Induction of viable but non-culturable state in vibrio cholerae O139 by temperature and its pathogenicity

Abstract

In this study, the effect of temperature on the formation of viable but non-culturable (VBNC) pathogenic Vibrio cholerae O139 was examined. Furthermore, both the virulence and cellular protein expression of the bacterial cells under culturable, non-culturable and resuscitated states were determined. Following the incubation of bacterial cells in river water microcosms (RWM) with 0.5% NaCl at 4°C for 35-45 days, plate counts declined to less than 1 CFU/mL; but viable counts dropped to 104-105 cells/mL during the first 2 weeks and maintained at this level till the end of incubation. However, the entry of cells into a VBNC state was not detected after the cells were cultured at either 15 or 25°C. When the 4°C -induced VBNC samples were either inoculated to a plate supplemented with H2O2-degrading compounds or shifted to 28°C, non-culturable O139 cells regained their culturability. The adhesion test showed that the number of O139 cells adhering to HeLa cells was highest in culturable cells and significantly lower in either resuscitated cells and non-culturable cells. As for the cytotoxicity test, all HeLa cells died after incubation with culturable O139 cells, whereas 32.1 ± 4.6% and 100% of target cells died after incubation with resuscitated cells for 3 and 5 hr, respectively. No difference was observed between the percentages of cell death after incubation with non-culturable cells and with sterile culture medium. When mice were challenged with culturable O139 cells, the mortality was 80%. However, all mice survived after challenge with either VBNC or resuscitated cells.