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

The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.Cholera is a harsh disease, the fundamental clinical feature of which is severe dehydrating diarrhea that can lead to rapidly progressing dehydration and death. The recent cholera epidemics that occurred in South America (7), Asia (8), and sub-Saharan Africa (18) affected millions of people, with a high mortality rate. The World Health Organization (WHO) annual figures on global cholera incidence (26), which are based on official cases reported by affected countries, are believed to be underestimated due to limitations related to a lack of adequate surveillance systems. In addition, the actual number of cholera cases globally is estimated to be much higher than officially reported (22) because outbreaks are often not reported to avoid the risk of travel and trade embargoes on the affected country.Prompt and accurate diagnosis of Vibrio cholerae is a key step in cholera outbreak surveillance that can greatly influence rapid intervention and prevention to minimize disease spread and mortality. Conventional culture methods (CMs) currently used for diagnosis of V. cholerae remain the gold standard, but this procedure is not precise and requires highly skilled technicians and laboratory infrastructure. In remote settings where cholera is endemic and modern laboratory facilities are often nonexistent, simple dark-field microscopy to detect cells showing characteristic darting motility is used to identify V. cholerae in stool specimens. Diagnostic tests known as cholera dipstick (DS) assays, which involve either cholera toxin (3) or lipopolysaccharide...

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Yearwise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 %

BackgroundAlthough endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal.MethodsV. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE).ResultsAll V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498–511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.ConclusionsIn 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.