Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.     

Characteristics of Vibrio parahaemolyticus O3:K6 from Asia

A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of V. parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains.     

Characteristics of Vibrio parahaemolyticus O3:K6 from Asia

A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of V. parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains.     

Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil

Aims:  To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil.
Methods and Results:  Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl , tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S–23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S–23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh ^– Kanagawa-negative isolates.
Conclusions:  V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless.
Significance and Impact of the Study:  Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.     

副溶血性弧菌Vibrio parahaemolyticus O3:K6大流行克隆的溯源

副溶血性弧菌(Vibrio Parahaemolyticus)是一种革兰氏阴性嗜盐性海洋细菌。1950年从日本一次暴发性食物中毒中首次分离发现。作为一种食源性人鱼共患致病菌,副溶血性弧菌在全球的河口、海洋和沿海广泛传播,由其引起的食物中毒已跃居其它病原菌之首。副溶血性弧菌在进化过程中通过基因重组和基因水平转移逐渐改善其对环境的适应性,因而与其它所有致病微生物相比,副溶血性弧菌的基因型和血清型都具有高度的多样性。本文就副溶血性弧菌,特别是1996年后在世界范围内出现的O3:K6新血清型流行株(形成所谓的O3:K6大流行克隆Pandemic clone)的发现及流行特征、变异分子流行病学特征、在我国的分布及研究进展进行综述,以期为O3:K6大流行克隆的溯源提供更多依据。     

Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Characterization of new O3:K6 strains and phylogenetically related strains of Vibrio parahaemolyticus isolated in Taiwan and other countries

AIMS: We analysed the genetic divergence in the pandemic new O3:K6 and phylogenetically related (new O3:K6-like) strains and compare these two groups in terms of virulence and other biological traits. METHODS AND RESULTS: A total of 160 new O3:K6, new O3:K6-like and other strains of Vibrio parahaemolyticus isolated in Taiwan and other countries were collected and their clonal relationships analysed using SfiI-pulsed-field gel electrophoresis. All of the new O3:K6 and new O3:K6-like strains were grouped in cluster I with five new patterns identified. A O6:K18 strain was identified as a new member of the new O3:K6-like strains in addition to O4:K68, O1:KUT and O1:K25 strains. All of the lipopolysaccharide preparations of the selected strains exhibited closely spaced quadruplet banding patterns with similar mobility. The two groups of strains exhibited 100% sequence identity in the internal sequences of the toxR and laf genes, and also displayed similar virulence properties as determined with a suckling mouse model. CONCLUSIONS: The new O3:K6 and new O3:K6-like strains were highly similar in virulence and in several other phenotypical and genotypical traits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated the spread and divergence of the pandemic and related clone of V. parahaemolyticus with similar virulence.     

Isolation of a Pandemic O3:K6 Clone of a Vibrio parahaemolyticus Strain from Environmental and Clinical Sources in Thailand

Application of an immunomagnetic enrichment method selective for Vibrio parahaemolyticus serovar K6 allowed isolation of a strain belonging to the pandemic O3:K6 clone of V. parahaemolyticus from fresh shellfish not implicated in a clinical case in southern Thailand. Arbitrarily primed PCR profiles of this strain, clinical O3:K6 strains isolated from sporadic diarrhea cases in the same area, and a standard pandemic O3:K6 strain were indistinguishable.     

Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.     

Outbreak of gastroenteritis caused by the pandemic Vibrio parahaemolyticus O3:K6 in Mexico

During 2003 and during late September of 2004, more than 1230 cases of gastroenteritis were reported in the south of Sinaloa State, north-western Mexico. All cases were attributed to the consumption of raw or undercooked shrimp collected at the Huizache-Caimanero lagunary system. Vibrio parahaemolyticus was identified by standard biochemical methods, and many strains were positive for PCR amplifications of the tlh and tdh genes and negative for the trh gene. A representative strain belonged to the O3:K6 serogroup. This is the first outbreak of gastroenteritis caused by the pandemic strains of O3:K6 V. parahaemolyticus in México.     

A filamentous phage of Vibrio parahaemolyticus O3:K6 isolated in Laos.

A filamentous phage, 'lvpf5,' of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10² ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10³ cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.     

Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Comparative Phenotypic, Molecular, and Virulence Characterization of Vibrio parahaemolyticus O3:K6 Isolates

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a “new” group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.     

Comparative phenotypic,molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.     

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

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.