Molecular Identification And Typing Of Vibrio Parahaemolyticus Isolates From Seafood

Vibrio parahaemolyticus is a Gram negative, halophilic, rod-shaped bacterium that distributed worldwide in the estuanne environment, especially in fishes, shellfish and seafood products. It has been considered as one of the most important seafood-borne pathogens in the countries and regions with long coastlines. Humans caused by this organism are almost exclusive with consumption of raw or inadequately cooked seafood, or any food cross-contaminated by handling raw seafood in the same environment or by rinsing with contaminated seawater. A period of time at room temperature is generally necessary to allow multiplication of organisms to the infectious level. The common symptoms are usually presented as gastroenteritis characterized mainly by watery diarrhea and abdominal cramps or occasionally by a dysentery-like illness with bloody or mucoid stools. Most outbreaks occurred during the warmer months. Apparently, V. parahaemolyticus is a pathogen of great concern to the seafood industry as well as to the consumers because of the diversity of its presence in many steps of the seafood processing lines. Therefore, to improve a reliable, rapid and convenient methods for identification and typing of V. parahaemolyticus isolates from seafoods and processing plants are crucial to identify the principal contamination points along the processing lines and establish the critical control points thereafter.A duplex PCR method was developed for rapid, specific and sensitive identification of Vibrio parahaemolyticus from seafoods using primers targeting the thermolabile hemolysin gene (tl) and Gyrase B gene (gyrB) specific for the bacterium. The procedure could amplify two fragments, one is 285bp specific for gyrB, and the other 450bp specific for tl. It could differentiate between V. parahaemolyticus and other vibrio spp. The duplex PCR could detect as low as 2.5xl02cfu/ml of V. parahaemolyticus in pure cultures. The detection limit for artificially contaminated samples was similar to that of pure culture, and could be lower (2.5xlO皛10'cfu/g) if combined with 3-6h of further incubation. Thus, the duplex PCR may be used forThesis for the Degree of Master of Science, Zhejiang Universityspecific identification of V. parahaemolyticus directly from shrimp samples.A total of 182 fresh and frozen seafoods and environmental samples along the shrimp processing lines from 4 seafood-processing factories and retail markets were examined for the presence of Vibrio parahaemolyticus using duplex PCR. The putative V. parahaemolyticus isolates (n=46) from selective agar plates were identification by conventional methods followed by duplex PCR, and finally screened for the virulence factor thermostable direct hemolysin (tdh) by single PCR. Both methods identified 32 isolates as V. parahaemolyticus. The gene tdh was not found in all V. parahaemolyticus isolates. This study suggests the potential risk of ingesting raw or undercooked frozen seafood products due to high contamination rate of potentially pathogenic V. parahaemolyticus.Pulsed-field gel electrophoresis (PFGE) was used for typing of eleven V. parahaemolyticus isolates (including a reference strain) after digestion of the genomic DNA by the restriction enzyme Apal. Hierarchical cluster analysis revealed that these isolates could be arbitrarily grouped into three major PFGE types A, B and C. The type C could further be subdivided into Cl, C2 and C3. A wide rang of PFGE types were identified in isolates from different origin (included reference strain). Moreover, the PFGE types were not specifically associated with the origin of kind of seafood. These results reveal high genetic diversity in V. parahaemolyticus isolated from seafoods.In summary, the present studies have provided some bases for rapid identification of V. parahaemolyticus in seafood as well as tracking of the source of its contamination along the seafood processing lines.