Development and Evaluation of Polymorphic Locus Sequence Typing for Epidemiological Tracking of Vibrio...

Vibrio parahaemolyticus is a common inhabitant of coastal estuaries, and can accumulate to high levels in the shellfish that populate those waters. Human gastrointestinal infection occasionally follows ingestion of raw oysters, and it can lead to extended closures of implicated oyster beds with serious economic consequences. To track down the source of human infection, and to monitor strain variation in the environment, a user-friendly and affordable typing method that provides sufficient resolution for epidemiological analysis is needed. Polymorphic locus sequence typing (PLST) is based on conventional PCR and dideoxynucleotide sequencing of the one or two most phylogenetically informative genomic loci. Bioinformatic analyses of GenBank databases identified the V. parahaemolyticus polymorphic tandem repeat-containing loci VpMT1 and VpMT2 on chromosomes 1 and 2, respectively, as promising PLST targets, yielding diversity indexes of 0.99. Phylogenetic analysis identified multiple clusters representing strains known or likely to be epidemiologically related. Correlations with serotype and multilocus sequence type were strong but resolution was higher; for example, North American ST36 strains yielded 16 VpMT1 alleles. In the laboratory, VpMT1 and VpMT2 were robust, resolving 16 of 17 strains following PCR and sequencing directly from heat-killed colonies. Finally, 4 of 13 retail oyster enrichments yielded VpMT sequences that were unique but closely related to previously characterized clinical or environmental V. parahaemolyticus isolates.