Detection, Enumeration, and Isolation of Vibrio parahaemolyticus and V. vulnificus from Seafood: Development of a Multidisciplinary Protocol

Abstract

Vibrio parahaemolyticus and V. vulnificus are bacterial foodborne pathogens that can cause illnesses in humans after ingestion or exposure to contaminated seafood or coastal waters. A procedure that combines microbiological, biochemical, and molecular methods was designed and optimized for the detection, enumeration, isolation, and characterization of these clinically significant Vibrio spp. Initially, microbiological culturing is used to resuscitate and isolate presumptive Vibrio spp. from chilled seafood samples. Biochemical tests are then used to analyze and select presumptive isolates at the species level, and, lastly, molecular methods, such as PCR targeting species-specific hemolysin genes, are used to confirm identification and assess the potential pathogenicity of presumptive isolates. By using artificially contaminated molluscan homogenates with known numbers of V. parahaemolyticus, this method yielded, on average, 90% recovery on complete agar media and 88% recovery on selective media. For V. vulnificus, the recovery rates were 86% (complete media) and 84% (selective media). Linearity of recovery of Vibrio spp. from artificially contaminated seafood homogenates supported the applicability of this method. Overall, this performance-tested protocol is easy to use, cost-effective, and fit-for-purpose, with potential for routine use in basic microbiological facilities.