Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Abstract

Vibrio cholerae persists in aquatic environments predominantly in a nonculturable state. In this study coccoid, nonculturable V. cholerae O1 in biofilms maintained for 495 days in Mathbaria, Bangladesh, pond water became culturable upon animal passage. Culturability, biofilm formation, and the wbe, ctxA, and rstR2 genes were monitored by culture, direct fluorescent antibody (DFA), and multiplex PCR. DFA counts were not possible after formation of biofilm. Furthermore, wbe, but not ctxA, were amplifiable, even after incubation for 54 and 68 days at room temperature ( approximately 25 degrees C) and 4 degrees C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4 degrees C, compared with approximately 25 degrees C, suggesting biofilm production to be temperature dependent and linked to loss of culturability. Small colonies appearing after incubation in microcosms for 54 and 68 days at 25 degrees C and 4 degrees C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTXphi. The coccoid V. cholerae O1 observed as free cells in microcosms incubated for 495 days could not be cultured, but biofilms in the same microcosms yielded culturable cells. It is concluded that biofilms can act as a reservoir for V. cholerae O1 between epidemics because of its long-term viability in biofilms. In contrast to biofilms produced in Mathbaria pond water, V. cholerae O1 in biofilms present in cholera stools and incubated under identical conditions as the Mathbaria pond water biofilms could not be cultured after 2 months, indicating that those V. cholerae cells freshly discharged into the environment are significantly less robust than cells adapted to environmental conditions.

Fig. 1.

Micrographs showing V. cholerae O1 in MW microcosms at different stages of incubation at room temperature (RT) (MW-RT). Cells were collected from microcosms, and smears were prepared on clean glass slides, air-dried, stained with crystal violet, washed, and visualized by using a phase-contrast microscope (model BX2; Olympus, Tokyo, Japan). Cells at day 1 were observed to be arranged in microcolonies (A), with larger aggregations in dark blue, an indication of biofilm, at day 7 (B). Cells appeared as multiple layers, tightly attached to thick biofilm surrounding the aggregations of cells at day 27 (C) and as aggregates of thick biofilm intensifying with time at day 45 (D).

Fig. 2.

Epifluorescence micrographs of nonculturable V. cholerae O1 in microcosms MW-RT (A and B) and MW-4C (C and D). Samples were stained with DFA reagent specific for V. cholerae O1 (New Horizon Diagnostics) and visualized with an epifluorescence microscope (Olympus, model BX51, BX2 Series) (4). Microscopic images were captured digitally (Olympus, DP 20) and processed by using Photoshop Version 5 (Adobe Systems, San Jose, CA). (A and B) Coccoid V. cholerae O1 cells attached to a thin biofilm at 450 days and 495 days after inoculation into MW-RT, respectively. (C and D) Coccoid V. cholerae O1 cells in biofilm 450 days and 495 days after inoculation into MW-4C, respectively.

Fig. 3.

Epifluorescence micrographs of nonculturable V. cholerae O1 in MW-4C microcosms. Samples were pretreated with 0.025% yeast extract and 0.002% nalidixic acid, stained by using cholera DFA (New Horizon Diagnostics), visualized under an epifluorescence microscope (Olympus, model BX51) (1, 4, 17). Images were captured digitally (Olympus DP 20) and processed by using Photoshop Version 5. (A and B) Elongation of V. cholerae O1 in a biofilm that had been incubated for 450 days after initial inoculation. (C and D) Elongation of coccoid V. cholerae O1 cells in a biofilm 495 days after inoculation into MW-4C.