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.     

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.     

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.     

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.     

Uptake of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by, and depuration of mussels (Mytilus galloprovincialis)

The uptakes of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by mussels (Mytilus galloprovincialis) and the times for depuration were investigated in order to determine the most useful indicator of vibrio contamination. The mussels were maintained in tanks of static seawater contaminated with bacteria at 5 log10 CFU/ml for bioaccumulation. Depuration was carried out by circulating fresh seawater through the tanks. Each organism was presented alone and with others to mussels, at temperatures of 14 and 21 degrees C. In water contaminated with either single or mixed organisms, the bacteria accumulated rapidly in the mussels reaching high concentrations after 1 h. With both single and mixed organisms, the maximum numbers of E. coli in mussels were 6.6 log10 CFU/g at 14 degrees C and 5.4 log10 CFU/g at 21 degrees C. Both V. cholerae non-O1 and E. durans alone or with other organisms reached a number ranging from 6.5 to 7 log10 CFU/g at both temperatures. During depuration the numbers of all the organisms slowly decreased, with E. coli alone, numbers ranged from 2.8 to 2 log10 CFU/g after 72 h at both 14 and 21 degrees C, and the organisms were undetectable after 144 h. With mixed organisms at 14 degrees C E. coli became undetectable after 168 h but at 21 degrees C no E. coli were recovered after 72 h. At 14 degrees C V. cholerae non-O1 alone also was undetectable after 168 h, but at 21 degrees C and with mixed organisms at both temperatures. V. cholerae was recovered after 168 h at numbers about 1 log10 CFU/g. After 168 h numbers of E. durans alone ranged from 2.6 log10 CFU/g at 14 degrees C to 1.5 log10 CFU/g at 21 degrees C, and with mixed organisms the numbers ranged from 2.3 to 2.0 log10 CFU/g at both temperatures. Of the three bacteria of faecal origin, E. durans is quickly acquired by mussels and released more slowly than the others, while E. coli quickly becomes undetectable. The results suggest that, for this kind of seafood, enterococci may be a more appropriate indicator than E. coli of risks to consumers from vibrios.     

INFECTIVE DOSE OF FOODBORNE PATHOGENS IN VOLUNTEERS: A REVIEW

Risk assessment and impact of foodborne pathogens on the health of different populations was one of the goals identified in the Presidential Food Safety Initiative three-year plan. This entailed estimation of dose-response relationship for foodborne pathogens to humans, either by feeding studies or from outbreaks. For certain pathogens, such as Listeria monocytogenes and Escherichia coli O157:H7, there are no feeding studies due to ethical reasons, and the results from outbreaks are normally used to estimate the infectious dose. The focus of this review is to compile dose-response information in volunteers for several foodborne pathogens including Salmonella, Shigella spp. , Campylobacter jejuni, Vibrio spp. , Escherichia coli, Cryptosporidium parvum and Entamoeba coli. The infectious dose for different serovars of Salmonella and strains of E. coli was quite large (> 10⁵ organisms), while the infectious dose for some Shigella spp. seemed to be as low as less than 10 organisms. Toxigenic V. cholerae (O1 and O139 serotypes) were infective at a dose of 10⁴ organisms; a non-O1 strain was infective at a much higher dose (10⁶ organisms). C. jejuni, C. parvum and Entamoeba coli appeared to have infectious doses as low as 500 organisms, 10 oocysts, and 1 cyst, respectively. The infectious dose and the dose response are dependent upon the strains used, and the age and physical condition of the individuals, and can therefore show wide variations. In addition, since many of the volunteer studies are carried out by feeding the organisms in a nonfood matrix after neutralizing the stomach acidity, results obtained may not reflect the true dose response.     

Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other Asian countries. A total of 371 isolates of V. parahaemolyticus collected from patients involved in foodborne illness outbreaks in Taiwan from 1992 to 1995 were characterized. These isolates had typical biochemical characteristics and only 4% were urease positive. The most frequently isolated serovars were O5:K15 (18.5%), O4:K8 (16.2%), O3:K29 (12.5%), O1:K56 (8.3%), O2:K3 (6.5%), and O4:K12 (6.0%). Most of the isolates were susceptible to nalidixic acid, tetracycline, tobramycin, cephalothin, and gentamicin. About 10% of the isolates were resistant to seven or more antibiotics. Approximately 92.4% of these V. parahaemolyticus showed beta-hemolysis on Wagatsuma blood agar plate and approximately 62.1% of these isolates exhibited detectable amounts of thermostable direct hemolysin. Most of the isolates examined exhibited two copies of tdh genes on the 1.3- and 2.5-kb HindIII-digested chromosome fragments with several variations on other fragments. A pulsed-field gel electrophoresis (PFGE) subspecies typing scheme was used to analyze these domestic isolates and the O3:K6 strains from Japan, Korea, and Taiwan. Fifty seven patterns were differentiated with A, B, C, E, and H being the major domestic types (cumulatively 76% of isolates), while O3:K6 strains (PFGE type I), abruptly occurring since 1996, were genetically distant from the major domestic types.     

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.     

OCCURRENCE OF POTENTIALLY PATHOGENIC VIBRIOS IN BIVALVE MOLLUSCS (MUSSELS AND CLAMS) FROM RETAIL OUTLETS IN THE NORTH OF SPAIN

Forty-three samples of shellfish (22 mussels and 21 clams) purchased from retail outlets from February through October were tested for the presence of vibrios associated with human disease . Vibrio spp. was found in 51.16% of the samples . V. alginolyticus was the commonest Vibrio species found in the samples, followed by V. parahaemolyticus, V. fluvialis and V. cholerae non-01. All V. parahaemolyticus isolated were Kanagawa-phenomenon negative. Hemolytic activities were shown in all isolates of V. fluvialis and V. cholerae. Bacterial indicators of quality and safety were within permitted limits by authorities. The results indicate the potential risks of food poisoning associated with the consumption of raw or undercooked shellfish .     

Occurrence of Vibrio and other pathogenic bacteria in Mytilus galloprovincialis (mussels) harvested from Adriatic Sea, Italy.

Sixty-two samples of Mytilus galloprovincialis (mussels) harvested from approved shellfish waters in the Adriatic Sea were examined for the presence of Vibrio, Salmonella, Campylobacter, and verocytotoxin producing Escherichia coli. Vibrio spp. were isolated from 48.4% of samples; the species most frequently found were V. alginolyticus (32.2%) and V. vulnificus (17.7%), followed by V. cincinnatiensis (3.2%), V. parahaemolyticus (1.6%), V. fluvialis (1.6%) and V. cholerae non-O1 (1.6%). V. parahaemolyticus resulted negative to Kanagawa-phenomenon and to PCR amplification of tdh gene. V. cholerae resulted negative to PCR amplification of sto gene. No Salmonella, Campylobacter, or E. coli verocytotoxin-producing strains were isolated. The results of this study suggest the potential risk of ingesting raw or undercooked mussels due to the frequent presence of potentially pathogenic Vibrio species.     

2001—2013年苏州市食源性疾病暴发事故流行病学分析

目的了解苏州市食源性疾病暴发事故流行病学特征和趋势。方法收集2001—2013年苏州市各地报告的食源性疾病暴发资料,并进行描述性流行病学分析。结果 2001—2013年共发生食源性疾病暴发343起,发病7 213人,无死亡病例。总体呈下降趋势;食源性疾病暴发高峰期为第三季度,占总起数的46.9%(161/343);食源性疾病暴发多发生在集体用餐场所,占总起数的56.6%(194/343)。报告明确或可疑致病因子的214起事故中由微生物或可疑微生物引起的占70.1%(150/214),毒素引起的占16.4%(35/214),化合物引起的占13.6%(29/214)。在实验室检出致病因子的168起暴发事故中,83.3%(140/168)的事故由10种致病因子引起,其中副溶血性弧菌导致的最多,达61起(36.3%),发病人数达1 436人。2009—2013年食源性暴发事故报告的109起中查明原因食品的共72起(66.1%)。原因食品被归因为5类食品,分别为水产品(33.3%,24/72)、肉制品(23.6%,17/72)、其他食品(15.3%,11/72)、混合食品(13.9%,10/72)、蔬菜(13.9%,10/72)。在暴发事故中较明确的致病因子-食品组合有:红细胞凝集素和皂甙-蔬菜(菜豆)、毒蘑菇及其他植物毒素-野生植物、组胺和河鲀鱼毒素-水产品、农药-蔬菜、亚硝酸盐-调味料、副溶血性弧菌-水产品、金黄色葡萄球菌-肉制品。导致暴发的污染环节最多的是交叉污染(44.2%,50/113)。结论对苏州市13年报告的食源性疾病暴发分析有利于加深对食源性疾病暴发流行病学特征的了解,食品安全监管部门、食品生产经营者和消费者可以利用此资料预防食品生产或经营中的污染,减少食源性疾病发生。完善报告信息管理系统有助于提高报告率。     

食源性副溶血弧菌脉冲场凝胶电泳分型

副溶血弧菌(Vibrio parahaemolyticus)是-种革兰氏阴性嗜盐杆菌,广泛分布于近岸海水、海底沉积物和海产品中,是引起食源性疾病的主要病源之一.尤其在我国沿海地区,由副溶血弧菌引发的食物中毒的发生规模及人群暴露规模呈明显上升趋势.因此,快速准确的检测鉴定副溶血弧菌成为控制其引起的食源性疾病的关键.本文以从海产品中分离的副溶血弧菌为研究对象,利用脉冲场凝胶电泳技术对副溶血弧菌进行分子分型,为副溶血弧菌引起的食源性疾病溯源及副溶血弧菌的分子流行病学研究提供技术基础.     

Vibrio cholerae and enteric bacteria in oyster-producing areas of two urban estuaries in Australia

Three sampling sites in oyster-producing areas of 2 estuaries were monitored at intervals of about 2 weeks for 1 year. Oysters (Crassostrea commercialis), water and sediment were examined for Vibrio cholerae, Escherichia coli and Salmonella. V. cholerae was detected in 20, 30 and 11% of oyster, water and sediment samples respectively. The highest incidence was in the autumn (March-May), with few isolations from July to October. Most isolates were non-O1 serotypes. The presence of V. cholerae and the enteric bacteria appeared to be influenced by different, but perhaps overlapping, sets of factors in these high salinity waters. There was no relationship between rainfall or salinity and the detection of V. cholerae, whereas high counts of E. coli in oysters and the presence of Salmonella were correlated wtih rainfall and, to a lesser degree, reduced salinity. High counts of E. coli were correlated with V. cholerae isolations from water and with the presence of Salmonella. Oysters concentrated E. coli effectively. The counts of E. coli in oysters were 7.3 times higher than those in water. Examination of 8 batches of purified and unpurified oysters indicated that purification reduces the incidence of V. cholerae. However V. cholerae was detected in 3 of 25 market samples of oysters, demonstrating that it can be present in oysters throughout the distribution system. The highest V. cholerae count observed in oysters was 3/g.     

Adhesion and colonization of Vibrio cholerae O1 on shrimp and crab carapaces

The potential of Vibrio cholerae O1 to attach to and colonize the carapaces of shrimp and crabs was evaluated. One million cells of V. cholerae O1 were spread within a circle on the external surfaces of separated carapaces and stored at 22 +/- 0.2 degrees C in a moist environment to permit adherence. Attached vibrios were counted directly by an immunofluorescence technique and by the pour plate technique after detachment of the cells. To study the colonization process, rifampicin-resistant strains of V. cholerae O1 were used. V. cholerae O1 strains, including those resistant to rifampicin, were able to attach to shrimp and crab carapaces. Dorsal crab carapaces showed higher levels of attachment than ventral carapaces. Colonization of V. cholerae O1 on these carapaces was also demonstrated. Both attachment and colonization on the shrimp exoskeleton were optimal at a salinity of 1.0 to 1.5%, a pH of 6.0 to 7.0, and a temperature of 37 degrees C. Less than 2% attachment at 3 degrees C contrasted with >20% attachment at 37 degrees C. Even at 3% NaCl, some attachment was observed. Although attachment percentages may appear low (2 to 20%), they represent significant numbers, about 3.7 to 5.6 log10 CFU per carapace. A rugose V. cholerae O1 strain attached to and colonized the shrimp carapace in a fashion very similar to that of the smooth strain from which it was derived. The ability of V. cholerae O1 to attach to and colonize exoskeletons of edible crustaceans provides a potential means of survival in aquatic environments. Concentrations of vibrios that may be reached on a single crab or shrimp carapace are clearly of concern with regard to public health.     

2013年山东省食源性疾病暴发事件 流行病学特征分析

目的分析2013年山东省食源性疾病暴发事件的流行病学特征,为制定食源性疾病预防控制措施提供依据。方法对2013年通过国家食源性疾病暴发报告系统上报的30起食源性疾病暴发事件进行整理分析。结果 2013年共发生食源性疾病暴发事件30起,发病人数654人,死亡2人。食源性疾病暴发事件的主要发病季节是第三季度,全年食源性疾病暴发主要发生在4~10月份,其中以8月份最高;16~60岁人群是暴发事件发生的主要人群;饮食服务单位是发生食源性疾病暴发事件的主要场所,其次是集体食堂;加工不当与交叉污染是引起食源性疾病暴发事件的主要原因:微生物是引起食源性疾病暴发事件发生的主要致病因素,不同致病因素之间导致的罹患率不同(P0.05);引起食源性疾病暴发事件的主要原因食品是混合食品。结论加强对高发季节、高发因素、高发污染环节的监控;加强食源性疾病暴发事件的调查处置;加大防控食源性疾病暴发事件的宣传力度等,是预防和控制食源性疾病暴发事件的有效措施。     

2001~2018年苏州市学校食源性疾病暴发事件分析

目的 为预防和控制苏州市学校食源性疾病暴发事件的发生提供科学决策。方法 对2001～2018年苏州市学校食源性疾病暴发事件报告和调查报告进行统计分析,主要分析不同年份食源性疾病暴发情况、致病因子、月份、学校类别、事件起数、发病人数等。结果 2001～2018年苏州市学校中食源性疾病暴发26起,占4.69%(26/554),发病985例,占8.87%(985/11104)。致病微生物及其毒素是导致学校食源性疾病暴发的主要致病因子,9月份是事件的高发月份,且小学暴发事件最多。粮食类及肉类是暴发的主要食物,加工人员污染和加工销售污染是暴发的主要原因。结论 缩短食品加工到就餐的时间,防止加工环节和加工人员污染能有效防止食源性疾病的暴发。     

2011年中国大陆食源性疾病暴发监测资料分析

目的 分析2011年中国食源性疾病暴发事件的流行病学特征。方法 对我国食源性疾病暴发监测系统收集的2011年食源性疾病暴发数据进行统计分析。结果 2011年29个省、自治区、直辖市共上报食源性疾病暴发事件809起,累计发病14 057人,死亡113人,微生物性因素引起的事件数和发病人数最多,分别占26.2%(212/809)和37.6%(5 292/14 057),化学性因素引起的死亡人数最多,占39.8%(45/113)。结论 微生物性因素是导致我国食源性疾病暴发的主要原因,化学性因素是导致死亡的主要原因。此外,应不断加强我国食源性疾病监测网络建设,增强报告意识。     

2015-2019年云南省家庭食源性疾病暴发事件分析

目的 分析2015-2019年云南省家庭食源性疾病暴发事件的流行病学特点,为制定家庭食源性疾病防控措施提供参考.方法 对2015-2019年云南省食源性疾病暴发监测系统中报告的家庭食源性疾病暴发事件数据进行统计分析.结果 2015-2019年云南省共报告家庭食源性疾病3 159起,发病12 402人,死亡229人,病死...     

Salmonella and Vibrio cholerae in brackishwater cultured tropical prawns.

The occurrence of Salmonella and Vibrio cholerae in brackishwater ponds was monitored over a 2-year period in one of the major prawn exporting countries in Southeast Asia. The principal production areas were identified and regular samples taken for Salmonella and V. cholerae analysis. Results demonstrated that brackishwater ponds and cultured prawns were inherently contaminated with both bacterial pathogens. Salmonella spp. were present in 16.0% of prawns and 22.1% of mud/water samples from ponds; and V. cholerae present in 1.5% of prawns and 3.1% of mud/water samples. Culturing by intensive methods tended to favour contamination by these pathogens, which is most likely due to the accumulation of waste and increase in the volume of sediments in ponds. Typical environmental factors such as water temperature, pH, and salinity were all favourable for growth of microorganisms. The incidence of the pathogens increased during the wet season and was marginally higher when ponds were located close to urban areas. S. weltevreden was identified as the principal serotype found in ponds, and to a lesser extent S. anatum (11%) S. wandsworth (8%) and S. potsdam (8%). The V. cholerae belonged to the non-O1 serogroup.     

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.