Foodborne disease outbreaks caused by sucrose-nonfermenting and beta-galactosidase-deficient variants of Vibrio cholerae

We reported four foodborne disease outbreaks in Taiwan caused by sucrose-nonfermenting and by beta-galactosidase-deficient variants of non-O1, non-O139 Vibrio cholerae. The sucrose-nonfermenting vibrios collected from three outbreaks were biochemically identified to be V. mimicus and the beta-galactosidase-deficient vibrios from an outbreak to be V. alginolyticus. However, molecular methods including DNA-DNA hybridization, fatty acid profile analysis, and sequence analysis of 16S rRNA, oriC, pyrH, recA, and rpoA indicated that these vibrios should be V. cholerae. These V. cholerae variants carried two hemolysin genes, hlyA and hlx, but contained neither cholera toxin gene, ctx, V. mimicus hemolysin gene, vmh, nor thermo-directed hemolysin, tdh. The sucrose-nonfermenting variants of V. cholerae shared a high level of genetic relatedness; they could derive from a common clone. In our record from 1995 to date, this was the first time that V. cholerae variants were discovered as etiologic agents for foodborne disease outbreaks in Taiwan.     

Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.     

Comparison of a Chromogenic Medium with Thiosulfate-Citrate-Bile Salts-Sucrose Agar for Detecting Vibrio parahaemolyticus

ABSTRACT: The widely used most probable number (MPN) method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus on the thiosulfate-citrate-bile salts-sucrose agar (TCBS). Presumptive positive colonies grown onTCBS need to be confirmed with lengthy biochemical tests. This study compared a chromogenic medium, Bio-Chrome Vibrio medium (BCVM), with TCBS for detecting V. parahaemolyticus in seawater, sediment, and oysters using a 3-tube MPN method. Among the 296 samples tested, 136 and 92 samples produced presumptive positive results on TCBS and BCVM, respectively. Biochemical tests and a multiplex polymerase chain reaction (PCR) assay confirmed 74 of 83 samples that were presumptive positive on both TCBS and BVCM as V. parahaemolyticus . Although false-positive results were reported when either medium was used, there were 62 reported for TCBS whereas only 15 were reported for BCVM. The specificities of TCBS and BCVM for V. parahaemolyticus detection were determined to be 77% and 94%, respectively. The accuracies of detecting V. parahaemolyticus were 54% for TCBS and 84% for BCVM. The Bio-Chrome Vibrio medium can be used in the MPN method to reduce the number of biochemical tests needed for V. parahaemolyticus confirmation.     

Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticust

The thiosulfate-citrate-bile salts-sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.     

The occurrence of Vibrio species in tropical shrimp culture environments; implications for food safety

The occurrence of various Vibrio species in water, sediment and shrimp samples from multiple shrimp farm environments from the east and west coast of India was studied. The relative abundance was higher in west coast farms (ca. 10(4) cfu/ml water) when compared to the east coast (ca. 10(2) cfu/ml water). Vibrio alginolyticus (3-19%), V. parahaemolyticus (2-13%), V. harveyi (1-7%) and V. vulnificus (1-4%) were the predominant Vibrio species identified by standard biochemical testing. In some cases, V. cholerae could be found, but all isolates were negative for the cholera toxin (ctx) gene that is associated with choleragenic strains. The biochemical identification of V. parahaemolyticus, the other human pathogen among the species mentioned above, was confirmed by PCR targeting the toxR gene and a 387 bp chromosomal locus specific for this species. Furthermore, the presence of the virulence-associated tdh (thermostable direct haemolysin) and trh (TDH-related haemolysin) genes in the V. parahaemolyticus isolates was also detected by PCR. Only 2 out of 47 isolates were tdh positive and one contained the trh gene. However, since V. cholerae, V. parahaemolyticus and V. vulnificus species are recognized as a major cause of seafood-borne illness, it is important to pay attention to post-harvest handling and adequate cooking.     

Prevalence and molecular characterization of Vibrio cholerae from ice and beverages sold in Jakarta, Indonesia, using most probable number and multiplex PCR

Vibrio cholerae is well recognized as the causative agent of cholera, an acute intestinal infection characterized by watery diarrhea that may lead to dehydration and death in some cases. V. cholerae is a natural inhabitant of the aquatic environment in the tropical regions. Jakarta has the highest percentage of individuals affected by sporadic diarrheal illness compared with other areas in Indonesia. Inadequate safety measures for drinking water supplies, improper sanitation, and poor hygiene can increase the risk of cholera outbreaks. Few studies have been conducted on the prevalence of these bacteria in ice and beverages that are popularly sold and consumed in Jakarta. In this study, we detected and quantified V. cholerae from ice and beverages collected from several areas in five regions of Jakarta. Levels of V. cholerae in both ice and beverages were determined with the three-tube most-probable-number (MPN) method and ranged from < 0.3 to > 110 MPN/ml. The presence of regulatory and virulence gene sequences was determined by using uniplex and multiplex PCR assays. Of 110 samples tested, 33 (30%) were positive for V. cholerae; 21 (64%) were ice samples and the remaining 12 (36%) were beverages. A total of 88 V. cholerae strains were isolated, based on the presence of the toxR gene sequence identified by PCR. Other genetic markers, such as hlyA (59%), ompU (16%), and ctxA (19%), also were found during the search for potential pathogenic strains. The detection and isolation of potentially harmful V. cholerae from ice and beverages in Jakarta indicate that these products pose a health risk from choleragenic vibrios, particularly because of the emergence of classical biotypes of V. cholerae O1 and potentially harmful non-O1 serovars of this species.     

食品中拟态弧菌变性高效液相色谱检测技术研究

应用PCR 结合变性高效液相色谱(DHPLC)技术对食品中致病菌--拟态弧菌进行检测,探讨其灵敏度特异性,建立快速高通量检测食品中拟态弧菌的新方法。利用DHPLC 在非变性温度下进行双链DNA 分离的原理,应用DHPLC 技术分析拟态弧菌特异性PCR 扩增产物。在优化的分析条件下,对样品洗脱峰排列形成的聚类分析图进行分析,得到拟态弧菌的特征峰型图谱。结果表明该方法有很好的特异性。与传统检测方法进行比较该方法准确度为100%。本实验建立的食品中拟态弧菌PCR-DHPLC 检测技术,特异性灵敏,准确度高,操作快速、简便,在食品安全检测中具有重要应用价值。     

Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on selective media

Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

Risk of Vibrio transmission linked to the consumption of crustaceans in coastal towns of Côte d'Ivoire

The purpose of this study was to assess the risk of Vibrio spp. transmission from crustaceans to humans in two coastal towns of C?te d'Ivoire. Bacteriologic analysis was performed on 322 crustacean samples obtained from six markets in Abidjan and one in Dabou. Suspected Vibrio colonies were identified by morphological, cultural, biochemical, and molecular tests and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. PCR assays were used to further characterize Vibrio strains. A survey on consumption of crustaceans was conducted among 120 randomly selected households in Abidjan. Overall, Vibrio spp. were isolated from 7.8% of the crustacean samples studied, at levels as high as 6.3 log CFU/g. Of the Vibrio strains identified, 40% were V. alginolyticus, 36% were V. parahaemolyticus, and 24% were nontoxigenic V. cholerae; the latter two species can cause mild to severe forms of seafood-associated gastroenteritis. Among interviewed households, 11.7% reported daily consumption of crustaceans, confirming the high probability of exposure of human population to Vibrio spp., and 7.5% reported symptoms of food poisoning after consumption of crustaceans. The absence of genes encoding major virulence factors in the studied strains, i.e., cholera toxin (ctxA and ctxB) in V. cholerae and thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) in V. parahaemolyticus, does not exclude the possibility of exposure to pathogenic strains. However, human infections are not common because most households (96.7%) boil crustaceans, usually for at least 45 min (85.9% of households) before consumption.     

基于弧菌毒力基因多重PCR法的建立及其在副溶血弧菌毒力基因检测中的应用

针对副溶血弧菌常见的11种毒力基因(tox R、Collagenase、tox S、trh、tdh、tlh、Ure R、Fla A、omp W、Asp A、fur),建立了两套六重PCR检测体系,应用于副溶血弧菌环境分离株和水产品分离株的毒力基因分布情况调查。在调查的248株副溶血弧菌中,鞭毛丝蛋白基因Fla A、外膜蛋白基因omp W和铁吸收调节蛋白基因fur的分布最广(100%),其次为碱性丝氨酸蛋白酶基因Asp A(99.60%),胶原蛋白酶基因Collagenase、不耐热性溶血毒素基因tlh以及毒力调控基因tox R和tox S的分布率均在90%以上且tox R和tox S的分布极为相似,尿素酶基因Ure R的分布极少(1.21%),而耐热直接溶血素基因tdh和耐热相关溶血素基因trh在这248株副溶血弧菌中没有检出。本研究建立的多重PCR检测体系能快速、高效地检测多个毒力基因的分布情况,为副溶血弧菌的毒力机制研究和风险评估提供方法和依据。     

Real-time PCR von virulenten Vibrio parahaemolyticus in Fisch- und Krebstierprodukten

Pathogenic V. parahaemolyticus strains cause gastroenteritis. Contaminated fi sh products and seafood represent the most important sources of infection. V. parahaemolyticus strains from patients differ from environmental isolates by their production of the haemolysins TDH and TRH that represent their main virulence factors. 710 specimens of fish and crayfish products were examined by an evaluated PCR-/real-time PCR. This detection method of virulent V. parahaemolyticus isolates reduces the working time by one day after a selective enrichment of vibrios with alkaline saline peptone water.

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Zusammenfassung: Pathogene Vibrio parahaemolyticus rufen Gastroenteritis hervor. Die Hauptinfektionsquellen stellen kontaminierte Fischprodukte und Meeresfrüchte dar. V. parahaemolyticus- Patientenisolate unterscheiden sich von Umweltisolaten durch die Anwesenheit der H?molysine TDH und TRH als deren wichtigste Virulenzfaktoren. Evaluierte PCR-/real-time PCR-Verfahren wurden zur Untersuchung von 710 Proben Fisch- und Krebstierprodukte eingesetzt. Nach der Selektivanreicherung von Vibrionen in alkalischem, salinischem Peptonwasser führen die real-time PCR-Verfahren der Gene von TDH und TRH zu einer Ersparnis von mindestens einem Arbeitstag beim Nachweis virulenter V. parahaemolyticus-Isolate.

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Eingegangen: 18. Januar 2007     

Real-time PCR von virulenten Vibrio parahaemolyticus in Fisch- und Krebstierprodukten

Pathogenic V. parahaemolyticus strains cause gastroenteritis. Contaminated fi sh products and seafood represent the most important sources of infection. V. parahaemolyticus strains from patients differ from environmental isolates by their production of the haemolysins TDH and TRH that represent their main virulence factors. 710 specimens of fish and crayfish products were examined by an evaluated PCR-/real-time PCR. This detection method of virulent V. parahaemolyticus isolates reduces the working time by one day after a selective enrichment of vibrios with alkaline saline peptone water.     

Occurrence of Vibrio spp. in fish and shellfish collected from the Swiss market

The genus Vibrio includes gram-negative bacteria that inhabit estuarine ecosystems. V. cholerae, V. parahaemolyticus, and V. vulnificus pose a considerable public health threat as agents of sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated fish or shellfish. In this study, we analyzed 138 fish and shellfish samples collected from the Swiss market (fish fillets [n = 102], bivalves [n = 34], and squid [n = 2]). Microbiological analysis was done according to International Organization for Standardization method 21872-1/21872-2:2007, using thiosulfate citrate bile sucrose agar and chromID Vibrio agar as selective agar. Presumptive-positive colonies on thiosulfate citrate bile sucrose agar or chromID Vibrio agar were picked and were identified by the API 20E and species-specific PCR systems. V. cholerae isolates were tested further by PCR for the presence of the cholera toxin A subunit gene (ctxA). V. parahaemolyticus isolates were tested by PCR for genes encoding for thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). V. cholerae was isolated from three samples and V. parahaemolyticus from eight samples. None of these strains harbored species-specific virulence factors. Further, V. alginolyticus was isolated from 40 samples, and V. fluvialis was isolated from 1 sample. Our study provides, for the first time, data for the assessment of exposure to Vibrio spp. in raw fish and bivalves consumed in Switzerland.     

Prevalence and population analysis of Vibrio parahaemolyticus in retail aquatic products from the southern Fujian coast,China

Vibrio parahaemolyticus is one of the most hazardous pathogens causing seafood-borne diseases in the southern Fujian coast, China. From June to October 2016, a total of 250 samples were collected from retail markets in the Xiamen, Quanzhou, and Zhangzhou regions. Seventy-seven V. parahaemolyticus isolates were identified using polymerase chain reaction (PCR). Then, molecular typing was performed using repetitive extragenic palindromic-PCR (REP-PCR). The distribution of seven virulence genes was detected by PCR. In aquatic products, the prevalence of V. parahaemolyticus was 30.8%, and the prevalence of tdh⁺ and trh⁺ was 2.6 and 1.3%, respectively. The prevalence of type III secretion system-2 (T3SS2) and the ureC gene was 5.2 and 3.9%, respectively. All 77 strains and the reference strain V. parahaemolyticus ATCC 17802 were classified into seven molecular types using REP-PCR. Thus, our findings demonstrated that the prevalence of V. parahaemolyticus was severe in the southern Fujian coast and that the regulations for aquatic food safety should be strengthened.     

Prevalence of potentially pathogenic Vibrio species in the seafood marketed in Malaysia

Seafood samples obtained in seafood markets and supermarkets at 11 sites selected from four states in Malaysia were examined for the presence of nine potentially pathogenic species from the genus Vibrio between July 1998 and June 1999. We examined 768 sample sets that included shrimp, squid, crab, cockles, and mussels. We extensively examined shrimp samples from Selangor State to determine seasonal variation of Vibrio populations. Eight potentially pathogenic Vibrio species were detected, with overall incidence in the samples at 4.6% for V. cholerae, 4.7% for V. parahaemolyticus, 6.0% for V. vulnificus, 11% for V. alginolyticus, 9.9% for V. metschnikovii, 1.3% for V. mimicus, 13% for V. damsela, 7.6% for V. fluvialis, and 52% for a combined population of all of the above. As many as eight Vibrio species were detected in shrimp and only four in squid and peel mussels. The overall percent incidence of any of the eight vibrios was highest (82%) in cockles (Anadara granosa) among the seafoods examined and was highest (100%) in Kuching, Sarawak State, and lowest (25%) in Penang, Pulau Penang State, among the sampling sites. Of 97 strains of V. cholerae isolated, one strain belonged to the O1 serotype and 14 to the O139 serotype. The results indicate that the various seafood markets in Malaysia are contaminated with potentially pathogenic Vibrio species regardless of the season and suggest that there is a need for adequate consumer protection measures.     

市售水产品中副溶血性弧菌的毒力及药敏性分析

为了解市售水产品中副溶血性弧菌毒力基因携带情况及药敏性情况,本文采用《GB4789.7-2013食品安全国家标准食品微生物学检验副溶血性弧菌检验》分离培养方法,利用VITEK 2 compact全自动细菌鉴定系统及荧光PCR从市售水产品中分离鉴定出60株副溶血性弧菌分离株,通过常规PCR对60株分离株中四种毒力基因的分布进行筛查,并用VITEK 2 compact全自动细菌鉴定系统对副溶血性弧菌分离株进行药敏性试验。结果显示,市售水产样品的60株副溶血性弧菌分离株中tlh基因携带率为100.00%、tox R/S基因携带率为100.00%、tdh基因携带率为95.00%,trh基因携带率为65.00%,多重毒力基因携带率高,其中四重毒力基因携带率为61.67%;分析60株分离株对19种抗菌药物的药敏情况发现对氨苄西林有极高的耐药性,耐药率为96.67%,多重耐药现象不突出,仅有4株分离株对2种药物耐药。结果表明,市售水产样品中副溶血性弧菌分离株毒力基因携带率较高,且多数携带多重毒力基因;分离株耐药情况不突出,但对抗菌类药物氨苄西林的耐药率较高,需警惕水产食品中副溶血性弧菌的潜在威胁。     

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.     

The international standard ISO/TS 21872-1 to study the occurence of total and pathogenic Vibrio parahaemolyticus and Vibrio cholerae in seafood: ITS improvement by use of a chromogenic medium and PCR

During two surveys conducted in 2008 and 2009, the culture method described in the international standard ISO/TS 21872-1 was applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae in 112 living bivalve mollusc samples, with a chromogenic medium used in addition to the TCBS agar, as second selective isolation medium and for enumeration of V. parahaemolyticus and V. cholerae by surface inoculation. A PCR method for detection of these 2 Vibrio species and the hemolysin genes tdh and trh, was applied in parallel. In 2009, the survey was extended to finfish fillets and crustaceans. PCR was also used for species confirmation of characteristic colonies. The identity of the PCR products, specifically targeting V. parahaemolyticus, was checked by sequencing. Occurrence of V. parahaemolyticus and V. cholerae isolates in living bivalve molluscs ranged from 30.4% to 32.6% and from 1.4% to 4.7% respectively. In frozen crustaceans (2009 survey) V. parahaemolyticus and V. cholerae isolates were respectively found in 45% and 10% of the samples. No V. parahaemolyticus or V. cholerae was detected in frozen fish fillets, neither by the ISO method nor by PCR. In 2009, enteropathogenic V. parahaemolyticus (trh+) was isolated from 4 out of 43 oyster samples while the trh gene was present in V. alginolyticus strains and in samples where V. parahaemolyticus was not detected (9 over 112 samples). The ISO method failed to isolate V. parahaemolyticus in 44% to 53% of the living bivalve molluscs where PCR detected the toxR gene specific of V. parahaemolyticus (Vp-toxR). Our results highlighted the need for a revision of the ISO/TS 21872-1 standard, at least, for analysis of living bivalve molluscs, and confirmed the increasing concern of enteropathogenic V. parahaemolyticus in French bivalve molluscs. Enrichment at 41.5°C was questioned and some reliable solutions for the improvement of the ISO/TS 21872-1 method, such as the PCR method for screening of positive samples and confirmation of colonies, were pointed out.