Use of Whole Genome Phylogeny and Comparisons in the Development of a Multiplex-PCR Assay to Identify Sequence Type 36 Vibrio parahaemolyticus

Vibrio parahaemolyticus sequence type (ST) 36 strains that are native to the Pacific Ocean have recently caused multi-state outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern US sources, were used to identify diagnostic loci: one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present only in one clade of closely-related strains, of ST36, ST59 and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two non-clade isolates, and cps in four. Based on the distribution of these loci in sequenced genomes, prp could identify clade strains with \u3e99% accuracy, but the addition of one more locus would increase accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus, and determines presence of both the tdh and trh hemolysin-encoding genes which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a four year period in three Northeastern US, and 87 environmental isolates, revealed the prp and cps amplicons were only detected in clinical isolates identified as belonging to the ST36-clade, and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections