Phage predation of Vibrio cholerae has recently been reported to be a factor that influences seasonal epidemics of cholera in Bangladesh.To understand more about this phenomenon, we studied the dynamics of the V. cholerae-phage interaction during a recent epidemic in Dhaka. Because the outbreak strain causing this epidemic was resistant to multiple antibiotics, including streptomycin, we used a selective medium containing streptomycin to monitor accurately the abundance of this strain in the environment. The changing prevalence in the environment of the epidemic V. cholerae O1 strain and a particular lytic cholera phage (JSF4) to which it was sensitive was measured every 48 -72 h for 17 weeks. We also monitored the incidence of phage excretion in stools of 387 cholera patients during the epidemic. The peak of the epidemic was preceded by high V. cholerae prevalence in the environment and was followed by high JSF4 phage levels as the epidemic ended. The buildup to the phage peak in the environment coincided with increasing excretion of the same phage in the stools of cholera patients. These results suggest that patients toward the end of the epidemic ingested both JSF4 phage and the outbreak V. cholerae strain. Host-mediated phage amplification during the cholera epidemic likely contributed to increased environmental phage abundance, decreased load of environmental V. cholerae and, hence, the collapse of the epidemic. Thus, in vivo phage amplification in patients and subsequent phage predation in the environment may explain the self-limiting nature of seasonal cholera epidemics in Bangladesh.Vibrio cholerae ͉ vibriophage T oxigenic strains of Vibrio cholerae belonging to the O1 and O139 serogroups cause cholera, a devastating diarrhea disease that occurs frequently as epidemics in many developing countries (1). Epidemics of cholera occur regularly in the Ganges Delta region of Bangladesh and India. In Bangladesh, outbreaks usually occur twice during a year, with the highest number of cases just after the monsoon and a somewhat smaller number of cholera cases during the spring. The occurrence of epidemics are known to coincide with increased prevalence of the causative V. cholerae strain in the aquatic environment (2). A variety of physical and biological parameters are likely to influence the survival and abundance of V. cholerae as a species in the environment, but these factors do not exclusively modulate the prevalence of toxigenic V. cholerae O1 and O139 strains. In contrast, bacterial viruses (phage or bacteriophage) in the environment have recently been found to inversely correlate with the abundance of toxigenic V. cholerae in water samples and the incidence rates of cholera (3). These data strongly suggest that phage predation in the environment likely influences the temporal dynamics of cholera epidemics. Phages also play a role in the emergence of pathogenic clones and may also be involved in territorialism between different strains of V. cholerae (3-5). For example, cholera toxin genes are transferred ...

The factors that enhance the waterborne spread of bacterial epidemics and sustain the epidemic strain in nature are unclear. Although the epidemic diarrheal disease cholera is known to be transmitted by water contaminated with pathogenic Vibrio cholerae, routine isolation of pathogenic strains from aquatic environments is challenging. Here, we show that conditionally viable environmental cells (CVEC) of pathogenic V. cholerae that resist cultivation by conventional techniques exist in surface water as aggregates (biofilms) of partially dormant cells. Such CVEC can be recovered as fully virulent bacteria by inoculating the water into rabbit intestines. Furthermore, when V. cholerae shed in stools of cholera patients are inoculated in environmental water samples in the laboratory, the cells exhibit characteristics similar to CVEC, suggesting that CVEC are the infectious form of V. cholerae in water and that CVEC in nature may have been derived from human cholera stools. We also observed that stools from cholera patients contain a heterogenous mixture of biofilm-like aggregates and free-swimming planktonic cells of V. cholerae. Estimation of the relative infectivity of these different forms of V. cholerae cells suggested that the enhanced infectivity of V. cholerae shed in human stools is largely due to the presence of clumps of cells that disperse in vivo, providing a high dose of the pathogen. The results of this study support a model of cholera transmission in which in vivo-formed biofilms contribute to enhanced infectivity and environmental persistence of pathogenic V. cholerae. cholera epidemic ͉ Vibrio cholerae ͉ conditionally viable environmental cells ͉ waterborne disease T oxigenic Vibrio cholerae of the O1 or O139 serogroup are the causative agents of cholera and belong to a group of organisms whose major habitat is aquatic ecosystems (1-3). Epidemics of cholera occur frequently in many developing countries of Asia, Africa, and Latin America (4, 5), and these occurrences coincide with increased prevalence of the causative V. cholerae strain in the aquatic environment (6). However, the concentration of toxigenic V. cholerae usually detected in surface water by standard culture techniques is far less than that required to induce infection and cause clinical disease under experimental conditions in volunteers challenged with V. cholerae (7,8). This discrepancy between the required infectious dose and the apparent concentration of V. cholerae in surface water fostering an epidemic has not been adequately explained.Various hypotheses have been proposed to explain the mode of persistence of pathogenic V. cholerae in the aquatic environment and to suggest that the true prevalence of the pathogen in natural water may be considerably higher than that observed by standard culture methods. For example, it has been proposed that V. cholerae can exist in the aquatic environment in a viable but nonculturable state, which by definition is a condition in which cells are incapable of forming a colony on commonly used medi...

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.Vibrio cholerae O1 has two biotypes, namely, classical and El Tor, which are believed to have evolved from separate lineages (7,8), and these biotypes have traditionally been differentiated by a number of phenotypic traits. Comparative genomic analyses have recently revealed a high degree of conservation among diverse strains of V. cholerae but have also shown genes that differentiate the classical biotype from the El Tor biotype (3). Apart from these phenotypic and genetic differences, there are also dissimilarities in the infection patterns of disease caused by the two biotypes. These include the occurrence of more asymptomatic than symptomatic carriers of El Tor strains, who outnumber active patients by a ratio of up to 50:1 (14), better survival of El Tor strains in the environment and in the human host, and more efficient host-to-host transmission of El Tor strains than of classical strains (5). There is firm evidence that the fifth and sixth pandemics of cholera were caused by the classical biotype, while the ongoing seventh pandemic is caused by the El Tor biotype, which has now globally replaced the classical biotype.Cholera toxin (CT), the principal toxin produced by V. cholerae O1 and O139, is responsible for most of the manifestations of the disease cholera. Based on the B subunit of CT, two immunologically related but not identical epitypes have been described: CT1 is the prototype elaborated by classical biotype strains and by U.S. Gulf Coast strains, while CT2 is produced by the El Tor biotype and O139 strains (4). Another classification identifies three types of ctxB genes based on three nonrandom base changes resulting in changes in the deduced amino acid sequence. Genotype 1 is found in strains of the classical biotype worldwide and in U.S. Gulf Coast strains, genotype 2 is found in El Tor biotype strains from Australia, and genotype 3 is found in El Tor biotype strains from the seventh pandemic and the Latin American epidemic (12). Thus, the V. cholerae O1 El Tor biotype of the ongoing seventh pandemic produces CT of the CT2 epitype and genotype 3, while the classical biotype CT belongs to the CT1 epitype and genotype 1. In this study, we examined a collection of clinical V. cholerae O1 strains isolated in Bangladesh during the past four and a half decades, using monoclonal antibodies (MAbs) produced to classical and El Tor CTs, and found that V. cholerae O1 El Tor strains isolated since 2001 in Bangladesh produce the CT subtype of the classical biotype.One hundred eighty-five strains of V. cholerae O1, consisting of 31 strains of the classical biotype isolated between 1960 and 1990 and 113 strains of the El Tor biotype and 41 hybrid strains of V. cholerae O1 (strains that could ...

Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.