniens: A randomized, controlled, compara-
tive pilot study. Dermatology 226(1):47–51.
Cavaleiro, C., Pinto, E., Gonçalves, M.J., and Salguerio, L. 2006. Antifungal activity of Juniperus essen-
tial oils against dermatophyte, Aspergillus and Candida strains. J. Appl. Microbiol. 100:1333–1338.
Chan, M.M.-Y. 2002. Antimicrobial effects of resveratrol on dermatophytes and bacterial pathogens
of the skin. Biochem. Pharmacol. 63:99–104.
Chauhan, V.S., Suthar, A., Naik, V., and Salkar, K. 2012. Screening of plants for antidermatophyte
activity. Int. J. Pharm. Sci. Res. 3:1502–1506.
Cos, P., Vlietinck, A., Vanden Berghe, D., and Maes, L. 2006. Anti-infective potential of natural prod-
ucts: How to develop a stronger in vitro “proof-of-concept”. J. Ethnopharmacol. 106:290–302.
Das, K., Tiwari, R.K.S., and Shrivastava, D.K. 2010. Techniques for evaluation of medicinal plant prod-
ucts as antimicrobial agent: Current methods and future trends. J. Med. Plants Res. 4:104–111.
De Campos, M.P., Cechinel Filho, V., Da Silva, R.Z., Yunes, R.A., Zacchino, S., Juarez, S., and Bella
Cruz, A. 2005. Evaluation of antifungal activity of Piper solmsianum C. DC. var. solmsianum
(Piperaceae). Biol. Pharm. Bull. 28:1527–1530.
Del Palacio, A., Garau, M., Gonzalez-Escalada, A., and Calvo, M.T. 2000. Trends in the treatment of
dermatophytosis. Rev. Iberoam. Micol. 16:148–158.
Devi, W.R., Singh, S.D.B., and Singh, C.B. 2013. Antioxidant and anti-dermatophytic properties leaf
and stem bark of Xylosma longifolium Clos. BMC Complement. Altern. Med. 13:155.
Dias, M.F.R.G., Quaresma-Santos, M.V.P., Schectman, R.C., Bernardes-Filho, F., Da Fonseca Amorim,
A.G., and Azulay, D.R. 2013. Treatment of superficial mycoses: Review-part II. Ann. Bras.
Dermatol. 88:937–944.
Dicle, Ö. and Özkesici, B. 2013. Tinea kapitis. Turk. J. Dermatol. 7:1–8.
Dorman, H.J.D. and Deans, S.G. 2000. Antimicrobial agents from plants: Antibacterial activity of
plant volatile oils. J. Appl. Microbiol. 88:308–316.
Duraipandiyan, V. and Ignacimuthu, S. 2007. Antibacterial and antifungal activity of Cassia fistula L.:
An ethnomedicinal plant. J. Ethnopharmacol. 112(3):590–594.
Engelmeier, D. and Hadacek, F. 2006. Antifungal natural products: Assays and applications. In:
Advances in Phytomedicine Series. Vol. III: Naturally Occurring Bioactive Compounds, eds. M. Rai
and M.C. Carpinella, pp. 23–43. Amsterdam, the Netherlands: Elsevier.
Falahati, M., Tabrizib, N.O., and Jahaniani, F. 2005. Antidermatophyte activities of Eucalyptus
camaldulensis in comparison with griseofulvin. Iran. J. Pharmacol. Ther. 4:80–83.
Favel, A., Kemertelidze, E., Benidze, M., Fallague, K., and Regli, P. 2005. Antifungal activity of steroi-
dal glycosides from Yucca gloriosa L. Phytother. Res. 19:158–161.
Fenner, R., Sortino, M., Rates, S.K., Dall’Agnol, R., Ferraz, A., Bernardi, A.P., and Zacchino, S. 2005.
Antifungal activity of some Brazilian Hypericum species. Phytomedicine 12:236–240.
Ferheen, S., Ahmed, E., Afza, N., Malik, A., Shah, M.R., Nawaz, S.A., and Choudhary, M.I. 2005.
Haloxylines a and b, antifungal and cholinesterase inhibiting piperidine alkaloids from
Haloxylon salicornicum. Chem. Pharm. Bull. 53:570–572.
Freixa, B., Vila, R., Ferro, E.A., Adzet, T., and Cañigueral, S. 2001. Antifungal principles from Piper
fulvescens. Planta Med. 67:873–875.
Freixa, B., Vila, R., Vargaz, L., Lozano, N., Adzet, T., and Cañigueral, S. 1998. Screening for antifungal
activity of nineteen Latin American plants. Phytother. Res. 12:427–430.
Garcıa, V.N., Gonzalez, A., Fuentes, M., Aviles, M., Rios, M.Y., Zepeda, G., and Rojas, M.G. 2003.
Antifungal activities of nine traditional Mexican medicinal plants. J. Ethnopharmacol. 87(1):85–88.
Global Action Fund for Fungal Infections. 2015. http://www.gaffi.org/why/fungal-disease-frequency.
Gonzales, M., Zamilpa, A., Marquina, S., Navarro, V., and Alvarez, L. 2004. Antimycotic spirostanol
saponins from Solanum hispidum leaves and their structure-activity relationships. J. Nat. Prod.
67:938.
Gullo, F.P., Sardi, J.C., Santos, V.A., Sangalli-Leite, F., Pitangui, N.S., Rossi, S.A., de Paula, E.S.A.C.,
Soares, L.A., Silva, J.F., Oliveira, H.C., Furlan, M., Silva, D.H., Bolzani, V.S., Mendes-Giannini,
M.J., and Fusco-Almeida, A.M. 2012. Antifungal activity of maytenin and pristimerin.
e-CAM 1–6.
Gupta, A.K. and Cooper, E.A. 2008. Update in antifungal therapy of dermatophytosis. Mycopathologia
166:353–367.
Hammer, K.A., Carson, C.F., and Riley, T.V. 2002. In vitro activity of Melaleuca alternifolia (tea tree) oil
against dermatophytes and other filamentous fungi. J. Antimicrob. Chemother. 50:195–199.
Hean, C.C., Rahim, I.N.A.B.A., Harn, G.L., Halimi, N.A.B.A., Hamzah, M.H.B., Aisyah, T.A.F., and
Said, N.F.B. 2011. Fungal disease and therapy. Webmed Central Pharm. Sci. 2(12):WMC002693.
Higgins, E.M., Fuller, L.C., and Smith, C.H. 2000. Guidelines for the management of tinea capitis.
Br. J. Dermatol. 143:53–58.
Ho, K. and Cheng, T. 2010. Common superficial fungal infections—A short review. The Hong Kong
Med. Diary 15:23–27.
Houghton, P., Patel, N., Jurzysta, M., Biely, Z., and Cheung, C. 2006b. Antidermatophyte activity of
Medicago extracts and contained saponins and their structure-activity relationships. Phytother.
Res. 20:1061–1066.
Houghton, P.J., Ismail, K.M., Maxia, L., and Appendino, G. 2006a. Antidermatophytic prenylated
coumarins from asafetida. Planta Med. 72:S-008.
Jacob, M.R. and Walker, L.A. 2005. Natural products and antifungal drug discovery. In: Methods in
Molecular Medicine, Vol. 118: Antifungal Agents: Methods and Protocols, eds. E.J. Ernst, and P.D.
Rogers. Totowa, NJ: Humana Press Inc.
Jacyk, W.K. 2004. Four common infectious skin condition: Tinea corporis, pityriasis versicolor, sca-
bies, larva migrans. S. Afr. Fam. Pract. 46:13–16.
Jamalian, A., Shams-Ghahfarokhi, M., Jaimand, K., Pashootan, N., Amani, A., and Razzaghi-
Abyaneh, M. 2012. Chemical composition and antifungal activity of Matricaria recutita flower
essential oil against medically important dermatophytes and soil-borne pathogens. J. Mycol.
Med. 22:308–315.
Jantan, İ., Yassin, M.S.M., Chin, C.B., Chen, L.L., and Sim, N.L. 2003. Antifungal activity of the essen-
tial oils of nine Zingiberaceae species. Pharm. Biol. 41:392–397.
Karakoca, Y., Endoğru, E., Turgut Erdemir, A., Kiremitçi, Ü., Gürel, M.S., and Güçin, Z. 2010.
Generalized inflammatory tinea corporis. J. Turk. Acad. Dermatol. 4:1–3.
Khaled, A., Mbarek, L.B., and Kharfi, M. 2007. Tinea capitis favosa due to Trichophyton schoenleinii.
Acta Dermatoven. APA 16:34–36.
Khattak, S., Saeed, P., Ullah, H., Ahmad, W., and Ahmad, M. 2005. Biological effects of indigenous
medicinal plants Curcuma longa and Alpinia galanga. Fitoterapia 76:254–257.
Khosravi, R.A., Shokri, H., Farahnejat, Z., Chalangari, R., and Katalin, M. 2013. Antimycotic efficacy
of Iranian medicinal plants towards dermatophytes obtained from patients with dermatophy-
tosis. Chin. J. Nat. Med. 11:43–48.
Kishore, N., Chansouria, J.P.N., and Dubey, N.K. 1996. Antidermatophytic action of the essential oil
of Chenopodium ambrosioides and an ointment prepared from it. Phytother. Res. 10:453–455.
Koroishi, A.M., Foss S.R., Cortez, D.A.G., Ueda-Nakamura, T., Nakamurad, C.V., and Filho, B.P.D.
2008. In vitro antifungal activity of extracts and neolignans from Piper regnellii against derma-
tophytes. J. Ethnopharmacol. 117:270–277.
Kosalec, I., Pepeljnjak, S., and Kustrak, D. 2005. Antifungal activity of fluid extract and essential oil
from anise fruits (Pimpinella anisum L., Apiaceae). Acta Pharm. 55:377–385.
Kuiate, J.R., Bessière, J.M., Vilarem, G., and Amvam Zollo, P.H. 2006. Chemical composition and
antidermatophytic properties of the essential oils from leaves, flowers and fruits of Cupressus
lusitanica mill from Cameroon. Flavour Frag. J. 21:693–697.
Kuiate, J.R., Mouokeu, S., Wabo, H.K., and Tane, P. 2007. Antidermatophytic triterpenoids from
Syzygium jambos (L.) Alston (Myrtaceae). Phytother. Res. 21:149–152.
Kusuma, I.W., Arung, E.T., Rosamah, E., Purwatiningsih, S., Kuspradini, H., Astuti, J., and Shimizu,
K. 2010. Antidermatophyte and antimelanogenesis compound from Eleutherine americana
grown in Indonesia. J. Nat. Med. 64:223–226.
Lang, G. and Buchbauer, G. 2012. A review on recent research results (2008–2010) on essential oils as
antimicrobials and antifungals. A review. Flavour Frag. J. 27:13–39.
Lock de Ugaz, O., Costa, J., Sanchez, L., Ubillas Sanchez, R.P., and Tempesta, M.S. 1991. Flavonoids
from Andira inermis. Fitoterapia 62:89–90.
Loizzo, M. R., Statti, G. A., Tundis, R., Conforti, F., Bonesi, M., Autelitano, G., and Menichini, F. 2004.
Antibacterial and antifungal activity of Senecio inaequidens DC and Senecio vulgaris L. Phytother.
Res. 18:777–779.
Lopes, G., Sousa, C., and Silva, L.R. 2012. Can phlorotannins purified extracts constitute a novel
pharmacological alternative for microbial infections with associated inflammatory condi-
tions? PLoS One 7(2):e31145.
Mabona, U., Viljoen, A., Shikanga, E., Marston, A., and Vuuren, S.V. 2013. Antimicrobial activity of
southern African medicinal plants with dermatological relevance: From an ethnopharmaco-
logical screening approach, to combination studies and the isolation of a bioactive compound.
Machado de Souza, A.C., Kato, L., Conceição da Silva, C., Cidade, A.F., Alves de Oliveira, C.M., and
Silva, M.R.R. 2009. Antimicrobial activity of Hymenaea martiana towards dermatophytes and
Cryptococcus neoformans. Mycoses 53:500–503.
Mann, A., Banso, A., and Clifford, L.C. 2008. An antifungal property of crude plant extracts from
Anogeissus leiocarpus and Terminalia avicennioides. Tanzan. J. Health Res. 10:34–38.
Mares, D., Romagnoli, C., Tosi, B., Andreotti, E., Chillemi, G., and Poli, F. 2005. Chicory extracts from
Cichorium intybus L. as potential antifungals. Mycopathologia 160:85–92.
Masoko, P., Picard, J., and Eloff, J.N. 2005. Antifungal activities of six South African Terminalia spe-
cies (Combretaceae). J. Ethnopharmacol. 99:301–308.
Moodahadu-Bangera, L.S., Martis, J., Mittal, R., Krishnankutty, B., Kumar, N., Bellary, S., and Rao,
P.K. 2012. Eberconazole-pharmacological and clinical review. Indian J. Dermatol. 78:217–222.
Moriarty, B., Hay, R., and Morris-Jones, R. 2012. The diagnosis and management of tinea. Br. Med. J.
345:1–10.
Mugnaini, L., Nardoni, S., Pistelli, L., Leonardi, M., Giuliotti, L., Benvenuti, M.N., and Mancianti, F.
2013. A herbal antifungal formulation of Thymus serpillum, Origanum vulgare and Rosmarinus
officinalis for treating ovine dermatopytosis due to Tricophyton mentagrophytes. Mycoses
56:333–337.
Muschietti, L., Derita, M., Sülsen, V., de Dios Muñoz, J., Ferraro, G., Zacchino, S., and Martino, V.
2005. In vitro antifungal assay of traditional Argentine medicinal plants. J. Ethnopharmacol.
102:233–238.
Natarajan, V., Venugopal, P.V., and Menon, T. 2003. Effect of Azadirachta indica (Neem) on the growth
pattern of dermatophytes. Indian J. Med. Microbiol. 21:98–101.
Noumedem, J.A.K., Tamoko, J.D., Teke G.N., Momo, R.C.D., Kuete, V., and Kuiate J.R. 2013.
Phytochemical analysis, antimicrobial and radical-scavenging properties of Acalypha manni-
ana leaves. Springer Plus 2:503.
Odom, R. 1993. Pathophysiology of dermatophyte infections. J. Am. Acad. Dermatol. 28:2–7.
Phongpaichit, S., Subhadhirasakul, S., and Wattanapirosakul, C. 2005. Antifungal activities of
extracts from Thai medicinal plants against opportunistic fungal pathogens associated with
AIDS patients. Mycoses 48:333–338.
Pinto, E., Pina-Vaz, C., Salgueiro, L., Gonçalves, M.J., Costa-de-Oliveira, S., Cavaleiro, C., and
Martinez-de-Oliveira, J. 2006. Antifungal activity of the essential oil of Thymus pulegioides on
Candida, Aspergillus and dermatophyte species. J. Med. Microbiol. 55:1367–1373.
Policegoudra, R.S., Goswami, S., Aradhya, S.M., Chatterjee, S., Datta, S., Sivaswamy, R., and Singh, L.
2012. Bioactive constituents of Homalomena aromatica essential oil and its antifungal activity
against dermatophytes and yeasts. J. Mycol. Med. 22:83–87.
Ponnusamy, K., Petchiammal, C., Mohankumar, R., and Hopper, W. 2010. In vitro antifungal activ-
ity of indirubin isolated from a South Indian ethnomedicinal plant Wrightia tinctoria R. Br. J.
Ethnopharmacol. 132:349–354.
Portillo, A., Vila, R., Freixa, B., Adzet, T., and Cañigueral, S. 2001. Antifungal activity of Paraguayan
plants used in traditional medicine. J. Ethnopharmacol. 6:93–98.
Portillo, A., Vila, R., Freixa, B., Ferro, E., Parella, T., Casanova, J., and Cañigueral, S. 2005. Antifungal
sesquiterpene from the root of Vernonanthura tweedieana. J. Ethnopharmacol. 97:49–52.
Premkumar, V.G. and Shyamsundar, D. 2005. Antidermatophytic activity of Pistia stratiotes. Ind. J.
Pharmacol. 37:126–128.
Rahman, A., Al-Reza, S.M., Siddiqui, S.A., Chang, T., and Kang, S.C. 2014. Antifungal potential of
essential oil and ethanol extracts of Lonicera japonica Thunb against dermatophytes. EXCLI J.
13:427–436.
Rai, M.K., Varma, A., and Pandev, A.K. 2004. Antifungal potential of Spilanthes calva after inocula-
tion of Piriformospora indica. Mycoses 47:479–481.
Rajendra Prasad, N., Anandi, C., Balasubramanian, S., and Pugalendi, K.V. 2004. Antidermatophytic
activity of extracts from Psoralea corylifolia (Fabaceae) correlated with the presence of a flavo-
noid compound. J. Ethnopharmacol. 91:21–24.
Rath, S. and Mohanty, R.C. 2013. Antifungal screening of Curcuma longa and Cassia tora on dermato-
phytes. Int. J. Life Sci. Pharma Res. 2(4):88–94.
Rebollo, N., López-Barcenas, A.P., and Arenas, R. 2008. Tinea capitis. Actas Dermosifiliogr. 99:91–100.
Ríos, J.L., Recio, M.C., and Villar, A. 1998. Screening methods for natural products with antimicro-
bial activity: A review of the literature. J. Ethnopharmacol. 23:127–149.
Rojas, R., Bustamante, B., Ventosilla, P., Fernádez, I., Caviedes, L., Gilman, R.H., and Hammond, G.B.
2006. Larvicidal, antimycobacterial and antifungal compounds from the bark of the peruvian
plant Swartzia polyphylla DC. Chem. Pharm. Bull. 54:278–279.
Rojas, R., Doroteo, V., Bustamante, B, Baver, J., and Lock, O. 2004. Antimicrobial and free radical
scavenging activity of Gentianella nitida. Fitoterapia 75:754–757.
Sabota, J., Brodell, R., Rutecki, G.W., and Hoppes, W.L. 1996. Severe tinea barbae due to Trichophyton
verrucosum infection in dairy farmers. Clin. Infect. Dis. 23:1308–1310.
Sanguinetti, M., Posteraro, B., Romano, L., Battaglia, F., Lopizzo, T., De Carolis, E., and Fadda, G.
2007. In vitro activity of Citrus bergamia (bergamot) oil against clinical isolates of dermato-
phytes. J. Antimicrob. Chemother. 59:305–308.
Satter, E.K. 2009. Tinea imbricata. Cutis 83:188–191.
Saxena, G. Farmer, S.W., Hancock, R.E.W., and Towers, G.H.N. 1996. Chlorochimaphilin: A new anti-
biotic from Moneses uniflora. J. Nat. Prod. 59:62–65.
Schmourlo, G., Mendonça-Filho, R.R., Alviano, C.S., and Costa, S.C. 2005. Screening of antifun-
gal agents using ethanol precipitation and bioautography of medicinal and food plants.
Shams-Ghahfarakhi, M., Goodarzi, M., Abvaneh, M.R., Al Tiraihi, T., and Sevedipovr, G. 2004.
Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and
Trichophyton mentagrophytes. Fitoterapia 75:645–655.
Shams-Ghahfarokhi, M., Shokoohamiri, M.R., Amirrajab, N., Moghadasi, B., Ghajari, A., Zeini, F.,
and Razzaghi-Abyaneh, M. 2006. In vitro antifungal activities of Allium cepa, Allium sativum
and ketoconazole against some pathogenic yeasts and dermatophytes. Fitoterapia 77:321–332.
Silva, M.R.R., Oliveira, J.G., Fernandes, O.F.L., Passos, X.S., Costa, C.R., Souza, L.K. H., and Paula, J.R.
2005. Antifungal activity of Ocimum gratissimum towards dermatophytes. Mycoses 48:172–175.
Singh, G., Kumar, P., and Joshi, C. 2014. Treatment of dermatophytosis by a new antifungal agent
‘apigenin’. Mycoses 57:497–506.
Singh, S. and Vidyasagar, G.M. 2014. In vitro antidermatophytic and preliminary phytochemical
studies of petroleum ether and inter-polar methanolic leaf extracts of Thevetia nerrifolia. J. Appl.
Pharma. Sci. 4(3):81–85.
Sipponen, P., Sipponen, A., Lohi, J., Soini, M., Tapanainen, R., and Jokinen, J.J. 2012. Natural conif-
erous resin lacquer in treatment of toenail onychomycosis: An observational study. Mycoses
56:289–296.
Stein, A.C., Sortino, M., Avancini, C., Zacchino, S., and Von Poser, G. 2005. Ethnoveterinary medi-
cine in the search for antimicrobial agents: Antifungal activity of some species of Pterocaulon
(Asteraceae). J. Ethnopharmacol. 99:211–214.
Svetaz, L., Agüero, M.B., Alvarez, S., Luna, L., Feresin, G., Derita, M., and Zacchino, S. 2007.
Antifungal activity of Zuccagnia punctata Cav: Evidence for the mechanism of action. Planta
Med. 73:1074–1080.
Tadeg, H., Mohammed, E., Asres, K., and Gebre-Mariam T. 2005. Antimicrobial activities of some
selected traditional Ethiopian medicinal plants used in the treatment of skin disorders.
Takahashi, T., Kokubo, R., and Sakaino, M. 2004. Antimicrobial activities of eucalyptus leaf extracts
and flavonoids from Eucalyptus maculata. Lett. Appl. Microbiol. 39:60–64.
Tiwari, T.N., Pandey, V.B., and Dubey, N.K. 2002. Plumieride from Allamanda cathartica as an antide-
rmatophytic agent. Phytother. Res. 16:393–394.
Trakranrungsie, N., Chatchawanchonteera, A., and Khunkitti, W. 2008. Ethnoveterinary study for
antidermatophytic activity of Piper betle, Alpinia galanga and Allium ascalonicum extracts in vitro.
Res. Vet. Sci. 84:80–84.
Vila, R., Freixa, B., and Cañigueral, S. 2013. Antifungal compounds from plants. In: Recent Advances
in Pharmaceutical Sciences III, eds. D. Muñoz-Torrero, A. Cortés, and E.L. Mariño, pp. 23–43.
Kerala, India: Transworld Research Network.
Weitzman, I. and Summerbell, R.C. 1995. The dermatophytes. Clin. Microbiol. Rev. 8:240–259.
Yenesew, A., Derese, S., Midiwo, J.O., Bii, C.C., Heydenreich, M., and Peter, M.G. 2005. Antimicrobial
flavonoids from the stem bark of Erythrina burttii. Fitoterapia 76:469–472.
Zacchino, S.A., López, S.N., Pezzenati, G.D., Furlán, R.L., Santecchia, C.B., Muñoz, L., and Enriz, R.D.
1999. In vitro evaluation of antifungal properties of phenylpropanoids and related compounds
acting against dermatophytes. J. Nat. Prod. 62:1353–1357.
Zamilpa, A., Tortoriello, J., Navarro, V., Delgado, G., and Alvarez, L. 2002. Five new steroidal sapo-
nins from Solanum chrysotrichum leaves and their antimycotic activity. J. Nat. Prod. 65:1815–1819.

Dhanasekaran, Dharumadurai PanneErselvam, A. Thajuddin, Nooruddin Antimicrobials Synthetic and Natural Compounds

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ANTIMICROBIALS

Synthetic and Natural Compounds

Chapter 1 Antibiotics: From discovery to journey................................................................1

Section I: Broad spectrum antimicrobial compounds from microorganisms

Chapter 2 Antimicrobial potential of marine actinobacteria: A review........................ 17

Chapter 3 Antimicrobial compounds from microorganisms: Production,

Chapter 4 Animal fecal actinomycetes: A new source for the discovery

Chapter 5 Potentially novel Actinobacteria-derived antibiotics from unique

Chapter 6 Antimicrobial agents from actinomycetes: Chemistry and applications....99

Chapter 7 Actinobacteria: A predominant source of antimicrobial compounds....... 117

Chapter 8 Novel antimicrobial and anticancer drugs from bacteria............................ 143

Chapter 9 Bacteriocin: A natural alternative to synthetic antibacterial antibiotics...... 155

Chapter 10 Protease inhibitors from marine organisms................................................... 175

Chapter 11 Ganoderma: A bioresource of antimicrobials.................................................189

Chapter 12 Marine cyanobacteria: A prolific source of

Chapter 13 Antimicrobial and natural compounds from edible mushrooms..............233

Section II: Broad spectrum antimicrobial compounds from animals

Chapter 15 Secondary metabolites from microorganisms isolated from

Section III: Broad spectrum antimicrobial compounds from

Chapter 16 Antimicrobial compounds and their chemical entities on therapeutic

Chapter 17 Role of antimicrobial compounds from Trichoderma spp. in plant

Chapter 18 Antimicrobial compounds from rhizosphere bacteria and their role

Section IV: Synthetic chemical compounds as broad spectrum antimicrobials

Chapter 19 Microbe-mediated synthesis of silver nanoparticles: A new drug of

Chapter 20 Nanomaterials: Source of antimicrobial products.........................................403

Chapter 21 Platinum-based anticancer therapeutics and their mechanistic

Section V: Narrow-spectrum antimicrobials

Chapter 22 Marine actinobacteria as potential drug storehouses: A future

Chapter 24 Bioactive compounds from actinomycetes and their antiviral

Chapter 25 Novel antidermatophytic drug candidates from nature...............................487

Prof. Nooruddin Thajuddin, PhD, is the dean, Faculty of

workshops, refresher courses, and DST-INSPIRE (Innovation in Science Pursuit for

Prof. Annamalai Panneerselvam, PhD, is an associate

Natarajan Amaresan Diana R. Cundell

Xueshi Huang Wen-Jun Li

Yi Jiang Mohammad F. Mehbub

K. Kannabiran W.M. Muiru

Ranjith N. Kumavath M. Nithya

Didem Deliorman Orhan Soumik Sarkar

V. Rajesh Kannan Nallanchakravarthula Srivathsa

Subhasish Saha Nooruddin Thajuddin

Govindaraj Vaijayanthi A. Vishnu Kirthi

Ramasamy Vijayakumar Wei Zhang

1.2 Classification of antibiotics

1. Mechanism of action: An antibiotic may be bactericidal or bacteriostatic. The bacteri-

(gentamicin) or synthesized in vitro (netilmicin, amikacin, arbekacin, and isepamicin)

Figure 1.1 Structure of streptomycin.

Structurally, aminoglycosides contain two or more amino sugars linked by glycosidic

1.2.1.1 Aminoglycoside mode of action

Figure 1.3 Structure of penicillin where “R” is the variable group.

Figure 1.5 Structure of (a) ampicillin and (b) amoxicillin.

1.2.2.1 Penicillin mode of action

Figure 1.6 Core structure of cephalosporins.

cephalosporins, besides the well-known first-, second-, and third-generation cephalospo-

1.2.3.1 Cephalosporin mode of action

Figure 1.10 Structure of the carbapenem backbone.

1.2.4.1 Carbapenem mode of action

Figure 1.11 Structure of vancomycin.

1.2.5.1 Glycopeptide mode of action

methicillin-resistant Staphylococcus aureus infections (Daum, 2007). Pirlimycin is active

1.2.6.1 Lincosamide mode of action

1.2.7.1 Macrolide mode of action

Broad spectrum antimicrobial

Chapter 15 Secondary metabolites from microorganisms isolated from

Section III: Broad spectrum antimicrobial compounds from

Chapter 16 Antimicrobial compounds and their chemical entities on therapeutic

3.6 Production and detection of antimicrobial

3.9 Identification and characterization

3.11 Challenges in the utilization of antimicrobial