Parahaemolyticus O3 Research Articles

Emergence and predominance of a new serotype of Vibrio parahaemolyticus in Huzhou, China

BackgroundDuring our surveillance period, we found a new type of Vibrio parahaemolyticus (V. parahaemolyticus) with serotype O10:K4, which had increased over the past 2 years in Huzhou and became the second most common serotype after O3:K6. MethodsStrains were isolated from stool samples of diarrhea patients in the First People's Hospital in Huzhou. The serotypes, virulence-associated genes, antimicrobial susceptibility, and pulsed field gel electrophoresis (PFGE) of these strains were analyzed. ResultsBetween January 2017 and December 2021, there were 598 (5.36%) V. parahaemolyticus-positive samples of 11,166 stool specimens. The V. parahaemolyticus detection rate was high in summer months. The O3:K6 was the dominant serotype in 2017–2020 and a new serotype, O10:K4, was the predominant serotype in 2021. The majority of isolates tested were resistant to ampicillin (86.8%). We randomly chose the strains with serotype O3:K6 and O10:K4 for PFGE to compare the genetic relationship between these two serotypes. The results showed that the PFGE profiles of V. parahaemolyticus O3:K6 and O10:K4 were genetically similar. The strains showed a tendency to cluster on the basis of their serotype profiles. However, some O3:K6 strains showed 100% similarity with O10:K4 strains. ConclusionA new serotype with pandemic potential of V. parahaemolyticus, O10:K4, was detected in 2020 and became dominant in 2021 in Huzhou.

In-silico analysis of bioactive compounds extracted from seaweed Amphiroa anceps on the pathogenicity of bacteria

Screening of biochemical compounds in marine algae is a gateway to understand the bioactive principles of antimicrobial activity and may be a significant tool in developing environment friendly herbicides and in drug discovery. In this study, the antibacterial activity of the seaweed Amphiroa anceps extracted with methanol and ethanol was determined against clinical isolates namely Vibrio cholerae 01 ogawa, Vibrio cholerae O139, Vibrio fluvialis, Vibrio parahaemolyticus, V. parahaemolyticus 81, V. parahaemolyticus O3:K6, salmonella enteritidis type 5, Enterotoxigenic E. coli, Bacillus cereus by disc diffusion method at different concentrations. Ethanolic extract showed the highest activity against V. parahaemolyticus, V. parahaemolyticus 81 and V. cholerae. The FTIR results showed the presence of major functional groups like amine, hydrogen bonded alcohols, alkanes and aromatic rings. Four major bioactive compounds were selected based on GC-MS data and used as ligands to dock in-silico against proteins of pathogenic bacteria that were used in antibiotic studies.

Efficiency of monovalent and polyvalent Vibrio alginolyticus and Vibrio Parahaemolyticus vaccines on the immune response and protection in gilthead sea bream, Sparus aurata (L.) against vibriosis

This experimental studies investigated the protective efficiencies and the potential immune mechanisms of vibrio monovalent and polyvalent autogenous formalin-inactivated whole-cell bacterins (FKC) in Gilthead sea bream (Sparus aurata) cultured in Egypt. Two months post-vaccination, the relative percentage survival (RPS) was estimated after challenge with the vaccine's homologues pathogenic strains. The survival values were 100% and 83.3% in groups immunized with monovalent V. alginolyticus or V. parahaemolyticus FKC bacterins, respectively. On the other hand, survival values were 91.75% and 75% in fish groups subjected to polyvalent (V. parahaemolyticus O11: K40 &V. alginolyticus) and (V. parahaemolyticus O3: K6 &V. alginolyticus) FKC bacterins, respectively. Overall, the tested vaccine preparations were significantly increased (P < 0.05) the agglutination antibody titer, phagocytic activity, respiratory burst activity, when compared to the non-immunized control group.The current results conclude that, autogenous Vibrio vaccines provoked a promising protection against vibriosis in Gilthead sea bream cultured in Egypt, it was superior in monovalent FKC V. alginolyticus vaccine and polyvalent FKC of V. parahaemolyticus O11: K40 with V. alginolyticus vaccine that could be useful means of prevention and control of vibriosis.

Isolation, Molecular Characterization and Antibiotic Susceptibility Pattern of Vibrio parahaemolyticus from Aquatic Products in the Southern Fujian Coast, China.

Vibrio parahaemolyticus is a major gastroenteritis-causing pathogen in many Asian countries. Antimicrobial resistance in V. parahaemolyticus has been recognized as a critical threat to food safety. In this study, we determined the prevalence and incidence of antimicrobial resistance in V. parahaemolyticus in the southern Fujian coast, China. A total of 62 isolates were confirmed in retail aquatic products from June to October of 2018. The serotype O3:K6 strains, the virulence genes tdh and trh, antibiotic susceptibility and molecular typing were investigated. Then plasmid profiling analysis and curing experiment were performed for multidrug-resistant strains. The results showed that the total occurrence of V. parahaemolyticus was 31% out of 200 samples. Five strains (8.1%) out of 62 isolates were identified as the V. parahaemolyticus O3:K6 pandemic clone. A large majority of isolates exhibited higher resistance to penicillin (77.4%), oxacillin (71%), ampicillin (66.1%) and vancomycin (59.7%). Seventy-one percent (44/62) of the isolates exhibited multiple antimicrobial resistance. All 62 isolates were grouped into 7 clusters by randomly amplified polymorphic DNA, and most of the isolates (80.6%) were distributed within cluster A. Plasmids were detected in approximately 75% of the isolates, and seven different profiles were observed. Seventy-six percent (25/33) of the isolates carrying the plasmids were eliminated by 0.006% SDS incubated at 42°C, a sublethal condition. The occurrence of multidrug-resistant strains could be an indication of the excessive use of antibiotics in aquaculture farming. The rational use of antimicrobial agents and the surveillance of antibiotic administration may reduce the acquisition of resistance by microorganisms in aquatic ecosystems.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Spreading of Pandemic Vibrio parahaemolyticus O3:K6 and Its Serovariants: A Re-analysis of Strains Isolated from Multiple Studies.

In China, V. parahaemolyticus has been a leading cause of foodborne outbreaks and bacterial infectious diarrhea since the 1990s, and most infections have been associated with the pandemic V. parahaemolyticus O3:K6 and its serovariants. However, a comprehensive overview of the sero-prevalence and genetic diversity of the pandemic V. parahaemolyticus clone in China is lacking. To compensate for this deficiency, pandemic isolates in both clinical and environmental Chinese samples collected from multiple studies were analyzed in this study. Surprisingly, as many as 27 clinical pandemic serovariants were identified and were widely distributed across nine coastal provinces and two inland provinces (Beijing and Sichuan). O3:K6, O4:K68, and O1:KUT represented the predominant clinical serovars. Only four environmental pandemic serovariants had previously been reported, and they were spread throughout Shanghai (O1:KUT, O3:K6), Jiangsu (O3:K6, O4:K48), Zhejiang (O3:K6), and Guangdong (O4:K9). Notably, 24 pandemic serovariants were detected within a short time frame (from 2006 to 2012). The pandemic isolates were divided into 15 sequence types (STs), 10 of which fell within clonal complex (CC) 3. Only three STs (ST3, ST192, and ST305) were identified in environmental isolates. Substantial serotypic diversity was mainly observed among isolates within pandemic ST3, which comprised 21 combinations of O/K antigens. The pandemic O3:K6 serotype showed a high level of sequence diversity, which was shared by eight different STs (ST3, ST227, ST431, ST435, ST487, ST489, ST526, and ST672). Antimicrobial susceptibility testing revealed that most isolates shared similar antibiotic susceptibility profiles. They were resistant to ampicillin but sensitive to most other drugs that were tested. In conclusion, the high levels of serotypic and genetic diversity of the pandemic clone suggest that the involved regions are becoming important reservoirs for the emergence of novel pandemic strains. We underscore the need for routine monitoring to prevent pandemic V. parahaemolyticus infection, which includes monitoring antimicrobial responses to avoid excessive misuse of antibiotics. Further investigations are also needed to delineate the specific mechanisms underlying the possible seroconversion of pandemic isolates.

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

Prevalence, antibiotic resistance, virulence and plasmid profiles of Vibrio parahaemolyticus from a tropical estuary and adjoining traditional prawn farm along the southwest coast of India

Vibrio parahaemolyticus, autochthonous to estuarine, marine, and coastal environments throughout the world, is a leading cause of foodborne gastroenteritis in Asia as well as in other countries. Considering their role in disease outbreaks, water and sediment samples from two different environments along the southwest coast of India, such as the Cochin estuary, which is a tourist hot spot and a traditional prawn farm, were examined for the detection of V. parahaemolyticus. A total of 120 presumptive vibrios were further screened using a chromogenic medium. The isolates producing the specific colour for V. parahaemolyticus on the chromogenic medium were confirmed by molecular methods. Using a PCR assay targeting the species-specific tlh gene, 75 isolates were confirmed as V. parahaemolyticus. The incidence of V. parahaemolyticus was found to be 71.6 % in the Cochin estuary and 53.3 % in the shrimp farm. The virulence genes tdh and trh were detected in two isolates from the Cochin estuary by multiplex PCR. Using 16S rRNA sequence analysis, our isolate exhibited 100 % similarity to the V. parahaemolyticus O3:K6 pandemic clone. The isolates were evaluated for their susceptibility towards 16 different antibiotics. All the isolates exhibited multiple antibiotic resistance (MAR). The MAR index of the isolates from the Cochin estuary ranged from 0.312 to 0.75 and that from the shrimp farm ranged from 0.1875 to 0.5. The plasmid profiles of the isolates were also checked. About 50.6 % of the isolates harboured plasmids and ten different profiles were observed. No correlation was observed between the antibiotic resistance patterns and plasmid profiles.

A pandemic Vibrio parahaemolyticus O3:K6 clone causing most associated diarrhea cases in the Pacific Northwest coast of Mexico.

Between September and October of 2004, more than 1230 cases of gastroenteritis due to pandemic O3:K6 strains of Vibrio parahaemolyticus (V. parahaemolyticus) were reported in the relatively small geographical area of Southern Sinaloa, a state located in Northwest Mexico. Since then, V. parahaemolyticus-associated gastroenteritis cases have gradually increased in prevalence spreading from south to north. The present study conducted an epidemiological surveillance of V. parahaemolyticus strains in both environmental and clinical samples along the Pacific coast of Sinaloa from 2011 to 2013. The genetic relatedness, serotype dominance and antibiotic resistance of isolates were investigated. A total of 46 strains were isolated from environmental samples (e.g., sediment, seawater and shrimp), whereas 249 strains were obtained from stools of patients with gastroenteritis. Nine different O serogroups and 16 serovars were identified. Serovars O3:K6 and O6:K46 were identified in both environmental and clinical strains. Whereas most environmental isolates carried the tdh gene (71.74%, 33/46), only three (6.52%) belonged to pandemic clones (O3:K6, O3:KUT and OUT:KUT). In contrast, 81.1% (202/249) of clinical isolates belonged to pandemic serotypes, with O3:K6 (tdh, toxRS/new, and/or orf8) representing the predominant serovar (97%, 196/202). This prevalence of pathogenic (tdh and/or trh positive) and O3:K6 pandemic V. parahaemolyticus isolates in this study were similar to those found from 2004 to 2010. As investigated by REP-PCR, genetic lineages of selected O3:K6 strains isolated in this study and some isolated earlier were nearly identical. Antimicrobial susceptibility testing showed that most strains (93.8%) were resistant to ampicillin but sensitive to chloramphenicol (98.8%). Multidrug resistance significantly increased from 8.6% (2004–2010) to 22.93% (2011–2013; p < 0.05). Our data indicate that pandemic O3:K6 clone has endemically established in the Pacific Coast of Mexico.

Detection of trh Positive Vibrio parahaemolyticus Serotype O1:K56 with Possible Eco‐pathological Impact from Freshwater Sediments around Dhaka City, Bangladesh

Vibrio parahaemolyticus is a well‐known marine bacterium with a pandemic effect of causing enteric diseases. Present study attempted to detect the possible presence of this enteropathogen in the freshwater sediments with an objective to demonstrate its pathogenic divergence as well as the dissemination potential within the aquatic environment. Isolates of V. parahaemolyticus were screened from freshwater sediments by the river sides around Dhaka City, and their genotypic traits were chalked out through toxR, tdh, and trh gene‐specific amplification. Isolates were serotyped as O10:K66 (N = 1), O10:KUT (N = 1), and O1:K56 (N = 5), of which two of the O1:K56 serovariants showed the presence of trh gene. Pulsed field gel electrophoresis analysis detected 65% similarity between the reference strain V. parahaemolyticus O3:K6 and the O1:K56 isolates. Since O1:K56 is known to be a serovariant of O3:K6 which imparts the pandemic potential, the unlikely presence of O1:K56 strains in the freshwater samples in our study further demonstrates the possible eco‐pathological impact.

Predictive model of Vibrio parahaemolyticus O3:K6 growth on cooked Litopenaeus vannamei

Vibrio parahaemolyticus is the leading cause of seafood-derived illness in China and a possible mechanism leading to illness is cross contamination of cooked shrimp. A growth rate model of pandemic V. parahaemolyticus O3:K6 strain on cooked shrimp stored at isothermal temperatures was developed and validated. The maximum specific growth rate of this pandemic O3:K6 strain ranged from 0.11 to 1.47 (1/h) between 12 °C and 40 °C. A square root type model was then used to quantify the dependency between the maximum specific growth rate and temperature. The temperature at which the specific maximum growth rate = 0 was 1.0 °C. A goodness-of-fit measure, quantified by an average bias and accuracy factor indicated that the models of growth of V. parahaemolyticus O3:K6 on cooked shrimp under dynamic temperature regimes were reliable. Cooked shrimp also appears to be a better growth medium than TSB broth between 12–16 °C. Therefore relying on models developed in TSB broth for measuring the risk of V. parahaemolyticus will result in an underestimation of risk at these temperatures. Additionally, the growth models developed and validated in cooked shrimp gives fail-safe prediction of V. parahaemolyticus growth on fresh shrimp.

Pandemic Vibrio parahaemolyticus O3:K6 on the American continent

Vibrio parahaemolyticus is one of the most important seafood-borne bacterial in recent years and is the leading causal agent of human acute gastroenteritis, primarily following the consumption of raw, undercooked or mishandled marine products. Until 1996, infections caused by V. parahaemolyticus were generally associated with diverse serovars. However, in February 1996, a unique serovar (O3:K6) of V. parahaemolyticus with specific genetic markers (tdh, toxRS/New and/or orf8) appeared abruptly in Kolkata, India. In subsequent years, O3:K6 isolates similar to those isolated in Kolkata have been reported from food borne outbreaks in Southeast Asia, as well as in the Atlantic and Gulf coasts of the United States (U.S). More recently, there have been reports in Europe, Africa and Central and South America. Specifically, in the American continent, some countries have reported cases of gastroenteritis due to the pandemic O3:K6 strain and its serovariants; the pandemic strain was first detected in Peru (1996, >100 cases), subsequently spreading to Chile in 1998 (>16,804 human cases), to the U.S. in 1998 (>700 cases), to Brazil in 2001 (>18 cases) and to Mexico in 2004 (>1200 cases). The arrival of the pandemic clone on the American continent may have resulted in a significant shift on the epidemic dynamics of V. parahaemolyticus. However, although O3:K6 is the predominant serovar of the recognized clinical strains in some countries in the Americas, a decrease in clinical cases caused by O3:K6 and an increase in cases associated with a new serotype (O3:K59, Chile) have been recently reported. The emergence and worldwide dissemination of O3:K6 and other pandemic strains since 1996 have come to represent a threat to public health and should concern health authorities. This review focuses on the presence, distribution and virulence factors of the V. parahaemolyticus O3:K6 pandemic clone and its serovariants in clinical and environmental strains on the American continent.

Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.

Association of Pandemic Vibrio parahaemolyticus O3:K6 Present in the Coastal Environment of Northwest Mexico with Cases of Recurrent Diarrhea between 2004 and 2010

In 2004, more than 1,230 cases of gastroenteritis due to pandemic O3:K6 strains of Vibrio parahaemolyticus were reported in southern Sinaloa, a state in Northwestern Mexico. Recurrent sporadic cases arose from 2004 to 2010, spreading from the south to the north. In the present study, Vibrio parahaemolyticus was detected in both environmental samples and clinical cases along the Pacific coast of Sinaloa during 2004 to 2010. An evaluation was made of the serotypes, distribution of virulence genes, and presence of pandemic O3:K6 strains. A total of 144 strains were isolated from environmental samples (from sediment, seawater, and shrimp), and 154 clinical strains were isolated. A total of 10 O serogroups and 30 serovars were identified in the strains. Environmental strains (n = 144) belonged to 10 O serogroups and 28 serovars, while clinical strains (n = 154) belonged to 8 O serogroups and 14 serovars. Ten serovars were shared by both environmental and clinical strains. Among 144 environmental isolates, 4.1% (6/144) belonged to the pandemic clone, with 83.3% containing the orf8 gene and with O3:K6 accounting for 67%. On the other hand, pathogenic strains (tdh and/or trh) accounted for 52% (75/144) of the environmental isolates. Interestingly, among 154 clinical isolates, 80.5% (124/154) were pandemic strains, with O3:K6 (tdh, toxRS(new), and orf8) representing the predominant serovar (99.2%, 123/124). Overall, our results indicate that in spite of a high serodiversity and prevalence of pathogenic Vibrio parahaemolyticus in the environment, the pandemic strain O3:K6 caused >79% of reported cases between 2004 and 2010 in Sinaloa, Mexico.

Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6

BackgroundPandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy.ResultsWe generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in V. parahaemolyticus O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between gmhD and rjg, determines the K6-antigen but not the O3-antigen in V. parahaemolyticus. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen.ConclusionUnderstanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic V. parahaemolyticus. This report confirms the genetic location of K-antigen synthesis in V. parahaemolyticus O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non-Pseudomonas phage related to the PhiKMV-like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage-sensitive and -resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.

Systematic Functional Pandemic Strain–Specific Genes, Three Genomic Islands, Two T3SSs in Foodborne, and ClinicalVibrio parahaemolyticusIsolates in China

Vibrio parahaemolyticus is one of the most important pathogens capable of causing foodborne gastroenteritis in China, Japan, and other countries. Pathogenic V. parahaemolyticus has been known to produce either thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), or both. The emergence of a new clone in 1995, V. parahaemolyticus O3:K6, has resulted in the first documented pandemic spread of V. parahaemolyticus. In this study, 235 isolates from clinical and food sources were characterized by determining the presence of known virulence factors (tdh, trh), systematic genetic markers (toxRS/new, pandemic group-specific sequence [PGS], orf8) specific for V. parahaemolyticus O3:K6 clone and its clonal derivatives, three important genomic islands (GIs) (VPaI-1, VPaI-5, and VPaI-7), and two type III secretion systems (T3SS1 and T3SS2). Our results showed that all 235 isolates harbored all or part of the T3SS1 genes. All the 103 tdh-positive strains harbored all or part of the VPaI-7 and T3SS2 genes. A total of 91 isolates including six foodborne isolates belonged to a pandemic clone in which eight isolates lacked orf8. All pandemic strains harbored VPaI-1 and VPaI-5 except one O4:K68 strain that lacked VPaI-5 altogether. Twelve clinical pathogenic strains had VPaI-7 and T3SS2 but lacked VPaI-1 and VPaI-5. Thirteen nonpathogenic clinical strains and 119 foodborne strains, including six foodborne pathogenic trh-positive strains, only harbored T3SS1 genes. These results indicated that O3:K6 and its serovariants were the main pandemic clone in China. VPaI-1 and VPaI-5 genes were specifically correlated with pandemic strains while VPaI-7 and T3SS2 were closely associated with tdh-positive strains.

Development of a Predictive Program for Vibrio parahaemolyticus Growth under Various Environmental Conditions

In this study, we developed a predictive program for Vibrio parahaemolyticus growth under various environmental conditions. Raw growth data was obtained with a V. parahaemolyticus O3:K6 strain cultured at a variety of broth temperatures, pH, and salt concentrations. Data were analyzed with our logistic model and the parameter values of the model were analyzed with polynomial equations. A prediction program consisting of the growth model and the polynomial equations was then developed. After the range of the growth environments was modified, the program successfully predicted the growth for all environments tested. The program could be a useful tool to ensure the bacteriological safety of seafood.

Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique

The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.