Multiplex qPCR Detection of Nonpathogenic and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus from Oysters Cultured in Massachusetts and Rhode Island: The Reliability of MPN as an Indicator

Abstract

The leading cause of seafood-borne illness in the United States is Vibrio parahaemolyticus, whereas Vibrio vulnificus has the highest case fatality rate from wound infections of any foodborne pathogen. The Food and Drug Administration (FDA) uses a method of bacterial determination in oyster tissues termed “most probable number” (MPN) to enumerate Vibrio spp. abundance. This study used the development of two multiplex qPCR methods for the enumeration of total and pathogenic V. parahaemolyticus and V. vulnificus in oyster tissue using plasmid controls. Oysters, Crassostrea virginica, were collected from aquaculture farms in Wareham, MA, and Portsmouth, RI, from May to October 2014. Food and Drug Administration MPN and MPN-qPCR methods were compared and showed that FDA methods had higher values for both locations than MPN-qPCR methods. Throughout the summer at both locations, V. parahaemolyticus was most abundant with a majority being trh⁺ strains. Although serotypes were not directly tested for, tdh⁺ and trh⁺ strains were identified, suggesting a low possible occurrence of O4: K12 and no possible occurrence of O3:K6. In some samples, Vibrio alginolyticus was detected, indicating that trh⁺ percentages may be attributed to either V. parahaemolyticus or V. alginolyticus because of the similarity of the trh gene. The results from this study indicate the poor specificity and quantifiability of the FDA MPN method. This work also demonstrated that the use of plasmid controls for routine molecular methods to be used in a laboratory provided a reliable and consistent source of positive controls to compare against unknown samples for the abundance of V. parahaemolyticus and V. vulnificus.