Although food safety efforts have focused on reducing Vibrio parahaemolyticus and V. vulnificus illnesses, other species are increasingly being recognized as causes of foodborne vibriosis, especially associated with consumption of raw bivalve molluscan shellfish. These emerging causes of vibriosis include V. fluvialis, V. cholerae (non-O1/non-O139), V. mimicus, V. alginolyticus, and Grimontia hollisae, that are naturally occurring in estuarine waters where shellfish are grown. In order to better understand the prevalence, distribution, and environmental drivers of these emerging human pathogens, appropriate methods are needed to detect, isolate, and identify these species from shellfish, water, and sediment.
This study aimed to specifically isolate V. fluvialis and V. mimicus from oysters and sediment. Oyster homogenate or sediment was enriched overnight in Alkaline Peptone Water (APW) at 35° and also 41° C for V. mimicus, then streaked to multiple chromogenic agars to target V. fluvialis and V. mimicus. Typical colonies were purified and further characterized by streaking to a panel of chromogenic agars and TCBS to determine phenotypic profile. Isolates with a typical V. fluvialis and V. mimicus profile were then analyzed with newly developed real-time PCR assays. Select isolates that were positive by real-time PCR were then confirmed by whole genome sequencing (WGS).
Of the 32 V. fluvialis-typical colonies (example in Figure 1), 20 were positive by real-time PCR. Four isolates were selected for WGS, and all four isolates were confirmed as V. fluvialis. Of the 39 V. mimicus--typical isolates (example in Figure 2), 28 were positive by real-time PCR. Ten PCR-positive and 11 PCR-negative isolates were sequenced, with 100% agreement between PCR and WGS. The results demonstrate the utility of this approach for isolating and identifying V. fluvialis and V. mimicus. Future work will include evaluating the real-time PCR assays for screening of enrichments to aid in detection and isolation of these emerging Vibrio spp.