Identification ofVibrioIsolates by a Multiplex PCR Assay andrpoBSequence Determination

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Abstract
Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species—Vibrio cholerae,Vibrio parahaemolyticus, andVibrio vulnificus—account for the majority ofVibrioinfections in humans. Rapid and accurate identification ofVibriospecies has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae,V. parahaemolyticus,V. vulnificus, andV. mimicus). The assay was tested on a sample of 309Vibrioisolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional fourV. parahaemolyticusisolates among the other 119 isolates. Sequence analysis based onrpoBwas used to validate the multiplex results for these four isolates, and all clustered with otherV. parahaemolyticussequences. TherpoBsequences for 12 of 15 previously unidentified isolates clustered with otherVibriospecies in a phylogenetic analysis, and three isolates appeared to represent unnamedVibriospecies. The PCR assay provides a simple, rapid, and reliable tool for identification of the majorVibriopathogens in clinical samples, andrpoBsequencing provides an additional identification tool for other species in the genusVibrio.