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

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.

Keywords: Factor converting VBNC into culturable; VBNC Vibrio cholerae.

Figure 1

Map (from Google Maps) showing the sampling points in Kolkata, India. (▪), Points A and B; (▴), Point C; (♦), Point D.

Figure 2

Reduction in the number of culturable cells of Vibrio cholerae in a VBNC microcosm buffer. Approximately 1 × 10⁸ cells mL⁻¹ of V. cholerae O1 (N16961) and V. cholerae O139 (VC-280) in VBNC microcosm buffer were incubated at 4°C in the dark without shaking. The number of culturable cells in each VBNC microcosm buffer was determined in duplicate. Representative results of three experiments are presented. (•), V. cholerae O1 (N16961); (▴), V. cholerae O139 (VC-280).

Figure 3

Heat stability of FCVC from HT-29 cells. (A) Aliquots (0.1 mL) of FCVC from HT-29 cells were treated at various temperatures for 5 min and the treated samples were subjected to 10-fold serial dilution. An aliquot (0.05 mL) of each diluted sample was added to 0.3 mL of 1.5-fold-condensed APW, to which 0.1 mL of VBNC Vibrio cholerae O139 (VC-280) had been added. After incubation at 37°C for 16 h, turbidity was observed by the naked eye and recorded. When the alkaline peptone water (APW) became turbid, it was plated on NA plates and randomly selected colonies were confirmed to be V. cholerae O139 by serotyping. Each experiment was repeated three times. (B) Aliquots (0.1 mL) of FCVC from HT-29 cells were incubated at 37°C for the indicated time periods and the incubated samples were subjected to 10-fold serial dilution. Further experimental procedures were as described in (A).

Figure 4

Colony formation of VBNC Vibrio cholerae O139 on F-TCBS plates. Aliquots (0.1 mL) of VBNC V. cholerae O139 (VC-280) were plated on F-TCBS (A) and TCBS (B) plates, and incubated at 37°C for 16 h.

Figure 5

Monthly isolation of VBNC Vibrio cholerae from environmental water samples in slum areas of Kolkata. The sums of the numbers of ompW-positiveV. cholerae (•) and ompW-positive, ctx-positive V. cholerae O1 (▪) observed during each month were plotted.