Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm

Aim: To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4 degrees C) and starvation. Methods and Results: Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4 degrees C. The cells became coccoid, rugose and subsequently nonculturable by day 16 on tryptic soy agar (TSA) and by day 23 on TSA-SP, while 87 and 65% of the cells retained their membrane integrity, respectively. Viable cells were observed until day 30 using direct fluorescent antibody-direct viable count method. In vitro resuscitation was demonstrated by temperature upshift. Utilizing 16S rRNA as an endogenous control, the DNA pol II (27.43-fold), fliG (12.44-fold), ABC transporter (27.11-fold), relA (60.76-fold) and flaC (15.29-fold) were significantly up-regulated in VBNC cells, while the expression of fadL-3 was comparable. The expression of DNA pol II, fliG, ABC transporter, relA and flaC was 3.3, 1.1, 5.9, 5.8 and 1.2-fold, respectively, for resuscitated cells. VBNC cells were found to be virulent, as ctxA and tcpA were expressed. Conclusions: Vibrio cholerae undergoes both phenotypic alteration and genotypic modulation to protect itself from stress in freshwater. Significance and Impact of the Study: Induction and resuscitation of the VBNC state in freshwater is important for an understanding of the epidemiology of cholera in the freshwater environment.