Ciência Rural, SantaAmerican Maria, clinical v.49:01, e20180744, Genotyping of South isolates of Pythium2019 insidiosum based on single nucleotide polymorphism-based multiplex PCR. 1 http://dx.doi.org/10.1590/0103-8478cr20180744 ISSNe 1678-4596 MICROBIOLOGY Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR Maria Isabel de Azevedo4 Lara Baccarin Ianiski2 Paula Cristina Stibbe1 Carla Weiblen1 Daniela Isabel Brayer Pereira5 Régis Adriel Zanette6 Luís Antônio Sangioni1 Rodolfo Rivero7 Janio Morais Santurio3 Sônia de Avila Botton1,2* Programa de Pós-graduação em Medicina Veterinária (PPGMV), Departamento de Medicina Veterinária Preventiva (DMVP), Universidade Federal de Santa Maria (UFSM), Centro de Ciências Rurais (CCR), Santa Maria, RS, Brasil. 2 Programa de Pós-graduação em Ciências Farmacêuticas (PPGCF), Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil. 3 Departamento de Microbiologia e Parasitologia (Demip), Universidade Federal de Santa Maria (UFSM), Centro de Ciências da Saúde (CCS), Santa Maria, RS, Brasil. 4 Faculdade de Medicina Veterinária, Universidade Federal de Minas Gerais (UFMG), Minas Gerais, RS, Brasil. 5 Departamento de Microbiologia e Parasitologia, Instituto de Biologia (IB), Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brasil. 6 Programa de Pós-graduação em Ciências Biológicas: Farmacologia e Terapêutica, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil. 7 Laboratorio Regional Noroeste DILAVE “Miguel C. Rubino”, ministério de Ganadería Agricultura e Pesca Casilla de Correo, 97105-900, Santa Maria, RS, Brasil. E-mail: sabott20@gmail.com. *Corresponding author. 1 ABSTRACT: We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates. Key words: Pythium insidiosum, Pythiosis, molecular detection, genotype, single nucleotide polimorphisms. Genotipagem de isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único baseado em PCR multiplex RESUMO: O objetivo deste estudo foi genotipar isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único (SNP) de sequências de rDNA. Anteriormente, um multiplex-PCR baseado em SNP foi capaz de distinguir P. insidiosum em três diferentes clados. Dessa forma, utilizamos este método para avaliar isolados clínicos de P. insidiosum da América do Sul (n=32), cepas padrão da Costa Rica (n=4), Tailândia (n=3), Japão (n=1) e Índia (n=1), uma cepa padrão de Pythium aphanidermatum e isolados ambientais brasileiros de Pythium torulosum; Pythium rhizo-oryzae e Pythium pachycaule voucher (n=3). Os isolados analisados foram alocados aos clados: I (americanos), II (isolados da Índia, Japão e Tailândia), e III (um isolado tailandês). P. aphanidermatum, P.torulosum, P.rhizo-oryzae e P.pachycaule voucher não foram amplificados. Pela primeira vez, um isolado de P. insidiosum do Uruguai foi incluído em análises moleculares. Através da multiplex-PCR baseada em SNP, foi possível realizar a identificação e genotipagem dos isolados sulamericanos de P. insidiosum, demonstrando características genéticas semelhantes entre esses isolados. Palavras-chave: Pythium insidiosum, Pitiose, detecção molecular, genótipo, polimorfismos de nucleotídeo único. INTRODUCTION Pythium genus is an ecofriendly oomycete found in a varied ecosystem. Most species are saprobic or pathogens of plants, algae, fishes, insects, and mammals (ADHIKARI et al., Received 09.11.18 2013). Pythium insidiosum causes pythiosis, a relevant infectious disease in human and animals that is widely distributed throughout the world (GAASTRA et al., 2010). In Brazil, this oomycete is present predominantly in the swampy areas of Pantanal Mato-Grossense and Rio Grande do Sul Approved 11.09.18 Returned by the author 12.09.18 CR-2018-0744.R1 Ciência Rural, v.49, n.1, 2019. 2 Weiblen et al. State (RS) (SANTOS et al., 2014; WEIBLEN et al., 2016). Little is known about the presence of pythiosis in some countries of South America, such as Uruguay, where the first case in an equine was recently reported in Costas del Tacuarí, Departamento de Treinta y Tres (LABORATORIO REGIONAL ESTE DE DILAVE, 2012). Due to the difficulty of diagnosing pythiosis and the high costs for laboratory identification of P. insidiosum, as well as the similarity to other agents, especially other oomycetes and filamentous fungi, there is a need for diagnostic tools that can identify rapidly this relevant microorganism. VILELA et al. (2015) proposed a biochemical assay for identification of oomycetes; however, this technique should still be used carefully for the evaluation of P. insidiosum isolates (KRAJAEJUN et al., 2018). Molecular biology tools have been successfully employed for diagnosing pythiosis, mainly using polymerase chain reaction (PCR) targeting the P. insidiosum internal transcribed spacer (ITS) of the rRNA locus, i.e., the ribosomal DNA (rDNA region) that consists of 18S rRNA, internal transcribed spacer 1 (ITS1), 5.8S rRNA, internal transcribed spacer 2 (ITS2), and 28S rRNA (GROOTERS & GEE, 2002). Phylogenetic studies of P. insidiosum have already been used with different genetic markers to elucidate aspects related to epidemiology, pathogenesis, and hosts (SCHURKO et al., 2003a,b; KAMMARNJESADAKUL et al., 2011; AZEVEDO et al., 2012; RIBEIRO et al., 2017). The first phylogenetic analyses grouped P. insidiosum in three clusters: cluster I North, Central, and South America; cluster II Australia, North America, Southeast Asia, and Thailand; and cluster III North America and isolates from Thailand (SCHURKO et al., 2003a,b). However, these analyses have limitations owing to the high costs, time required for DNA sequencing, and the delay in obtaining results. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers have been used for studies of diversity and relationship in different microorganisms, including P. insidiosum (SUPABANDHU et al., 2007; RUJIRAWAT et al., 2017). The purpose of our study was genotyping American clinical isolates of P. insidiosum using a fast, simple, and low-cost tool based on SNP multiplex PCR. Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1) were analyzed (Table1). Additionally, one standard strain of Pythium aphanidermatum and three environmental species of Pythium (P.torulosum, P.rhizooryzae and P.pachycaule voucher) were included in this research. All isolates were cultivated and submitted to total DNA extraction and amplification of rDNA region were according to AZEVEDO et al. (2012) using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-CTTCCGTCAATTCCTTTAAG-3′) (WHITE et al., 1990). The primers used for SNP multiplex PCR amplification were ITS1, R1 (5′-CCTCACATTCTGCCATCTCG-3’), R2 (5′-ATACCGCCAATAGAGGTCAT-3′), and R3 (5′-TTACCCGAAGGCGTCAAAGA-3′) (RUJIRAWAT et al., 2017). Amplifications were performed according to RUJIRAWAT et al. (2017) with modifications. Briefly, in a final volume of 25μL, the PCR reaction contained 1μM of the forward primer ITS1, 0.5μM each of the reverse primers (R1, R2, and R3), 1.5 units of Taq DNA polymerase (Invitrogen), 200μM of each deoxynucleotide, 1.5mM MgCl2, 1x enzyme buffer, and 100ng of DNA sample. The amplifications were carried out in a programmable thermal cycler (PTC-100, MJ Research), with initial denaturation at 95°C for 5min, 20 cycles of denaturation at 95°C for 30s, annealing at 53°C for 30s, and extension at 72°C for 45s, and then a final extension at 72°C for 10min. A 5µL aliquot of the PCR product was submitted to electrophoresis on 1% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. The PCR products of rDNA region presenting a single band with the desired length (approximately 500-800pb) were purified with PureLink PCR Purification Kit (Invitrogen), and the DNA was sequenced in an automatic sequencer (ABIPrism 3500 Genetic Analyzer) using the primers ITS1 and ITS4 (WHITE, 1990). Phylogenetic analysis for rDNA region was conducted by Neighbor-joining (NJ) method with 10,000 bootstrap replicates. All rDNA sequences from P. insidiosum clinical isolates from South America and standard strains of Costa Rica, India, Japan and Thailand, as well as P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher and Phytopythium vexans (outgroup) were used to construct the phylogenetic tree. MATERIALS AND METHODS Based on the multiplex PCR targeting the three SNPs identified in the rDNA region, all the thirty-six South and Central American isolates of P. insidiosum and the five standard strains from Thirty-one clinical isolates of P. insidiosum from Brazil, one from Uruguay, standard strains from RESULTS AND DISCUSSION Ciência Rural, v.49, n.1, 2019. Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR. 3 Thailand, India and Japan were grouped in their respective clades, as suggested by RUJIRAWAT et al. (2017) (Table 1, Figure 1). We observed that the American clinical isolates, grouped in clade I, generated amplicons of approximately 490 and 660bp when using the primers ITS1/R1 and ITS1/R2, respectively. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher were not amplified since these isolates do not belong to any P. insidiosum clade. The multiplex PCR targeting the three SNPs identified in the rDNA region was developed by RUJIRAWAT et al. (2017) and has many advantages, such as 100% of sensitivity, and specificity, rapid and cost-effective identification, and genotyping of P. insidiosum. As these authors evaluated only one Brazilian isolate of P. insidiosum in their study, we proposed to evaluate an expressive number of P. insidiosum clinical isolates from South America using this technique. The molecular phylogeny obtained for the rDNA region showed P. insidiosum as paraphyletic in relation to other Pythium species. However, it was observed that South and Central American P. insidiosum isolates were grouped together, forming a monophyletic group. In addition, isolates from other countries formed a basal-positioning group in relation to the American isolates (Figure 2). These results were consistent with AZEVEDO et al. (2012) and RIBEIRO et al (2017) that used rDNA (ITS) and cytochrome c oxidase subunit II as molecular markers and exo-1,3-β glucanase gene in phylogenetic analyses of Brazilian P. insidiosum isolates, respectively. Moreover, all isolates of P. insidiosum from India, Japan and Thailand were grouped in different clades as proposed by SCHURKO et al. (2003 a,b) and lately supported by SUPABANDHU et al. (2008). According to RUJIRAWAT et al. (2017) multiplex PCR targeting the three SNPs identified in the rDNA (ITS) region were able to allocate P. insidiosum to clade-I provided two amplicons (approximately 490 and 660bp), whereas the cladesII and –III showed only one amplicon (approximately 660 and 800bp, respectively). The same results were obtained in this study, allowing to assign each American P. insidiosum isolates to clade I, isolates from India, Japan, and Thailand to clade II, and one Thai isolate to clade III. In addition, P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher were not amplified. Thus, these results evidenced that this molecular biology methodology is specific and sensitive for identification and genotyping of P. insidiosum, in agreement with RUJIRAWAT et al. (2017). The genome sequences of P. insidiosum recently available can be a useful genetic resource for exploring aspects related the biology and evolution Figure 1 - Agarose gel electrophoresis of PCR amplification of the rDNA sequences of Pythium insidiosum from strains of each clade and controls. The amplicon size generated by SNP were: primers ITS1/R1 (~490bp) + primers ITS1/R2 (~660bp)=Clade I - 1: P. insidiosum 0-44, 2: P. insidiosum 138, 3: P. insidiosum 219, 4: P. insidiosum 247, 5: P. insidiosum 260, 6: P. insidiosum 152, 7: P. insidiosum 152, primers ITS1/R2 (~660bp)=Clade II - 8: P. insidiosum 1H, 9: P. insidiosum 2H, 10: P. insidiosum 6H, 11: P. insidiosum 7H, primers ITS1/R3 (~800bp) = Clade III - 12: P. insidiosum 8H, 13: P. aphanydermatum, 14: P. tolurosum, 15: Negative control: ddH2o (PCR grade), 16: 100-bp DNA ladder marker. Ciência Rural, v.49, n.1, 2019. Weiblen et al. 4 Table 1 - Isolates of Pythium. insidiosum (n=36) and other species of Pythium (n=4) used for evaluation of the multiplex PCR assay and their information of GenBank acession number of rDNA sequence, isolate source, geographic origin and phylogenetic clade. Isolate GenBank# Isolate source Geographic origin Amplicon (bp)## Clade 0-44 118 121 123 126 135 137 138 142 143 152 156 178 187 210 219 223 232 247 252 254 259 260 268 271 290 291 293 295 296 337 339 ATCC 58637 CBS 57485 3H - CBS 57685 4H- CBS 57585 1H-CBS 119453 2H-CBS 119455 6H- CBS 70283 7H- CBS 77784 8H- CBS 119454 P.aphanidermatum CBS 128995 P.torulosum P.rhizo-oryzae P.pachycaule voucher MF767408 JN126280 JN126282 JN126283 JN126286 MH813295 MH813296 JN126289 MH813297 JN126290 MH813298 JN126293 JN126295 JN126296 JN126298 JN126299 JN126300 JN126302 JN126304 MH813299 MH813300 JN126306 JN126307 JX675977 MH813301 KJ176713 MH813302 MH813303 MH813304 MH813305 MH813306 MH813307 JN126310 AY598637 AB898106 AB971178 EF016853 EF016855 AY151170 AY151169 AB971185 JF412451 MH813308 MH813309 MH813310 Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Equine Canine Equine Equine Equine Equine Canine Equine Equine Equine Equine Equine Equine Equine Human Human Equine Mosquito larva Human Human Environmental Environmental Environmental Uruguay Jaguari* Santa Maria* Cachoeira do Sul* Corumbá* Corumbá* Corumbá* Corumbá* Corumbá* Corumbá* Santa Maria* Santa Maria* Corumbá* Jari* Uruguaiana* São Lourenço do Sul* Cachoeira do Sul* Uruguaiana* Restinga Seca* Uruguaiana* Pelotas* Santa Vitória do Palmar* Santa Vitória do Palmar* Canguçu* Silveira Martins* Santa Maria* Pelotas* Rio Grande* Pelotas* Pelotas* Jaguari* Uruguaiana Costa Rica Costa Rica Costa Rica Costa Rica Thailand Thailand Japan India Thailand Afghanistan Capão do Leão* Santa Vitória do Palmar* Santa Vitória do Palmar* ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~490 and 660pb ~660 ~660 ~660 ~660 ~800 NA NA NA NA I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II II II III - # GenBank acession number corresponding to rDNA (ITS) sequences. ##Amplicon size generated by SNP (base pair) using: Primers ITS1/R1 (~490 bp) + Primers ITS1/R2 (~660bp)=Clade I; Primers ITS1/R2 (~660bp)=Clade II; Primers ITS1/R3 (~800bp)=Clade III; and no amplification)=No P. insidiosum genotype. =This isolate does not belong to P. insidiosum clade. *Municipality of Brazil. Ciência Rural, v.49, n.1, 2019. Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR. 5 Figure 2 - Neighbor-joining tree based on sequence analysis of the rDNA ITS showing relationships among clinical isolates of P. insidiosum from South America (Brazil (BR) and Uruguay (Uy) and standard strains of Costa Rica (CR), India (IN), Japan (JP) and Thailand (TH), as well as P. aphanidermatum and environmental Pythium spp. (P. pachycaule voucher, P. rhizo oryzae and P. torulosum isolates). Bootstrap values expressed in percentages based on 10,000 replicates are present at their corresponding clades. P.insidiosum and other oomycetes since independently assessed genes may not provide much information when compared to genomes. However, genome analyses are still recent, expensive and laborious when compared to the available molecular analyses (RUJIRAWAT et al., 2015; TANGPHATSORNRUANGA et al., 2016). For the first time, a P. insidiosum isolate from Uruguay were included in phylogenetic analysis. A single case of equine pythiosis has been reported by the Laboratorio Regional Este de DILAVE (2012). It is of note there is still little knowledge about pythiosis in Uruguay. However, we are aware of other cases of equine pythiosis in that country (unpublished data). Additionally, MACHADO et al. (2018) suggested that P. insidiosum is a generalist pathogen that has the potential to move between the borders of southern Brazil, e.g., RS (the southernmost state in Brazil) Ciência Rural, v.49, n.1, 2019. Weiblen et al. 6 and Uruguay. P. insidiosum isolate from Uruguay was grouped in clade I, together with isolates from Brazil. This was evidenced by both multiplex PCR and phylogenetic analysis, proving that the South American isolates are grouped in the same clade, as previously suggested (SCHURKO et al., 2003a,b; KAMMARNJESADAKUL et al., 2011; AZEVEDO et al., 2012; RIBEIRO et al., 2017). CONCLUSION The SNP-based multiplex-PCR methodology has benefits (i.e., fast, simple, and lowcost) and was possible to carry out the identification and genotyping of the South American isolates of P. insidiosum. For the first time a P. insidiosum isolate from equine in Uruguay was identified and genotyped. Furthermore, the American P. insidiosum isolates evaluated showed similar genetic characteristics. ACKNOWLEDGMENTS The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: 442020/2014-7), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (PqG/FAPERGS: 27293.414.15435.20062017) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (finance code 001) for scientific, financial support and student’s scholarships. DECLARATION INTERESTS OF CONFLICTING The authors declare no conflict of interest. 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