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 (Ϸ25°C) and 4°C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4°C, compared with Ϸ25°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°C and 4°C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTX⌽. 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. Bangladesh ͉ bacteriophage CTX⌽ ͉ DFA ͉ multiplex-PCR ͉ ctxA C holera continues to pose a serious health threat globally, notably in those countries where clean drinking water is not available to local populations. Vibrio cholerae serogroups O1 and O139 are associated with epidemic and pandemic cholera. Cholera is endemic in the Ganges delta, occurring twice yearly in epidemic form (1). It is also a major health problem for countries of Africa, Latin America, and Asia (2). V. cholerae O1 is native to both marine and fresh water environments and exists in association with plankton (3). In general, it can be isolated from only 1% of water samples collected during epidemic periods and rarely, if ever, between epidemics (4). However, fluorescent antibody-based studies show that V. cholerae O1 is, nevertheless, present in aquatic environments throughout the year (5). Furthermore, the presence of nonculturable V. cholerae O1 is confirmed by molecular methods (6). The question of whether such nonculturable cells in aquatic environments are capable of returning to an actively growing state to initiate cholera epidemics has been debated.Extensive studies have shown that V. cholerae O1 becomes coccoid and enters into a nonculturable state in the environment when conditions are not conducive to active growth (5, 7). Some of...

Climate Variability and the Outbreaks of Cholera in Zanzibar, East Africa: A Time Series Analysis

“…5,8,12,14,[27][28][29][30] Warmer temperatures increase SST facilitating Vibrio reproduction and hence the probability of ingesting an infectious dose. 5,14 Our concurring findings are important in light of the current global warming phenomenon, Climatologists predict a 1.4°C/35°F to 5.8°C/42°F rise in mean temperature over the next 100 years.…”

Biofilms in water, its role and impact in human disease transmission

“…The number of cells in a biofilm may reach as high as 1.0 Â 10 9 cells per clump which, in most cases, can comprise an infectious dose of a pathogen. However, survivability of bacterial cells and retention of their potential to cause disease when present in a biofilm remains to be fully elucidated [4 ].…”

Origins of the current seventh cholera pandemic

“…The low levels of recombination that were observed during long-term survival of the organisms in the environment could be the result of cells of pathogenic V. cholerae strains existing primarily in a viable but noncultivable form (VBNC) when not in a host (48,50). In these experiments, organisms isolated from the stools of infected persons entered the VBNC state within 24 h of being placed in water.…”

“…Recombination in a seventh-pandemic strain has been observed under laboratory conditions when induced by the presence of chitin (48). Pandemic V. cholerae strains have also been associated with crustacean and other invertebrates, which could promote recombination, because their exoskeleton is mostly chitin.…”