Prevalence and distribution of a novel prophage in pandemic vibrio parahaemolyticus

Vibrio parahaemolyticus is endemic in marine ecosystems and is the most common cause of bacterial seafood-borne gastroenteritis worldwide. Recent years have shown a dramatic increase in global V. parahaemolyticus infections owing to the emergence and spread of the pandemic O3:K6 serotype. Further, clinical reporting has shown an expanding poleward range resulting from climate change. In the Pacific Northwest (PNW) region of the United States, virtually all clinical cases are caused by an endemic serotype (O4:K12) despite an environmental abundance of the pandemic O3:K6 serotype. Previous analysis of PNW V. parahaemolyticus revealed that O3:K6 strains were virulence-attenuated in a zebrafish model, and subsequent genomic analyses revealed that PNW O3:K6 strains carry a novel prophage (NW1) that may be associated with avirulent conversion. The purpose of this study was to broaden the understanding of where prophage NW1 is maintained in natural reservoirs (e.g., seawater, plankton, and oysters) in the PNW through the following objectives: 1) determine the prevalence and distribution of prophage NW1 from a collection of 731 environmental PNW V. parahaemolyticus strains by polymerase chain reaction (PCR), and 2) analyze draft genomes of NW1+ isolates to identify the prophage integration site and determine the genetic consequences of integration (e.g., gene gain or gene loss). Results show that the pandemic O3:K6 strain was prevalent among the PNW isolates (230/731 or 31.5%), more so than the endemic O4:K12 strain (64/731 or 8.8%), and the overwhelming majority of those pandemic isolates harbored the NW1 prophage (187/230 or 81.3%). Genome analyses confirmed the same integration site and gene loss of VP1884 through VP1890 in all sequenced NW1+ isolates (n=17). Interestingly, the pandemic O3:K6 NW1+ isolates were relatively abundant in seawater and plankton samples (110/248 or 44.4% and 65/253 or 25.7%, respectively) but absent from oyster samples (0/204). This disparity may explain the near absence of clinical O3:K6 cases in this region, although the mechanism behind this observation is yet unknown and warrants further study. In summary, these results demonstrate that prophage NW1 is prevalent in the PNW environment; the majority of O3:K6 isolates carry the prophage. However, this is not a geographically or temporally isolated phenomenon, as the prophage was detected in isolates from distant locations over the past two decades. Future analyses will involve challenging zebrafish with VP1884-VP1890 O3:K6 mutants to identify the gene or genes responsible for attenuated virulence. Future analyses will also involve challenging oysters with VP1884-VP1890 O3:K6 mutants to identify the gene or genes responsible for colonization deficiency.