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Environmental strains of Vibrio parahaemolyticus, a foodborne pathogen, were isolated from milkfish and grouper aquaculture facilities in southern Taiwan and characterized by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction targeting on the virulence-associated and genomic island (VPaI) marker genes. Analyses of 62 environmental isolates, including two putative pathogenic isolates, by NotI-PFGE revealed 11 pulse-type clusters with a similarity of 85%. Some of the T3SS2α-associated genes (vopB2, vopC and vopT) were not present in all of these two putative pathogenic isolates. Marker genes of VPaI-1 (MTase gene), VPaI-2 (VP0636) and VPaI-3 (VP1073 and VP1077) were detected in 14–100% of the environmental isolates examined, and the VP1073 and VP1077 of VPaI-3 marker genes were not detected. This study confirmed the high genetic variability of the environmental isolates including the putative pathogenic strains and identified some marker genes of VPaI in the environmental V. parahaemolyticus isolates.Vibrio parahaemolyticus is a prevalent seafood-borne enteropathogen with the appearance of pandemic O3:K6 strains in 1996. This study characterized the environmental nontoxigenic and toxigenic isolates by pulsed-field gel electrophoresis and the presence of marker genes of genomic islands. Results showed that the T3SS2α-associated genes are not present in all environmental tdh+ isolates, and the presence of movable elements may contribute to genetic variation in the environmental V. parahaemolyticus isolates.