A PCR Assay for Specific Detection of the Pandemic \u3cem\u3eVibrio parahaemolyticus\u3c/em\u3e O3:K6 Clone from Shellfish

The current standard method for identifying Vibrio parahaemolyticus serotype O3:K6, an emerging pathogen with apparent enhanced virulence characteristics, typically takes 4 to 6 d to complete and requires serotyping. To provide a more rapid strategy, we optimized a polymerase chain reaction (PCR)-based assay for specific detection of V. parahaemolyticus O3:K6. Of 78 V. parahaemolyticus isolates and other related species; only strains classified into the V. parahaemolyticus O3:K6 clonal group (n = 39) showed positive results in the PCR assay. The assay detected 2.3 cells/PCR reaction and 310 cells/g using bacterial cultures and inoculated oyster samples, respectively. Sensitive and specific detection of V. parahaemolyticus O3:K6 was possible following a 6-h enrichment

Comparative Phenotypic, Molecular, and Virulence Characterization of \u3cem\u3eVibrio parahaemolyticus\u3c/em\u3e O3:K6 Isolates

Historically, Vibrio parahaemolyticus infections have been characterizedby sporadic cases caused by multiple, diverse serotypes. However,since 1996, V. parahaemolyticus serotype O3:K6 strains havebeen associated with several large-scale outbreaks of illness,suggesting the emergence of a new group of organisms withenhanced virulence. We have applied three different molecularsubtyping techniques to identify an appropriate method for differentiatingO3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis(PFGE) following NotI digestion differentiated seven closelyrelated subtypes among O3:K6 and related strains, which weredistinct from PFGE patterns for non-O3:K6 isolates. Ribotypingand tdh sequencing were less discriminatory than PFGE, but furtherconfirmed close genetic relationships among recent O3:K6 isolates.In vitro adherence and cytotoxicity studies with human epithelialcells showed that O3:K6 isolates exhibited statistically higherlevels of adherence and cytotoxicity to host cells than non-O3:K6isolates. Epithelial cell cytotoxicity patterns were determinedwith a lactate dehydrogenase release assay. At 3 h postinfection,high relative cytotoxicities (\u3e50% maximum lactate dehydrogenaseactivity) were found among a greater proportion of recentlyisolated O3:K6 and closely related strains (75%) than amongthe non-O3:K6 isolates (23%). A statistically significant relationshipbetween adherence and cytotoxicity suggests that the pathogenicpotential of some isolates may be associated with increasedadherence to epithelial cells. Our findings suggest that enhancedadherence and cytotoxicity may contribute to the apparent uniquepathogenic potential of V. parahaemolyticus O3:K6 strains