Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g ؊1 . Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh ؉ V. parahaemolyticus than previously reported.Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that occurs naturally in estuarine environments worldwide (21). Its densities in the environment and seafoods vary greatly by season, location, sample type, fecal pollution, and analytical methodology (5, 8-10, 14, 21, 22, 36). Pathogenic V. parahaemolyticus generally produces a thermostable direct hemolysin (TDH), the product of the tdh gene (20). More than 90% of clinical V. parahaemolyticus isolates but fewer than 1% of food or environmental strains produce TDH or possess tdh (9,10,14,22,26,31,35). The frequency of TDH or tdh detection in environmental samples and seafoods ranges from 0 to 6% (5,14,22,26,31,34). Quantitative data for tdh ϩ V. parahaemolyticus have been reported in only one study and are based on one or two isolates from each of four oyster samples (10).A series of oyster-associated outbreaks of V. parahaemolyticus (3, 4, 6, 7) in 1997 (Washington State) and 1998 (Texas, New York and Connecticut, and Washington State) prompted the development of nonradioactive DNA probes (28, 29) and direct-plating methods for rapid and efficient determination of the abundance of total and pathogenic V. parahaemolyticus in oysters. The Food and Drug Administration (FDA) initiated a surveillance program using these new methods in Alabama in March 1999. The objective of this study was to correlate quantitative data on the abundance of total and pathogenic V. parahaemolyticus with each other and with environmental parameters such as water temperature and salini...

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n ‫؍‬ 37) and environmental (n ‫؍‬ 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.Vibrio parahaemolyticus is a natural inhabitant of coastal waters and is the leading cause of seafood-borne gastroenteritis (34). Since 1996 an increasing number of V. parahaemolyticus infections and outbreaks caused by strains belonging to a pandemic clonal complex have been observed throughout the world (2,8,10,17,18,20,32,33,38,39). The emergence of this clonal complex has elevated public health concerns for pandemic spread previously uncharacteristic of V. parahaemolyticus. Serology, the historic mainstay of V. parahaemolyticus surveillance, has been unreliable in tracking the spread of the epidemic clonal complex because in addition to the original O3:K6 serotype, at least 11 other serovariants have been identified (2, 7, 9). These new serovariants are highly similar to or indistinguishable from the original O3:K6 strains by a variety of molecular fingerprinting techniques, including arbitrarily primed PCR, pulsed-field gel electrophoresis, ribotyping, the intergenic spacer region between the 16S and 23S rRNA genes, and direct genome restriction enzyme analysis (8,18,20,33,38). Accordingly, the molecular subtyping techniques, which usually demonstrate a high degre...

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the tdh gene but not thetrh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150–3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh andtrh typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus Vibrio. The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The tdh-positive andtrh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of V. parahaemolyticus and reports a novel toxRS-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.

This investigation extends by 1000 km the northernmost documented source of oysters that caused illness due to V. parahaemolyticus. Rising temperatures of ocean water seem to have contributed to one of the largest known outbreaks of V. parahaemolyticus in the United States.

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...