Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Abstract

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.

FIG. 1.

Map of Bangladesh showing areas where environmental samples were collected. Mathbaria and Matlab are two of the important foci of cholera endemicity in this region.

FIG. 2.

Epifluorescent micrographs of V. cholerae O1 and O139 detected in environmental samples collected between March and December 2004. Fluorescent monoclonal antibodies (DFA) specific for V. cholerae serovar O1 or O139, obtained from New Horizon Diagnostic, were used, and the stains were visualized (18, 19) using an epifluorescence microscope (Olympus model BX51, BX2 series). Microscopic images were captured digitally (Olympus DP20) and processed by using Adobe Photoshop (version 5). (A and C) Typical single cells of V. cholerae O1 and O139, respectively; (B and D) cell clusters of V. cholerae O1 in water samples collected from Mathbaria and Matlab, respectively, during July 2004.

FIG. 3.

Epifluorescent micrographs of V. cholerae O1 detected in environmental samples collected in September and November 2004. Samples were stained by using DFA reagents specific for V. cholerae serovar O1, obtained from New Horizon Diagnostic, and visualized (18, 19) by using an epifluorescence microscope (Olympus model BX51, BX2 series). Microscopic images were captured digitally (Olympus DP20) and processed by using Adobe Photoshop version 5. (A and B) Different stages of coccoid V. cholerae O1 cells attached to thin film (biofilm) on surfaces, either free-floating (A) or attached to plankton or other aggregates (B) in samples collected in September from pond sites 1 and 3, respectively; (C and D) short rods of V. cholerae O1 cells in in situ biofilms in samples collected in November from ponds at sites 1 and 3, respectively.

FIG. 4.

Epifluorescent micrographs of V. cholerae O1 detected in environmental samples collected in December 2004. Samples were stained with direct fluorescent monoclonal antibody (DFA) specific for V. cholerae serovar O1 (obtained from New Horizon Diagnostic) and visualized (18, 19) by using an epifluorescence microscope (Olympus model BX51, BX2 series). Microscopic images were captured digitally (Olympus DP20) and processed by using Adobe Photoshop version 5. (A and B) V. cholerae O1 in the initial stage of in situ biofilm formation, either free-swimming (A) or attached to plankton or other aggregates (B) in samples collected from pond site 1; (C and D) V. cholerae O1 in the initial stage of in situ biofilm formation in a sample collected from ponds at sites 5 and 6, respectively.