Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India.

The levels of total and tdh(+)Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh(+)V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh(+)V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India.

Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.