A Mishra1, N Taneja, M Sharma. 1. Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Abstract
AIM: To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation. METHODS AND RESULTS: Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4°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.
AIM: To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation. METHODS AND RESULTS:Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4°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.