Efficient resuscitation of early-stage viable but non-culturable cells of <i>Vibrio cholerae</i> using treatment with proteolytic enzymes

<i>V. cholerae</i> enters into a viable but non-culturable (VBNC) state when exposed to low water temperatures. Contamination of food and drinking water with VBNC cells poses a critical public health risk, as these cells retain their virulence but cannot be detected by conventional methods. In this study, we demonstrated that VBNC cells of <i>V. cholerae</i> could be efficiently resuscitated by treatment with proteolytic enzymes such as proteinase K, enabling their detection through standard culture-based assays. Environmental brackish water samples were analyzed for <i>V. cholerae</i> density using the most probable number (MPN)-quantitative polymerase chain reaction (qPCR) method. While <i>V. cholerae</i> was not detected in untreated samples, proteinase K treatment revealed densities of 93 or 1,500 MPN/mL. Therefore, the combination of proteinase K treatment with the MPN-qPCR method offers a promising approach for detecting VBNC bacterial contamination in food, drinking water, and environmental water.