International Journal of Systematic and Evolutionary Microbiology (2005), 55, 437–442 DOI 10.1099/ijs.0.63071-0 Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia Marı́a E. Pavan,1 Raúl J. Franco,1 Juan M. Rodriguez,1 Patricia Gadaleta,2 Sharon L. Abbott,3 J. Michael Janda3 and Jorge Zorzópulos1,2 Correspondence Jorge Zorzopulos zorzopul@hotmail.com 1 Instituto de Investigaciones Biomédicas Fundación Pablo Cassará, Saladillo 2452, Buenos Aires (1440), Argentina 2 Departamento de Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina 3 Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Health Services, Richmond, CA, USA In order to assess the relationship between the genus Kluyvera and other members of the family Enterobacteriaceae, the 16S rRNA genes of type strains of the recognized Kluyvera species, Kluyvera georgiana, Kluyvera cochleae, Kluyvera ascorbata and Kluyvera cryocrescens, were sequenced. A comparative phylogenetic analysis based on these 16S rRNA gene sequences and those available for strains belonging to several genera of the family Enterobacteriaceae showed that members of the genus Kluyvera form a cluster that contains all the known Kluyvera species. However, the type strain of Enterobacter intermedius (ATCC 33110T) was included within this cluster in a very close relationship with the type strain of K. cochleae (ATCC 51609T). In addition to the phylogenetic evidence, biochemical and DNA–DNA hybridization analyses of species within this cluster indicated that the type strain of E. intermedius is in fact a member of the genus Kluyvera and, within it, of the species Kluyvera cochleae. Therefore, following the current rules for bacterial nomenclature and classification, the transfer of E. intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. is proposed (type strain, ATCC 33110T=CIP 79.27T=LMG 2785T=CCUG 14183T). Biochemical analysis of four E. intermedius strains and one K. cochleae strain independent of the respective type strains further indicated that E. intermedius and K. cochleae represent the same species and are therefore heterotypic synonyms. Nomenclatural priority goes to the oldest legitimate epithet. Consequently, Kluyvera cochleae Müller et al. 1996 is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005. INTRODUCTION Despite their great medical and economical importance, the bacteria included in the family Enterobacteriaceae are still The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of K. cochleae ATCC 51609T, K. georgiana ATCC 51603T, K. cryocrescens ATCC 33435T, K. ascorbata ATCC 33433T and Enterobacter intermedius ATCC 33110T are AF047187, AF047186, AF310218, AF310219 and AF310217, respectively. A phylogenetic tree showing that members of the genus Enterobacter are intertwined with members of other genera is available as supplementary material in IJSEM Online. 63071 G 2005 IUMS poorly understood phylogenetically. Furthermore, there is evidence suggesting the need for extensive revision of the taxonomic relationships among genera and species within this family. For example, based on 16S rRNA trees (Drancourt et al., 2001), it has been reported that the genus Klebsiella is heterogeneous and composed of species which form three clusters that include members of other genera. Kluyvera is a genus of small rod-shaped bacteria, thus conforming to the general definition of the family Enterobacteriaceae (Holt et al., 1994). Bacteria of this genus are mainly grouped in four known species, Kluyvera ascorbata, Downloaded from www.microbiologyresearch.org by IP: 34.227.94.161 On: Fri, 13 Apr 2018 15:27:57 Printed in Great Britain 437 M. E. Pavan and others Kluyvera cryocrescens, Kluyvera cochleae and Kluyvera georgiana (Farmer et al., 1981; Müller et al., 1996). The present study was undertaken to gain insight into the phylogenetic relationships among species within the genus Kluyvera and between the genus Kluyvera and related genera within the family Enterobacteriaceae, using 16S rRNA genebased trees, DNA–DNA hybridization analysis and phenotypic characterization. METHODS Bacterial strains. Culture collection strains used in this study were K. cochleae ATCC 51609T (=CDC 9514-94T=DSM 9406T) and ATCC 51717 (CDC 9532-94=DSM 9407), K. georgiana ATCC 51603T (=CDC 2891A-76T=CDC 2891-76T=DSM 9409T), K. ascorbata ATCC 33433T (=CDC 0648-74T), K. cryocrescens ATCC 33435T (=CDC 2065-78T) and Enterobacter intermedius ATCC 33110T (=CUETM 77-130T=CIP 79.27T=Gavini E 86T). Strains 77/123, 77/136 and 77/139 were received from D. Old (Ninewells Hospital and Medical School, Dundee, UK) and strain CDC 9011-82 was received from the Centers for Disease Control and Prevention (Atlanta, GA, USA) as Enterobacter intermedius. Culture was performed on Luria–Bertani medium at 35 uC under aerobic conditions. Biochemical studies. Enterobacter intermedius and Kluyvera strains were characterized phenotypically using a battery of 43 biochemical tests in conventional media. These tests included: triple-sugar iron agar reactions; pigmentation (25 uC); motility; production of cytochrome oxidase, nitrate reductase and indole; growth in KCN broth; urea hydrolysis (Christensen’s); utilization of malonate, citrate (Simmon’s), acetate and mucate; production of b-galactosidase (ONPG) and phenylpyruvic acid (phenylalanine deaminase); lysine decarboxylase, ornithine decarboxylase and arginine dihydrolase (Møeller’s) activities; elaboration of acetylmethylcarbinol (Voges– Proskauer); degradation of gelatin, corn oil (lipase), DNA and polypectate (25 uC); and aesculin hydrolysis (broth). Carbohydrate fermentation reactions were performed in extract broth against 1 % solutions of the following carbohydrate or carbohydrate-like compounds: adonitol, amygdalin, L-arabinose, D-arabitol, cellobiose, dulcitol, methyl a-D-glucopyranoside, D-glucose, glycerol, myoinositol, lactose, maltose, D-mannitol, melibiose, raffinose, Lrhamnose, D-sorbitol, salicin, sucrose, trehalose and D-xylose. All of these tests have been described previously (Abbott et al., 1992, 2003; Janda et al., 1996). Unless otherwise specified, tests were incubated at 35 uC for 2–4 days (7 days for carbohydrate fermentation and extracellular enzymes) and final results were recorded. Biochemical reactions presented in Tables 1 and 2 are at 48 h incubation. Bacterial DNA extraction and analysis. Bacterial DNA was extracted by standard procedures and analysed using electrophoresis in agarose gels of serial dilutions of K. cochleae ATCC 51609T and Enterobacter intermedius ATCC 33110T DNA of equal concentration and Southern blot capillary transfer to nylon membranes under alkaline conditions (Sambrook et al., 1989). DNA–DNA hybridization. The genetic relatedness among members of the genus Kluyvera was determined by DNA–DNA hybridization on nylon membranes (Johnson, 1991). Serial dilutions of cell suspensions of equal OD570 values in denaturation solution (0?5 M NaOH/1?0 M NaCl) were applied to nylon membranes (Pall Biodyne). The membranes were then neutralized with 1?5 M NaCl/ 0?5 M Tris/HCl (pH 8) and fixed by UV radiation for 15 min. Pre-hybridization (2 h) was performed at 65 uC in a buffer containing 0?15 M NaCl, 1 % SDS and 0?3 % non-fat dried milk, and 438 Table 1. Key reactions for Enterobacter intermedius, K. cochleae, K. georgiana, K. ascorbata and K. cryocrescens 1, E. intermedius ATCC 3310T; 2, K. cochleae ATCC 51609T; 3, K. georgiana ATCC 51603T; 4, K. ascorbata ATCC 33433T; 5, K. cryocrescens ATCC 33435T. Only variable results are shown in the table. The following tests gave identical results for the five type strains: triple-sugar iron (24 h) (acid/acid+gas); motility (+); urea hydrolysis (2); ONPG (24 h) (+); utilization of citrate (+) and acetate (+); degradation of mucate (+), DNA (2), corn oil (2) and gelatin (2); polypectate (+); Møeller’s reaction, arginine (2) and ornithine (+); phenylpyruvic acid test (2); fermentation of L-arabinose (+), D-glucose (+), L-rhamnose (+), D-xylose (+), cellobiose (+), lactose (+), maltose (+), sucrose (+), trehalose (+), raffinose (+), adonitol (2), myoinositol (2), D-mannitol (+), salicin (+), melibiose (+), amygdalin (+) and D-arabitol (2); aesculin hydrolysis (+); pigmentation (2); growth in KCN broth (+). Reactions are reported at 48 h. Test 1 2 3 4 5 Formation of indole Utilization of malonate Voges–Proskauer Lysine (Møeller’s reaction) Fermentation of: Dulcitol Erythritol Glycerol* D-Sorbitol Ascorbate Growth on CIND 2 + + 2 2 + + 2 + 2 2 + + + 2 + + 2 2 2 + 2 +G + + + + 2 +G + + + + 2 +w 2 + + 2 2 +w 2 + + 2 2 +G 2 2 2 *G, Gas production; W, weakly acidic. DCIN, Cefsulodin/irgasan/novobiocin. hybridization (18 h) was performed at 65 uC in a buffer containing 0?03 M NaCl, 1 % SDS and 0?3 % non-fat dried milk. The hybridization probe was chromosomal DNA from the indicated strain digested with AluI endonuclease and 32P-labelled with the Random Primer DNA Labelling System (Gibco, Life Technologies). The results were scored first by autoradiography and then by cutting the spots and measuring the radiation in a scintillation counter (Beckman). Hybridization levels were calculated as described by Johnson (1991). Sequencing of the 16S rRNA gene and phylogenetic analysis. DNA from bacterial strains was purified (Sambrook et al., 1989), and the 16S rRNA genes were amplified by PCR using the bacterial primers 27f (59-AGAGTTTGATCMTGGCTCAG-39), corresponding to positions 8–27 of forward Escherichia coli numbering, and 1492r (59-GGTTACCTTGTTACGACTT-39), corresponding to positions 1510–1492 of reverse Escherichia coli numbering. The following temperature programme was used: 94 uC for 5 min, 30 cycles of 94 uC for 60 s, 60 uC for 60s and 72 uC for 60 s, followed by a final 7 min incubation at 72 uC. The PCR product was purified using GFX-PCR DNA and a gel band purification kit (Amersham Biosciences) and sequenced completely by using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer) and an ABI Prism 377 DNA Sequencer (Perkin-Elmer). The Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 55 IP: 34.227.94.161 On: Fri, 13 Apr 2018 15:27:57 Phylogenetic relationships of the genus Kluyvera Table 2. Phenotypic comparison between K. cochleae and Enterobacter intermedius strains 1, K. cochleae ATCC 51716; 2, K. cochleae ATCC 51717; 3, Enterobacter intermedius ATCC 33110T; 4, E. intermedius CDC 9011-82; 5, E. intermedius 77-123; 6, E. intermedius 77-136; 7, E. intermedius 77-139; 8, E. intermedius 77-140. Only variable results are shown in the table. The following tests gave identical results for all the strains: triple-sugar iron (acid/acid+gas); motility (+); pigmentation (2); hydrolysis of urea (2) and aesculin (+); utilization of citrate (+) and malonate (+); growth in KCN broth (+); formation of indole (2), acetylmethylcarbinol (+) and phenylpyruvic acid (2); Møeller’s reactions, lysine (2), arginine (2) and ornithine (+); degradation of mucate (+), DNA (2), corn oil (2) and gelatin (2); nitrate reductase (+); fermentation of L-arabinose (+), D-glucose* (+), L-rhamnose (+), D-xylose (+), cellobiose* (+), lactose* (+), maltose (+), trehalose (+), raffinose (+), adonitol (2), glycerol* (+), myo-inositol (2), D-mannitol (+), D-sorbitol (+), salicin (+), melibiose (+), D-arabitol (2) and methyl a-D-glucopyranoside (+). *, Gas determined. Biochemical reactions are reported at 48 h; (+), delayed positive reactions (>48 h). Test 1 ONPG + Utilization of: Acetate + Degradation of: Polypectate + Fermentation of: Sucrose + Dulcitol + Amygdalin (+) 2 3 4 5 6 7 8 2 + + + + + + + + + (+) + + + + + + (+) + (+) (+) + + + (+) (+) (+) + + (+) (+) + + + + + (+) (+) (+) (+) (+) (+) obtained 16S rRNA gene sequences were aligned with those of type strains of bacterial genera related to the genus Kluyvera available in the EMBL and Ribosomal Database Project libraries (Maidak et al., 1997) by using the CLUSTAL W program (Thompson et al., 1994) with default parameters and optimized using a multiple sequence alignment editor (Galtier et al., 1996). Phylogenetic trees were constructed by both the neighbour-joining distance method (Kimura two-parameter model and jumble option) and the parsimony character method, using programs contained in the PHYLIP package (Felsenstein, 1989). The stability of the relationships was assessed by bootstrapping (1000 replicates), with programs included in the same package. The sequence of Aeromonas hydrophila ATCC 7966T (GenBank/EMBL/DDBJ accession no. X74677) was used as an outgroup to establish the root of the tree. RESULTS AND DISCUSSION In order to understand the phylogenetic relationships of members of the genus Kluyvera with other members of the family Enterobacteriaceae, the 16S rRNA genes of the type strains of K. cochleae (ATCC 51609T), K. georgiana (ATCC 51603T, K. cryocrescens (ATCC 33435 T) and K. ascorbata (ATCC 33433 T) were sequenced. Also, for reasons to be discussed below, the 16S rRNA gene of the type strain of Enterobacter intermedius (ATCC 33110T) was sequenced. A http://ijs.sgmjournals.org phylogenetic tree, constructed using the neighbour-joining distance method, of members of the genus Kluyvera and related genera is shown in Fig. 1. As can be observed, the four members of the genus Kluyvera clustered together in the tree. However, the type strain of Enterobacter intermedius was included in the Kluyvera cluster very close to the type strain of K. cochleae (bootstrap value 100 %). A tree constructed using the parsimony character method was in complete agreement with this (not shown). It should be also pointed out that, according to the base composition of its 16S rRNA gene, Enterobacter intermedius is distantly related to Enterobacter cloacae (similarity value 97?4 %), the type species of the genus Enterobacter, in comparison with its relationship to K. cochleae (similarity value 99?9 %). This strongly suggested that, if there was a misclassification of the Enterobacter intermedius type strain, many (if not all) strains of the same species may also be misclassified. A phenotypic analysis (Table 1) showed that Enterobacter intermedius ATCC 33110T is remarkably similar to the type strain of K. cochleae and that both strains are Voges–Proskauer-positive, differing from the type strains of the other Kluyvera species. Furthermore, Table 2 shows that this remarkable similarity is not exclusive to the type strains but can be extended to other available independently isolated strains of these two species. The DNA relatedness among the type strains of species in the genus Kluyvera and the type strain of Enterobacter intermedius was also studied (Table 3). As can be observed, this DNA relatedness was higher than the current minimal standard (70 % relatedness) accepted for the phylogenetic definition of a species (Wayne et al., 1987; Stackebrandt & Goebel, 1994) when labelled DNA from Enterobacter intermedius was used as a probe. However, the DNA relatedness was about 59 % when labelled DNA from K. cochleae was used as a probe. This difference may be due to the presence of extrachromosomal DNA in one or both of the reference strains. In agreement with this, Fig. 2 shows that cells of the type strain of Enterobacter intermedius have several high-copy-number plasmids, while the cells of the type strain of K. cochleae apparently do not have extrachromosomal elements. Using chromosomal DNA of the type strain of Enterobacter intermedius extracted from gels, the DNA relatedness with the DNA of the type strain of K. cochleae increased to more than 70 % (not shown). Thus, DNA–DNA hybridization assays indicate that the type strains of these bacterial species are included within the same species. Taken together, these results indicate that Enterobacter intermedius phenotypically and genotypically is a member of the genus Kluyvera. According to Rules 27(2) and 27(3) of the Bacteriological Code, we formally propose to transfer Enterobacter intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. The species name intermedia is proposed because Latin feminine adjectives must agree in gender with the feminine generic name Kluyvera (Farmer et al., 1981). DNA–DNA hybridization assays indicate that Downloaded from www.microbiologyresearch.org by IP: 34.227.94.161 On: Fri, 13 Apr 2018 15:27:57 439 M. E. Pavan and others Fig. 1. Phylogenetic tree derived from the analysis of the 16S rRNA gene sequences of members of the family Enterobacteriaceae according to the neighbour-joining distance method. The Kimura two-parameter model was used to correct the distances for multiple substitutions at a site. Bootstrap values are from 1000 replications and only those greater than 50 % are shown. Bar, substitutions per nucleotide position. Table 3. DNA relatedness (%) among type strains of species of the genus Kluyvera and the type strain of Enterobacter intermedius 1, K. ascorbata ATCC 33433T; 2, K. cochleae ATCC 51609T; 3, K. cryocrescens ATCC 33435T; 4, K. georgiana ATCC 51603T; 5, Enterobacter intermedius ATCC 33110T; 6, Escherichia coli ATCC 11775T (unrelated control). Results are expressed as the mean of three experiments; standard error <3 %. Labelled DNA probe K. ascorbata K. cochleae K. cryocrescens K. georgiana Enterobacter intermedius Escherichia coli 440 1 2 3 4 5 6 100 20 19 17 21 13 16 100 23 18 89 13 15 26 100 25 30 14 17 16 22 100 13 12 19 59 29 14 100 14 11 12 15 13 16 100 K. cochleae and K. intermedia are heterotypic synonyms. Rule 42 of the Bacteriological Code requires the oldest legitimate epithet be retained. Thus, the oldest legitimate epithet for these taxa is that of Izard et al. (1980). Therefore, K. cochleae Müller et al. 1996 is a later synonym of K. intermedia (Izard et al. 1980) Pavan et al. 2005. It is also worth noting that the 16S rRNA gene-based tree presented here strongly suggests that members of the genera Kluyvera and Buttiauxella, as defined by phenotypic assays, are monophyletic, in agreement with the conclusion of a previous report (Spröer et al., 1999). In contrast, members of the genus Enterobacter are intertwined with members of other genera (see Fig. A, available as supplementary material in IJSEM Online), a fact also suggested by trees constructed using groE genes (Harada & Ishikawa, 1997). This indicates the need for an extensive revision of the phenotypic criteria to classify bacteria within the genus Enterobacter. Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 55 IP: 34.227.94.161 On: Fri, 13 Apr 2018 15:27:57 Phylogenetic relationships of the genus Kluyvera Basonym: Enterobacter intermedius Izard et al. 1980. Fig. 2. Southern blot analysis of total DNA extracted from cells of K. cochleae ATCC 51609T and Enterobacter intermedius ATCC 33110T. (A) Results of the Southern blot analysis when the probe was labelled DNA from Enterobacter intermedius. (B) Results of the Southern blot analysis when the probe was labelled DNA from K. cochleae. Lanes 1, 2, 3, 4 and 5 correspond to 2?5, 1?25, 0?62, 0?31 and 0?15 mg of total DNA, respectively. The description is that given by Izard et al. (1980). Some characteristics are as follows. Cells are straight rods, 0?5– 0?762–3 mm, Gram-negative, motile by scant peritrichous flagella. Facultatively anaerobic and chemo-organotrophic, having both a respiratory and a fermentative type of metabolism. Colonies are circular, convex, greyish and smooth on nutrient agar, with growth at 30 and 37 uC. Catalase-positive. Oxidase-negative. Nitrate reductasepositive. Indole-negative. Voges–Proskauer-positive. Acid produced from amygdalin, L-arabinose, cellobiose, dulcitol, D-glucose, glycerol, lactose, maltose, D-mannitol, melibiose, raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, trehalose and D-xylose. Negative for urea hydrolysis. ONPG-positive. Grows in KCN broth. Utilizes citrate and acetate. Does not utilize adonitol, arabitol, erythritol or myo-inositol. Does not degrade gelatin, DNA (DNase-negative) or corn oil (lipase-negative), but degrades mucate. Arginine dihydrolase-negative. Does not form phenylpyruvic acid. Does not produce pigment at 25 uC. Polypeptate-positive. Isolated from molluscs, surface water, soil and a variety of human samples including stool, blood, wounds, bile and a gall bladder. The type strain is ATCC 33110T (=CIP 79.27T=LMG 2785T=CCUG 14183T). ACKNOWLEDGEMENTS We thank G. Pavan for her technical assistance. This work was partially supported by a grant from the CONICET to J. Z. There is a good correlation between the results of DNA– DNA hybridization studies presented previously (Farmer et al., 1981) and the results of the phylogenetic analysis presented herein. In both cases, Klebsiella, Enterobacter and Citrobacter species are close relatives of Kluyvera species. Salmonella, Escherichia, Shigella and Erwinia species are intermediate and Proteus and Yersinia species are the most distant relatives among the species analysed in both studies. In the Farmer et al. (1981) analysis, the large heterogeneity of the Enterobacter species observed in our study was also evident. In conclusion, the results presented herein indicate that there is good agreement between the grouping of species of the genus Kluyvera by 16S rRNA gene-based phylogenetic analysis and phenotypic clustering, and that strains of Enterobacter intermedius should be reclassified as proposed since they are by phylogenetic, phenotypic and DNA–DNA relatedness criteria members of the genus Kluyvera. 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