Quantitative Detection of Viable but Nonculturable Vibrio parahaemolyticus in Frozen Bivalve Molluscs

Abstract

Vibrio parahaemolyticus is a foodborne pathogen diffusely distributed in the marine environment and often isolated from raw seafood belonging to different species, mostly shellfish. Ingestion of under- or uncooked seafood contaminated by V. parahaemolyticus can cause severe gastrointestinal symptoms in humans. Due to its ability to withstand low temperatures, Vibrio spp. could survive in frozen seafoods for long periods by entering the viable but nonculturable state (VBNC) and may constitute an unrecognized source of food contamination and infection. In the present study, seventy-seven frozen bivalve molluscs (35 mussels; 42 clams) were subjected to the detection and enumeration of viable V. parahaemolyticus using standard culture methods. VBNC forms were detected and quantified by applying an optimized protocol based on Propidium Monoazide (PMA) and Quantitative PCR (qPCR). All samples were negative for both the detection and enumeration of V. parahaemolyticus by the standard culture methods. VBNC forms were detected in 11.7% of the samples (9/77), with values ranging from 1.67 to 2.29 Log CFU/g. Only clam samples were positive for the detection of VBNC forms. The results of this study highlighted that VBNC V. parahaemolyticus may be present in frozen bivalve molluscs. Further data on the prevalence of VBNC V. parahaemolyticus in frozen seafood are needed in order to perform a robust risk assessment.

Tools to Enumerate and Predict Distribution Patterns of Environmental Vibrio vulnificus and Vibrio parahaemolyticus

“…However, there is hope; examples of more efficient direct analyses of DNA are becoming more prevalent, such as direct qPCR [191], and metagenomics [192], where both of these types of methods by-pass the culture-dependent step completely. Sensitivity of qPCR-only studies is, like with methods containing a culture step, very good.…”