Abstract
Vibrio cholerae, the etiological agent of cholera, is ubiquitous in environmental brackish waters. Exposure to low water temperatures induces the bacterium to enter a viable but non-culturable (VBNC) state. In this study, a stepwise decrease in water temperature to 4°C was found to delay the transition to the non-culturable state compared to an abrupt temperature drop, suggesting that V. cholerae cells partially adapt to low temperatures. V. cholerae VBNC cells maintained at 4°C gradually lost their ability to revert to a culturable state. However, VBNC cells in the early stage of dormancy were efficiently resuscitated following treatment with proteolytic enzymes, including proteinase K. The abundance of culturable V. cholerae cells in brackish estuarine waters was quantified using the most probable number (MPN)-quantitative polymerase chain reaction (qPCR) method. Although culturable cells were undetectable in samples treated with bovine serum albumin, they were estimated at 93 and 1,500 MPN/mL in two water samples collected on different days and pre-incubated with proteinase K. Similarly, the abundance of Vibrio species increased markedly following treatment with this enzyme. Additionally, cells of Vibrio species were enumerated by the plating method using CHROMagar Vibrio plates. Consistent with the results of the MPN-qPCR method, treatment with proteinase K resulted in over a 100-fold increase in colony formation. Collectively, these findings suggest that treatment with proteinase K is effective for resuscitating and quantifying V. cholerae VBNC cells in environmental water samples. IMPORTANCE: V. cholerae 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 V. cholerae 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 V. cholerae density using the most probable number (MPN)-quantitative polymerase chain reaction (qPCR) method. While V. cholerae 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.