The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10 4 organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.Vibrio parahaemolyticus is a halophilic bacterium that occurs in estuarine environments worldwide (5,13,14,16,18,19). This organism was first discovered in Japan in 1950 in association with a food poisoning case (9). V. parahaemolyticus infection can cause gastroenteritis in humans, and the illness is most frequently associated with the consumption of raw or undercooked seafood and seafood recontaminated with the bacterium after cooking (31). V. parahaemolyticus accounts for about 70% of the gastroenteritis associated with seafood in Japan (14), and in India about 10% of the cases of gastroenteritis in patients admitted to the Infectious Diseases Hospital in Kolkata are due to V. parahaemolyticus (3). However, not all strains of V. parahaemolyticus are pathogenic. It has been demonstrated that the Kanagawa phenomenon, a beta-hemolysis in high-salt blood agar (Wagatsuma agar), is associated with most clinical strains but with very few environmental strains (32, 38). The hemolysis is due to the production of a thermostable direct hemolysin (TDH) (30). A TDH-related hemolysin (TRH) produced by a Kanagawa phenomenon-negative strain was discovered during investigation of an outbreak of gastroenteritis in the Maldives Islands in 1985 (10, 11). TDH and TRH, encoded by the tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus (31). It has been reported that more than 90% of clinical isolates but less than 1% of environmental isolates produce TDH (7,22,33). In recent years, the incidence of V. parahaemolyticus infection has been increasing in many parts of the world, and this has been attributed to the emergence of a new clone of the O3:K6 serotype carrying only the tdh gene (24). Although various serovars of the...

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Aims: To study the incidence of Vibrio parahaemolyticus in seafoods, water and sediment by molecular techniques vs conventional microbiological methods. Methods and Results: Of 86 samples analysed, 28 recorded positive for V. parahaemolyticus by conventional microbiological method, while 53 were positive by the toxR-targeted PCR, performed directly on enrichment broth lysates. While one sample of molluscan shellfish was positive for tdh gene, trh gene was detected in three enrichment broths of molluscan shellfish. Conclusions: Direct application of PCR to enrichment broths will be useful for the rapid and sensitive detection of potentially pathogenic strains of V. parahemolyticus in seafoods. Significance and Impact of the Study: Vibrio parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis world-wide. As, both pathogenic and non-pathogenic strains of V. parahaemolyticus exist in the seafood, application of PCR specific for the virulence genes (tdh & trh) will help in detection of pathogenic strains of V. parahaemolyticus and consequently reduce the risk of food-borne illness.

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Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.