Detection of pathogenic Vibrio parahaemolyticus through biochemical and molecular-based methodologies in coastal waters of the Gulf of Trieste (North Adriatic Sea)

Culturable vibrios were isolated from seawater collected during an annualsampling study performed along the Gulf of Trieste coast (Northern AdriaticSea), and conventional culturing and identification methods were used toinvestigate the presence of Vibrio parahaemolyticus. Biochemically selected Vibriostrains were subjected to phenotypical identification performed using Alsina’sscheme, API 20E and API 20NE. PCR and sequence analysis of the 16S rRNA geneand detection of the species-specific toxR and tlh genes were carried out on strainspresumptively identified as V. parahaemolyticus and on a set of unidentified strainsto confirm biochemical characterizations. In addition, PCR assays targeting thevirulence genes, tdh and trh, were carried out to detect pathogenic strains. PCRresults were compared with phenotypic characterizations to evaluate the accuracyof the biochemical methods applied. False-negative identifications were obtainedby all phenotypic-based procedures, while API 20E yielded only one false positive.Because the amplification of the 16S rRNA gene produced uncertain results, toxRand tlh gene detections were necessary to confirm the biochemical identifications.Finally, molecular characterization demonstrated the presence of V. parahaemolyticustrh-positive strains and underlined the difficulty in the recognition of thepathogenic environmental organism using conventional methods.