Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state.

Mitsutoshi Senoh,Shin-Ichi Miyoshi,Jayeeta Ghosh-Banerjee,Tamaki Mizuno,Yoshifumi Takeda,Takashi Hamabata,Sumio Shinoda,G Balakrish Nair

doi:10.1002/mbo3.164

Abstract

Previously, we reported that viable but nonculturable (VBNC) Vibrio cholerae was converted into a culturable state by coculture with several eukaryotic cell lines including HT-29 cells. In this study, we found that a factor converting VBNC V. cholerae into a culturable state (FCVC) existed in cell extracts of eukaryotic cells. FCVC was nondialyzable, proteinase K-sensitive, and stable to heating at <60°C for 5 min. We prepared thiosulfate citrate bile salts sucrose (TCBS) plates with FCVC (F-TCBS plates). After confirming that VBNC V. cholerae O1 and O139 formed typical yellow colonies on F-TCBS plates, we tried to isolate cholera toxin gene-positive VBNC V. cholerae from environmental water samples collected in urban slum areas of Kolkata, India and succeeded in isolating V. cholerae O1 El Tor variant strains harboring a gene for the cholera toxin. The possible importance of VBNC V. cholerae O1 as a source of cholera outbreaks is discussed.

Highlights

Cholera caused by Vibrio cholerae O1 and O139 continues to be a major cause of severe diarrhea, especially in developing countries (World Health Organization 2012)
Conversion of viable but nonculturable (VBNC) bacteria to a culturable state has been reported under several conditions, such as temperature upshift (Wai et al 1996; Gupte et al 2003), incubation in phosphate buffer (Dukan et al 1997), supplementation with catalase or sodium pyruvate (Mizunoe et al 2000), addition of heat-stable autoinducer of growth (Reissbrodt et al 2002), addition of resuscitation-promoting factor (Panutdaporn et al 2006), and presence of Acanthamoeba castellannii (Steinert et al 1997)
We reported the conversion of VBNC V. cholera and other enteric bacteria into a culturable state by coculture with several eukaryotic cell lines including HT-29 (Senoh et al, 2010; Senoh et al 2012)

Summary

Introduction

Cholera caused by Vibrio cholerae O1 and O139 continues to be a major cause of severe diarrhea, especially in developing countries (World Health Organization 2012). In cholera endemic areas like Kolkata, India (Nair et al 2010) where clean drinking water is not available, large numbers of people use environmental water from lakes, ponds, and rivers as a 2014 The Authors. It has been suggested that the cholera endemic continues because environmental water is contaminated by V. cholerae O1 and O139. The frequencies of isolating V. cholerae O1 and O139 from environmental water samples in cholera endemic areas are quite low even in epidemic seasons (Huq et al 1990; Islam et al 1994) and the numbers of the organisms, if isolated, are not sufficient to explain the cause of the disease (Levine et al 1988). Between epidemic seasons V. cholerae O1 and O139 are very rarely isolated (Roszak and Colwell 1987)

Methods

Results

Conclusion