Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a speciesspecific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10 4 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh ؉ and trh ؉ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.Vibrio parahaemolyticus is found worldwide in estuarine waters and is the leading cause of gastroenteritis from seafood in the United States, with most infections resulting from the consumption of raw or mishandled seafood (2, 29). The tlh (thermolabile hemolysin) gene is a species-specific marker for V. parahaemolyticus (42,26), while the tdh (thermostable direct hemolysin) (33, 34) and trh (thermostable-related hemolysin) (35) genes are pathogenicity markers for V. parahaemolyticus. Pathogenic V. parahaemolyticus bacteria in the environment and food samples typically comprise 0.3 to 3% of the total V. parahaemolyticus population (4,8,20,22,43). DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44). The preferential amplification of nonpathogenic members of the total V. parahaemolyticus population due to the muchhigher copy number of the target could preclude the detection of pathogenic strains pres...

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A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationaryphase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene.Vibrio parahaemolyticus is a gram-negative, estuarine bacterial species and is the leading cause of seafood-associated bacterial gastroenteritis worldwide (3, 4). During a V. parahaemolyticus outbreak associated with Alaskan oysters in 2004 (12), numerous V. parahaemolyticus strains were isolated from oysters. Many of these strains carried both thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) genes, which are associated with pathogenicity (1,11,13,17). The tdh gene can be subdivided by sequence similarities into five subtypes (tdh1 through tdh5), sharing 96 to 98% identity (14). In contrast, there exists among the trh genes a significant nucleotide sequence variation, and they can be clustered into two main subgroups, trh1 and trh2, which share 84% identity (9).During investigation of the Alaskan V. parahaemolyticus outbreak of 2004, two strains isolated from oysters tested positive for the presence of the trh gene but were negative for the presence of the tlh gene (thermolabile hemolysin), reportedly in all V. parahaemolyticus strains (2,21,22,26). These strains were also urease positive, indicating the presence of the ure gene, which is genetically linked to the trh gene in V. parahaemolyticus (6, 7). The trh gene was detected using an alkaline phosphatase-labeled DNA probe designed specifically for the detection of trh (16). The presence of the trh but not the tlh gene in those isolates was very unusual, since the trh gene has been reported only in V. parahaemolyticus. These results suggest that vibrios other than V. parahaemolyticus could be a reservoir for trh in the environment.In the present study, we described the characterization of a Vibrio alginolyticus strain isolated from Alaskan oysters that possesses and expresses a trh gene with 98% homology to the trh2 gene of V. parahaemolyticus.Bacterial strains and phenotypic characterization. V. parahaemolyticus strain 93A-5807 (clinical isolate) was obtained from the GCSL culture collection. This strain and those described below were originally isolated from thiosulfate citrate bile sucrose agar. Individual colonies were selected and streaked for purification on T1N1 agar as recommended in the FDA's Bacteriological Analytical Manual (BAM) (FDA/CFSAN; http: //www.cfsan.fda.gov/ϳebam/bam-9.html). Single-well-isolated colonies were selected for all phenotypic and genotypic assays. V. parahaemolyticus strains Vp 28 (AK 2228-1 0826), Vp 32 (AK 2162-1B 1054B), and Vp 33 (AK 2162-1A ...