Stepwise changes in viable but nonculturable Vibrio cholerae cells

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC‐state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT‐29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co‐cultivation with HT‐29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co‐cultivation with HT‐29. These characteristic changes in VBNC‐state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.