Genome sequence of the clinical O4:K12 serotype Vibrio parahaemolyticus strain 10329.
ABSTRACT: Vibrio parahaemolyticus is the leading cause of food-borne illnesses worldwide. Here, we report a draft genome of V. parahaemolyticus strain 10329 of the O4:K12 serotype. It belongs to the main U.S. West Coast clonal complex of V. parahaemolyticus (sequence type 36 [ST36]) causing oyster-associated human illness. It contains the virulence determinants tdh and trh but appears to infect at much lower doses than V. parahaemolyticus strains with these same determinants from other areas, such as the U.S. Gulf and Atlantic coasts.
Project description:<i>Vibrio parahaemolyticus</i> is an important cause of foodborne gastroenteritis globally. Thermostable direct haemolysin (TDH) and the TDH-related haemolysin are the two key virulence factors in <i>V. parahaemolyticus. Vibrio</i> pathogenicity islands harbour the genes encoding these two haemolysins. The serotyping of <i>V. parahaemolyticus</i> is based on the combination of O and K antigens. Frequent recombination has been observed in <i>V. parahaemolyticus</i>, including in the genomic regions encoding the O and K antigens. <i>V. parahaemolyticus</i> serotype O4:K12 has caused gastroenteritis outbreaks in the USA and Spain. Recently, outbreaks caused by this serotype of <i>V. parahaemolyticus</i> have been reported in China. However, the relationships among this serotype of <i>V. parahaemolyticus</i> strains isolated in different regions have not been addressed. Here, we investigated the genome variation of the <i>V. parahaemolyticus</i> serotype O4:K12 using the whole-genome sequences of 29 isolates. We determined five distinct lineages in this strain collection. We observed frequent recombination among different lineages. In contrast, little recombination was observed within each individual lineage. We showed that the lineage of this serotype of <i>V. parahaemolyticus</i> isolated in America was different from those isolated in Asia and identified genes that exclusively existed in the strains isolated in America. Pan-genome analysis showed that strain-specific and cluster-specific genes were mostly located in the genomic islands. Pan-genome analysis also showed that the vast majority of the accessory genes in the O4:K12 serotype of <i>V. parahaemolyticus</i> were acquired from within the genus <i>Vibrio</i>. Hence, we have shown that multiple distinct lineages exist in <i>V. parahaemolyticus</i> serotype O4:K12 and have provided more evidence about the gene segregation found in <i>V. parahaemolyticus</i> isolated in different continents.
Project description:Vibrio parahaemolyticus is the leading bacterial cause of food-borne illness due to the consumption of contaminated seafood. The aim of the present study was to determine the population of its subtypes and establish a better understanding of the various types of V. parahaemolyticus strains that are causing human illness in Canada. The subtypes for 100 human clinical isolates of V. parahaemolyticus collected between 2000 and 2009 were determined by performing serotyping, ribotyping, pulsed-field gel electrophoresis, and multilocus sequence typing. Within this panel of strains, there was a high level of diversity (between 22 and 53 subtypes per method), but the presence of predominant clones with congruent subtypes between the various methods was also observed. For example, all 32 isolates belonging to sequence type 36 (ST36) were from serogroup O4, while 31 of them were ribotype EcoVib235-287, and 24 of the 32 were SfiI pulsed-field gel electrophoresis (PFGE) pattern VPSF1.0001. With regard to the presence of known virulence genes, 74 of the 100 isolates were PCR positive for the presence of the thermostable direct hemolysin (tdh); and 59 of these 74 strains also contained the second virulence marker, the tdh-related hemolysin (trh). The detection of trh was more predominant (81%) among the clinical isolates, and only four (4%) of the clinical isolates tested negative for the presence of both tdh and trh. This database, comprising 100 clinical isolates of V. parahaemolyticus strains from Canada, forms a baseline understanding of subtype diversity for future source attribution and other epidemiologic studies.
Project description:In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2? or T3SS2? genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2? genes, and all isolates negative for tdh and positive for trh possessed all T3SS2? genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2?, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2? genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.
Project description:<h4>Background</h4>Vibrio parahaemolyticus is a common cause of foodborne disease. Beginning in 1996, a more virulent strain having serotype O3:K6 caused major outbreaks in India and other parts of the world, resulting in the emergence of a pandemic. Other serovariants of this strain emerged during its dissemination and together with the original O3:K6 were termed strains of the pandemic clone. Two genomes, one of this virulent strain and one pre-pandemic strain have been sequenced. We sequenced four additional genomes of V. parahaemolyticus in this study that were isolated from different geographical regions and time points. Comparative genomic analyses of six strains of V. parahaemolyticus isolated from Asia and Peru were performed in order to advance knowledge concerning the evolution of V. parahaemolyticus; specifically, the genetic changes contributing to serotype conversion and virulence. Two pre-pandemic strains and three pandemic strains, isolated from different geographical regions, were serotype O3:K6 and either toxin profiles (tdh+, trh-) or (tdh-, trh+). The sixth pandemic strain sequenced in this study was serotype O4:K68.<h4>Results</h4>Genomic analyses revealed that the trh+ and tdh+ strains had different types of pathogenicity islands and mobile elements as well as major structural differences between the tdh pathogenicity islands of the pre-pandemic and pandemic strains. In addition, the results of single nucleotide polymorphism (SNP) analysis showed that 94% of the SNPs between O3:K6 and O4:K68 pandemic isolates were within a 141 kb region surrounding the O- and K-antigen-encoding gene clusters. The "core" genes of V. parahaemolyticus were also compared to those of V. cholerae and V. vulnificus, in order to delineate differences between these three pathogenic species. Approximately one-half (49-59%) of each species' core genes were conserved in all three species, and 14-24% of the core genes were species-specific and in different functional categories.<h4>Conclusions</h4>Our data support the idea that the pandemic strains are closely related and that recent South American outbreaks of foodborne disease caused by V. parahaemolyticus are closely linked to outbreaks in India. Serotype conversion from O3:K6 to O4:K68 was likely due to a recombination event involving a region much larger than the O-antigen- and K-antigen-encoding gene clusters. Major differences between pathogenicity islands and mobile elements are also likely driving the evolution of V. parahaemolyticus. In addition, our analyses categorized genes that may be useful in differentiating pathogenic Vibrios at the species level.
Project description:BACKGROUND:V. parahaemolyticus is autochthonous to the marine environment and causes seafood-borne gastroenteritis in humans. Generally, V. parahaemolyticus recovered from the environment and/or seafood is thought to be non-pathogenic and the relationship between environmental isolates and acute diarrhoeal disease is poorly understood. In this study, we explored the virulence potential of environmental V. parahaemolyticus isolated from water, plankton and assorted seafood samples collected from the Indian coast. RESULTS:Twenty-two V. parahaemolyticus isolates from seafood harboured virulence associated genes encoding the thermostable-direct haemolysin (TDH), TDH-related haemolysin (TRH), and Type 3 secretion systems (T3SS) and 95.5% of the toxigenic isolates had pandemic strain attributes (toxRS/new+). Nine serovars, with pandemic strain traits were newly identified and an O4:K36 tdh-trh+V. parahaemolyticus bearing pandemic marker gene was recognised for the first time. Results obtained by reverse transcription PCR showed trh, T3SS1 and T3SS2? to be functional in the seafood isolates. Moreover, the environmental strains were cytotoxic and could invade Caco-2 cells upon infection as well as induce changes to the tight junction protein, ZO-1 and the actin cytoskeleton. CONCLUSION:Our study provides evidence that environmental isolates of V. parahaemolyticus are potentially invasive and capable of eliciting pathogenic characteristics typical of clinical strains and present a potential health risk. We also demonstrate that virulence of this pathogen is highly complex and hence draws attention for the need to investigate more reliable virulence markers in order to distinguish the environmental and clinical isolates, which will be crucial for the pathogenomics and control of this pathogen.
Project description:Vibrio parahaemolyticus sequence type 36 (ST36) strains that are native to the Pacific Ocean have recently caused multistate outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole-genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern U.S. sources, were used to identify diagnostic loci, one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present in only one clade of closely related strains of ST36, ST59, and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two nonclade isolates and cps in four. Based on the distribution of these loci in sequenced genomes, prp identified clade strains with >99% accuracy, but the addition of one more locus increased accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus and determines the presence of both the tdh and trh hemolysin-encoding genes, which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a 4-year period in three northeastern U.S. states and 87 environmental isolates revealed that the prp and cps amplicons were detected only in clinical isolates identified as belonging to the ST36 clade and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections.
Project description:<h4>Background</h4>Outbreak of V. parahaemolyticus infections occurred since 1996 was linked to a proposed clonal complex, the pandemic group. The whole genome sequence provides an unprecedented opportunity for dissecting genome plasticity and phylogeny of the populations of V. parahaemolyticus. In the present work, a whole-genome cDNA microarray was constructed to compare the genomic contents of a collection of 174 strains of V. parahaemolyticus.<h4>Results</h4>Genes that present variably in the genome accounted for about 22% of the whole gene pool on the genome. The phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the 174 strains. Strains were assigned into five complexes (C1 to C5), and those in each complex were related genetically and phylogenetically. C3 and C4 represented highly virulent clinical clones. C2 and C3 constituted two different clonal complexes 'old-O3:K6 clone' and 'pandemic clone', respectively. C3 included all the 39 pandemic strains tested (trh-, tdh+ and GS-PCR+), while C2 contained 12 pre-1996 'old' O3:K6 strains (trh+, tdh- and GS-PCR-) tested herein. The pandemic clone (post-1996 'new' O3:K6 and its derivates O4:K68, O1:K25, O1:KUT and O6:K18) might be emerged from the old-O3:K6 clone, which was promoted by acquisition of toxRS/new sequence and genomic islands. A phylogenetic intermediate O3:K6 clade (trh-, tdh- and GS-PCR+) was identified between the pandemic and old-O3:K6 clones.<h4>Conclusion</h4>A comprehensive overview of genomic contents in a large collection of global isolates from the microarray-based comparative genomic hybridization data enabled us to construct a phylogenetic structure of V. parahaemolyticus and an evolutionary history of the pandemic group (clone) of this pathogen.
Project description:Vibrio parahaemolyticus is a bacterial pathogen that can cause illness after the consumption or handling of contaminated seafood. The primary virulence factors associated with V. parahaemolyticus illness are thermostable direct hemolysin (TDH) and Tdh-related hemolysin (TRH). However, clinical strains lacking tdh and trh have recently been isolated, and these clinical isolates are poorly understood. To help understand the emergence of clinical tdh- and trh-negative isolates, a genomic approach was used to comprehensively compare 4 clinical tdh- and trh-negative isolates with 16 environmental tdh- and trh-negative isolates and 34 clinical isolates positive for tdh or trh, or both, with the objective of identifying genomic features that are unique to clinical tdh- and trh-negative isolates. The prevalence of pathogenicity islands (PAIs) common to clinical isolates was thoroughly examined in each of the clinical tdh- and trh-negative isolates. The tdh PAI was not present in any clinical or environmental tdh- and trh-negative isolates. The trh PAI was not present in any environmental isolates; however, in clinical tdh- and trh-negative isolate 10-4238, the majority of the trh PAI including a partial trh1 gene was present, which resulted in reclassification of this isolate as a tdh-negative and trh-positive isolate. In the other clinical tdh- and trh-negative isolates, neither the trh gene nor the trh PAI was present. We identified 862 genes in clinical tdh- and trh-negative isolates but not in environmental tdh- and trh-negative isolates. Many of these genes are highly homologous to genes found in common enteric bacteria and included genes encoding a number of chemotaxis proteins and a novel putative type VI secretion system (T6SS) effector and immunity protein (T6SS1). The availability of genome sequences from clinical V. parahaemolyticus tdh- and trh-negative isolates and the comparative analysis may help provide an understanding of how this pathotype is able to survive in vivo during clinical illness.
Project description:Vibrio parahaemolyticus is the main pathogen of food-borne diarrheal in coastal areas. Through the study of pathogen characteristics of 1870 V. parahaemolyticus, the isolation rate of O4:KUT had increased significantly since 2013. In this study, we analyzed virulence, antimicrobial susceptibility, molecular, and epidemiological characteristics of a new serotype named O4:KUT2. O4:KUT2 strains had tlh + tdh + trh - toxRS/new- characteristics and were prevalent during 2013-2015. The 91.5% O4:KUT2 serotype strains were resistant to ampicillin. The growth curves of O4:KUT2 strains were different with O4:K9, O4:K8, and O3:K6 serotype strains. O4:KUT2 strains belonged to ST332 where four strains had a large fragment inserted at recA. Compared the whole genomes of O4:KUT2 strains with O4:K9 strain which also belonged to ST322 isolated from acute diarrhea patients in Zhejiang province in 2012, no different alleles at 2249 loci was found. This finding implied that O4:KUT2 strains might be derived from O4:K9 strains. Clinical presentation of patients positive for V. parahaemolyticus O4:KUT2 were no significant difference with patients positive for O3:K6, although their genetic characteristics were different. The appearance and the increase of proportion of the new serotype O4:KUT2 strains was aware that we should not relax the monitoring of the pathogen spectrum of acute diarrheal patients.
Project description:<h4>Background</h4><i>Vibrio parahaemolyticus</i> is a causative agent of gastroenteritis. Most of the clinical isolates carry either <i>tdh</i> and/or <i>trh</i> genes which are considered as the major virulence genes of this pathogen. In this study, the clinical isolates of <i>V. parahaemolyticus</i> carrying <i>trh</i> gene (<i>n?</i>=?73) obtained from 1886 to 2012 from various countries were investigated for the urease production, haemolytic activity, and biofilm formation. In addition, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping among these isolates was investigated.<h4>Results</h4>In this study, no significant differences were observed in the urease production between <i>tdh</i> <sup>+</sup> <i>trh</i>1<sup>+</sup> and <i>tdh</i> <sup>+</sup> <i>trh</i>2<sup>+</sup> isolates (<i>p</i>?=?0.063) and between the <i>tdh</i> <sup>-</sup> <i>trh</i>1<sup>+</sup> and <i>tdh</i> <sup>-</sup> <i>trh</i>2<sup>+</sup> isolates (<i>p</i>?=?0.788). The isolates carrying only the <i>trh</i> gene showed variation in their haemolytic activity. The ratio of urease production and haemolytic activity between the <i>trh</i>1<sup>+</sup> and <i>trh</i>2<sup>+</sup> isolates and biofilm formation of <i>trh</i> <sup>+</sup> <i>V. parahaemolyticus</i> isolates were not significantly different. Sixteen of thirty-four tested isolates (47.0%) of <i>trh</i> <sup>+</sup> <i>V. parahaemolyticus</i> were positive for CRISPR detection. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the DI obtained by CRISPR typing alone.<h4>Conclusion</h4>The <i>tdh</i> and <i>trh</i> genes were not involved in urease production in the <i>trh</i> <sup>+</sup> <i>V. parahaemolyticus</i>, and variation of haemolytic activity detected in <i>V. parahaemolyticus</i> carrying only the <i>trh</i> gene might be correlated to the sequence variation within <i>trh</i>1 and <i>trh</i>2 genes. Additionally, biofilm production of <i>V. parahaemolyticus</i> was not associated with harboring of virulence genes. For genotyping, CRISPR sequences combined with virulence genes can be used as genetic markers to differentiate <i>trh</i> <sup>+</sup> <i>V. parahaemolyticus</i> strains.