Biofilms Comprise a Component of the Annual Cycle of Vibrio cholerae in the Bay of Bengal Estuary

Abstract

Vibrio cholerae, an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study of V. cholerae occurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. Toxigenic V. cholerae exhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenic V. cholerae, were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detect V. cholerae O1 cells throughout the year, as free-living cells, within clusters, or in association with plankton. V. cholerae O1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. Toxigenic V. cholerae O1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenic V. cholerae in the estuarine environment of Bangladesh.IMPORTANCEVibrio cholerae, the causative agent of cholera, is autochthonous in the estuarine aquatic environment. This study describes morphological changes in naturally occurring V. cholerae O1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh. V. cholerae O1 cells are predominantly coccoid within biofilms but are rod shaped as free-living cells and when attached to plankton or to particulate matter in interepidemic periods of the year. It is concluded that biofilms represent a stage of the annual life cycle of V. cholerae O1, the causative agent of cholera in Bangladesh.

Keywords: Vibrio cholerae; biofilms; cholera.

FIG 1

Map of Bangladesh showing the six sampling sites (stars) located in Mathbaria, a coastal village near the Bay of Bengal.

FIG 2

Total direct (AODC), culturable bacterial (HPC), and culturable V. cholerae O1 (VCO1) counts of water samples collected from six pond sites (S1 to S6) of the estuarine environment of Bangladesh between December 2005 and November 2006.

FIG 3

Percentages of samples positive for V. cholerae O1 (VCO1) determined by DFA and culture in water samples collected from the estuarine environment of Bangladesh between December 2005 and November 2006.

FIG 4

Micrographs of acridine orange-stained microbial communities occurring in the natural estuarine aquatic ecosystem of the mangrove forest, Sundarbans, Bay of Bengal, Bangladesh, where cholera is endemic. The micrographs provide in situ evidence of free-living bacteria in a natural aquatic ecosystem assembling and transforming into smaller and coccoid cells in the spring and fall (B and D) and forming biofilm consortia during the winter and monsoon months of the year (A and C). Scale bars in red indicate 10 µm.

FIG 5

Direct fluorescent monoclonal antibody (DFA) detection of V. cholerae O1 in the estuarine aquatic ecosystem of the Bay of Bengal, Bangladesh, during different seasons of the year. (A to D) Micrographs show biofilms of V. cholerae O1 in water samples collected during winter and monsoon months (A and C) and free-living V. cholerae O1 cells in water samples collected in spring and fall months (B and D). (Scale bars in red indicate 10 µm.)

FIG 6

(A to F) Micrographs of acridine orange-stained microcolonies of the bacterial communities, representing stages in bacterial biofilm formation, including cell assembly (A and B) and secretion of exopolysaccharide into packages to form small consortia (C to F). Scale bars in red indicate 10 µm.

FIG 7

(A to F) Observation of bacterial communities forming biofilms in situ. Acridine orange-stained biofilm consortia occur free-floating in the aquatic environment. Scale bars in red indicate 10 µm.

FIG 8

(A to F) Bacterial colonization of zooplankton in biofilms analogous to those formed by free-floating bacteria in water samples. Bacterial populations reside within or on the chitinous structures of zooplankton to form chitin-associated biofilms. (D and E) Typical biofilm formation of bacteria in association with a rotifer. Scale bars in red indicate 10 µm.

FIG 9

Acridine orange-stained biofilm consortia consisting of bacteria producing biofilms on both living and nonliving free-floating particulate matter. Arrows indicate circular and semicircular cavities in the biofilms. Scale bars in red indicate 10 µm.