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...

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.Toxigenic Vibrio cholerae O1 and O139 are causative agents of cholera (38), an acute dehydrating diarrhea, which occurs in epidemic (13, 31) and pandemic (23) forms. Since the first pandemic was recorded in 1817, as many as seven cholera pandemics have occurred (35). The most recent, the seventh pandemic, began in Indonesia (14), but cholera pandemics have usually begun in the Gangetic delta of the Indian subcontinent and then in other continents (10,40). Of the 206 O serogroups of V. cholerae, serovar O1 was the only recognized cause of cholera until late 1992. At that time an outbreak of acute watery diarrhea clinically resembling cholera erupted in India and southern Bangladesh (1, 10). The bacterium causing cholera-like diarrhea failed to agglutinate with any of the then existing 138 V. cholerae O antisera (1) and was thus designated O139 with the synonym "Bengal" to commemorate its emergence in the coast of the Bay of Bengal. Since then, O1 and O139 remain the two recognized serogroups causing epidemics of cholera.Epidemiological studies of V. cholerae O139, including its emergence, prevalence, and coexistence with O1 El Tor V. cholerae, have been conducted primarily in Bangladesh and India via systematic surveillance (19). In the Ganges delta region, cholera outbreaks occur seasonally (13,14), but variations in prevalence of the two epidemic serogroups O1 and O139 of V. cholerae are distinct ...

Bacteriophage predation selects for diverse antiphage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature is lacking, particularly in clinical contexts where there is need to inform phage therapy efforts and to understand how phages drive pathogen evolution. Using time-shift experiments, we uncovered fluctuations in Vibrio cholerae’s resistance to phages in clinical samples. We mapped phage resistance determinants to SXT integrative and conjugative elements (ICEs), which notoriously also confer antibiotic resistance. We found that SXT ICEs, which are widespread in γ-proteobacteria, invariably encode phage defense systems localized to a single hotspot of genetic exchange. We identified mechanisms that allow phage to counter SXT-mediated defense in clinical samples, and document the selection of a novel phage-encoded defense inhibitor. Phage infection stimulates high-frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistances.

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...

Forty-two episodes of Vibrio parahaemolyticus infections were detected in Beira, Mozambique, from January to May 2004. The majority of the isolates (81%) belonged to the pandemic serovars (O3:K6 and O4:K68) of V. parahaemolyticus. The pandemic serovars were positive by group-specific PCR (GS-PCR) and a PCR specific for open reading frame ORF8 (ORF8-PCR), which are molecular markers of the pandemic clone, and were positive for tdh but negative for trh. The remaining 19% of the strains also possessed the tdh gene but were GS-PCR and ORF8-PCR negative and did not belong to the pandemic serovars. Patients with V. parahaemolyticus infection were older (mean age, 27 years) than patients infected by other diarrheal agents (mean age, 21 years). Ten percent of diarrhea patients from whom no V. parahaemolyticus was cultured were severely dehydrated, but none of the V. parahaemolyticus cases were severely dehydrated. This is the first report of the isolation of pandemic strains of V. parahaemolyticus in sub-Saharan Africa and clearly indicates that the pandemic of V. parahaemolyticus has spread into the African continent.

Little is known about the physical and socioeconomic postpartum consequences of women who experience obstetric complications and require emergency obstetric care (EmOC), particularly in resource-poor countries such as Bangladesh where historically there has been a strong cultural preference for births at home. Recent increases in the use of skilled birth attendants show socioeconomic disparities in access to emergency obstetric services, highlighting the need to examine birthing preparation and perceptions of EmOC, including caesarean sections. Twenty women who delivered at a hospital and were identified by physicians as having severe obstetric complications during delivery or immediately thereafter were selected to participate in this qualitative study. Purposive sampling was used for selecting the women. The study was carried out in Matlab, Bangladesh, during March 2008–August 2009. Data-collection methods included in-depth interviews with women and, whenever possible, their family members. The results showed that the women were poorly informed before delivery about pregnancy-related complications and medical indications for emergency care. Barriers to care-seeking at emergency obstetric facilities and acceptance of lifesaving care were related to apprehensions about the physical consequences and social stigma, resulting from hospital procedures and financial concerns. The respondents held many misconceptions about caesarean sections and distrust regarding the reason for recommending the procedure by the healthcare providers. Women who had caesarean sections incurred high costs that led to economic burdens on family members, and the blame was attributed to the woman. The postpartum health consequences reported by the women were generally left untreated. The data underscore the importance of educating women and their families about pregnancy-related complications and preparing families for the possibility of caesarean section. At the same time, the health systems need to be strengthened to ensure that all women in clinical need of lifesaving obstetric surgery access quality EmOC services rapidly and, once in a facility, can obtain a caesarean section promptly, if needed. While greater access to surgical interventions may be lifesaving, policy-makers need to institute mechanisms to discourage the over-medicalization of childbirth in a context where the use of caesarean section is rapidly rising.

Vibrio cholerae serogroup O1 is responsible for epidemic and pandemic cholera and remains a global public health threat. This organism has been well established as a resident flora of the aquatic environment that alters its phenotypic and genotypic attributes for better adaptation to the environment. To reveal the diversity of clinical isolates of V. cholerae O1 in the Bay of Bengal, we performed whole-genome sequencing of isolates from Kolkata, India, and Dhaka, Bangladesh, collected between 2009 and 2016. Comparison with global isolates by phylogenetic analysis placed the current isolates in two Asian lineages, with lineages 1 and 2 predominant in Dhaka and Kolkata, respectively. Each lineage possessed different genetic traits in the cholera toxin B subunit gene, Vibrio seventh pandemic island II, integrative and conjugative element, and antibiotic-resistant genes. Thus, although recent global transmission of V. cholerae O1 from South Asia has been attributed only to isolates of lineage 2, another distinct lineage exists in Bengal. IMPORTANCE Cholera continues to be a global concern, as large epidemics have occurred recently in Haiti, Yemen, and countries of sub-Saharan Africa. A single lineage of Vibrio cholerae O1 has been considered to be introduced into these regions from South Asia and to cause the spread of cholera. Using genomic epidemiology, we showed that two distinct lineages exist in Bengal, one of which is linked to the global lineage. The other lineage was found only in Iran, Iraq, and countries in Asia and differed from the global lineage regarding cholera toxin variant and drug resistance profile. Therefore, the potential transmission of this lineage to other regions would likely cause worldwide cholera spread and may result in this lineage replacing the current global lineage.

Cholera outbreaks occur each year in the remote coastal areas of Bangladesh and epidemiological surveillance and routine monitoring of cholera in these areas is challenging. In this study, a total of 97 Vibrio cholerae O1 isolates from Mathbaria, Bangladesh, collected during 2010 and 2014 were analyzed for phenotypic and genotypic traits, including antimicrobial susceptibility. Of the 97 isolates, 95 possessed CTX-phage mediated genes, ctxA, ace, and zot, and two lacked the cholera toxin gene, ctxA. Also both CTX+ and CTX− V. cholerae O1 isolated in this study carried rtxC, tcpAET, and hlyA. The classical cholera toxin gene, ctxB1, was detected in 87 isolates, while eight had ctxB7. Of 95 CTX+ V. cholerae O1, 90 contained rstRET and 5 had rstRCL. All isolates, except two, contained SXT related integrase intSXT. Resistance to penicillin, streptomycin, nalidixic acid, sulfamethoxazole-trimethoprim, erythromycin, and tetracycline varied between the years of study period. Most importantly, 93% of the V. cholerae O1 were multidrug resistant. Six different resistance profiles were observed, with resistance to streptomycin, nalidixic acid, tetracycline, and sulfamethoxazole-trimethoprim predominant every year. Ciprofloxacin and azithromycin MIC were 0.003–0.75 and 0.19–2.00 μg/ml, respectively, indicating reduced susceptibility to these antibiotics. Sixteen of the V. cholerae O1 isolates showed higher MIC for azithromycin (≥0.5 μg/ml) and were further examined for 10 macrolide resistance genes, erm(A), erm(B), erm(C), ere(A), ere(B), mph(A), mph(B), mph(D), mef(A), and msr(A) with none testing positive for the macrolide resistance genes.