Rapid Detection and Fast Induction of Viable but Nonculturable Vibrio parahaemolyticus and Vibrio cholerae

Vibrio (V.) species, such as V. parahaemolyticus and V. cholerae, are commonly associated with foodborne infections and are frequently detected in seafood worldwide. Unfavorable environmental conditions and process-related factors can induce a shift from culturable Vibrio cells into viable but nonculturable (VBNC) cells. Conventional culture-based detection methods (ISO 21872-1:2023-06) cannot detect bacteria in the VBNC state, even though these cells remain metabolically active and pathogenic due to the expression of toxin-encoding genes. This study aimed to develop a detection method using viable quantitative PCR (vqPCR) to identify viable cells, including those in VBNC state. In parallel, a relatively rapid protocol for inducing the VBNC state to generate VBNC cell controls was established. The established vqPCR assays included a preliminary step to inhibit dead bacterial cells using a proprietary DNA intercalating dye (Reagent D) in combination with the detection of long gene fragments of groEL (510 bp) for V. parahaemolyticus and ompW (588 bp) for V. cholerae using previously published primers. These assays demonstrated a high sensitivity, detecting as low as 20 fg DNA = 3.5 V. parahaemolyticus cells and 30 fg DNA = 6.9 V. cholerae cells. An induction of Vibrio VBNC cells of ≈ 6.5 Log10 cells/ml was successfully achieved within one hour from an initial 7.3 Log10 viable Vibrio cells/ml by treating the cells with a solution containing 0.5 or 1.0% Lutensol A03 and 0.2 M ammonium carbonate. The results showed that the established vqPCR methods were able to detect V. parahaemolyticus and V. cholerae in up to 50% (2.6 to 4.2 Log10 cells/g) and 56% (2.8 to 5.2 Log10 cells/g) of retail samples, respectively, that were initially false-negative in culture-based tests. The use of vqPCR assays along with culture-based tests can significantly enhance the seafood safety assessment by enabling the detection of VBNC cells of the most important foodborne Vibrio pathogens. In addition, the induction assay can be used for a rapid production of VBNC cells to standardize and validate such detection methods.