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...

Cholera is a devastating disease, the epidemics of which, until 1992, were caused by Vibrio cholerae serogroup O1 biotype classical or El Tor. The classical biotype is believed to have caused the first six pandemics, which occurred in the Indian subcontinent and subsequently in other areas of the world between 1817 and 1923 (9, 27). V. cholerae O1 biotype El Tor was first reported in 1905 (30). However, it was not until the early 1960s that V. cholerae biotype El Tor displaced the sixth-pandemic V. cholerae O1 classical biotype (11,32). The emergence in 1992 of a V. cholerae non-O1 serovar, designated V. cholerae synonym O139 Bengal, in Bangladesh (2, 4) and India (28) and its subsequent appearance in Southeast Asia, displacing V. cholerae O1 El Tor, was considered a significant point in the history of cholera (33). V. cholerae O1 El Tor reemerged in 1994 to 1995, but V. cholerae O139 continues to coexist with V. cholerae O1 as indicated by its temporal quiescence and subsequent reemergence in 1997, 1999, and 2002 (11, 15). Of all of these outbreaks, the resurgence of V. cholerae O139 in a major outbreak, resulting in an estimated 30,000 cases in Dhaka, Bangladesh, caused more cases than the number attributed to V. cholerae O1 El Tor within a very short time (11,30). Since then, V. cholerae serogroup O139 has continued to cause a small number of cases of cholera in the subcentral parts of Bangladesh and southern Bangladesh (34). Despite its significance as a causal agent of cholera, little is known about the geographic distribution of V. cholerae O139 in the coastal areas of the Bay of Bengal.Despite being autochthonous to the aquatic environment (6, 7), toxigenic strains of V. cholerae O1 are only infrequently isolated from surface waters by culture methods (7,21) and are rarely isolated during interepidemic periods (18). It was when fluorescent-antibody (FA) and molecular-based detection methods were used that the presence of V. cholerae O1 in the environment was unequivocally demonstrated (3, 18) and its viable but not culturable state was discovered (5,7,18,29). V. cholerae O139 has been shown to behave similarly to V. cholerae O1, since detection and isolation of V. cholerae O139 from water samples were negative by culture methods between epidemics (19, 21).The correlation of sea surface temperature and sea surface height in the Bay of Bengal with the occurrence of cholera in Bangladesh has been established (8). Field studies in Bakerganj, which is located 70 km north of the Bay of Bengal coast, showed correlation of selected environmental parameters with the ecology and epidemiology of V. cholerae and cholera, respectively (19). In 1992, V. cholerae serogroup O139 was first isolated in Bangladesh in the vicinity of the Bay of Bengal. With the resurgence of V. cholerae O139 in 2002, the number of cholera cases caused by this serogroup surpassed the number caused by V. cholerae O1 in Bangladesh. This phenomenon is believed to have been the result of rapid genetic changes in

Antibiotic resistance in avian bacterial pathogens is a common problem in the Bangladesh poultry industry. The aim of the present study was to provide information on the present status of antibiotic resistance patterns in avian pathogenic Escherichia coli in Bangladesh. Of 279 dead or sick poultry of different ages, 101 pathogenic E coli strains isolated from broilers and layer hens with colibacillosis infections were screened to determine phenotypic expression of antimicrobial resistance against 13 antibiotics used in both veterinary and human medicine in Bangladesh. Of 101 pathogenic E. coli isolates, more than 55% were resistant to at least one or more of the tested compounds, and 36.6% of the isolates showed multiple-drug-resistant phenotypes. The most common resistances observed were against tetracycline (45.5%), trimethoprim-sulphamethoxazole (26.7%), nalidixic acid (25.7%), ampicillin (25.7%), and streptomycin (20.8%). Resistance to ciprofloxacin (12.9%), chlormaphenicol (8.9%), nitrofurantoin (2%), and gentamicin (2%) was also observed, and none of the isolates were resistant to tigecycline as well as extended spectrum beta-lactamase (ESBL) producers. One isolate was resistant to cefuroxime (1%), cefadroxil (1%), and mecillinam (1%) but was not an ESBL producer. Resistance rates, although significant in Bangladeshi isolates, were found to be lower than those reported for avian isolates from the Republic of Korea and clinical, avian, and environmental isolates from Bangladesh. The high level of antibiotic resistance in avian pathogens from Bangladesh is worrisome and indicates that widespread use of antibiotics as feed additives for growth promotion and disease prevention could have negative implications for human and animal health and the environment.

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