Environmental prevalence of toxigenic Vibrio cholerae O1 in Bangladesh coincides with V. cholerae non-O1 non-O139 genetic variants which overproduce autoinducer-2.

Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways

Quorum Regulated Resistance of Vibrio cholerae against Environmental Bacteriophages.

Predation by bacteriophages can significantly influence the population structure of bacterial communities. Vibrio cholerae the causative agent of cholera epidemics interacts with numerous phages in the aquatic ecosystem, and in the intestine of cholera patients. Seasonal epidemics of cholera reportedly collapse due to predation of the pathogen by phages. However, it is not clear how sufficient number of the bacteria survive to seed the environment in the subsequent epidemic season. We found that bacterial cell density-dependent gene expression termed "quorum sensing" which is regulated by signal molecules called autoinducers (AIs) can protect V. cholerae against predatory phages. V. cholerae mutant strains carrying inactivated AI synthase genes were significantly more susceptible to multiple phages compared to the parent bacteria. Likewise when mixed cultures of phage and bacteria were supplemented with exogenous autoinducers CAI-1 or AI-2 produced by recombinant strains carrying cloned AI synthase genes, increased survival of V. cholerae and a decrease in phage titer was observed. Mutational analyses suggested that the observed effects of autoinducers are mediated in part through the quorum sensing-dependent production of haemaglutinin protease, and partly through downregulation of phage receptors. These results have implication in developing strategies for phage mediated control of cholera

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh.

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera

Whole Genome PCR Scanning Reveals the Syntenic Genome Structure of Toxigenic Vibrio cholerae Strains in the O1/O139 Population

Vibrio cholerae is commonly found in estuarine water systems. Toxigenic O1 and O139 V. cholerae strains have caused cholera epidemics and pandemics, whereas the nontoxigenic strains within these serogroups only occasionally lead to disease. To understand the differences in the genome and clonality between the toxigenic and nontoxigenic strains of V. cholerae serogroups O1 and O139, we employed a whole genome PCR scanning (WGPScanning) method, an rrn operon-mediated fragment rearrangement analysis and comparative genomic hybridization (CGH) to analyze the genome structure of different strains. WGPScanning in conjunction with CGH revealed that the genomic contents of the toxigenic strains were conservative, except for a few indels located mainly in mobile elements. Minor nucleotide variation in orthologous genes appeared to be the major difference between the toxigenic strains. rrn operon-mediated rearrangements were infrequent in El Tor toxigenic strains tested using I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis and PCR analysis based on flanking sequence of rrn operons. Using these methods, we found that the genomic structures of toxigenic El Tor and O139 strains were syntenic. The nontoxigenic strains exhibited more extensive sequence variations, but toxin coregulated pilus positive (TCP+) strains had a similar structure. TCP+ nontoxigenic strains could be subdivided into multiple lineages according to the TCP type, suggesting the existence of complex intermediates in the evolution of toxigenic strains. The data indicate that toxigenic O1 El Tor and O139 strains were derived from a single lineage of intermediates from complex clones in the environment. The nontoxigenic strains with non-El Tor type TCP may yet evolve into new epidemic clones after attaining toxigenic attributes

Detection of Vibrio cholerae and Acanthamoeba species from same natural water samples collected from different cholera endemic areas in Sudan

<p>Abstract</p> <p>Background</p> <p><it>Vibrio cholerae </it>O1 and <it>V. cholerae </it>O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. <it>V. cholerae </it>and the free-living amoebae <it>Acanthamoeba </it>species are present in aquatic environments, including drinking water and it has shown that <it>Acanthamoebae </it>support bacterial growth and survival. Recently it has shown that <it>Acanthamoeba </it>species enhanced growth and survival of <it>V. cholerae </it>O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both <it>V. cholerae </it>and <it>Acanthamoeba </it>species from same natural water samples by polymerase chain reaction (PCR).</p> <p>Findings</p> <p>For the first time both <it>V. cholerae </it>and <it>Acanthamoeba </it>species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected <it>V. cholerae </it>was found with <it>Acanthamoeba </it>in same water samples.</p> <p>Conclusions</p> <p>The current findings disclose <it>Acanthamoedae </it>as a biological factor enhancing survival of <it>V. cholerae </it>in nature.</p

Inapparent infections and cholera dynamics

In many infectious diseases, an unknown fraction of infections produce symptoms mild enough to go unrecorded, a fact that can seriously compromise the interpretation of epidemiological records. This is true for cholera, a pandemic bacterial disease, where estimates of the ratio of asymptomatic to symptomatic infections have ranged from 3 to 100 (refs 1-5). In the absence of direct evidence, understanding of fundamental aspects of cholera transmission, immunology and control has been based on assumptions about this ratio and about the immunological consequences of inapparent infections. Here we show that a model incorporating high asymptomatic ratio and rapidly waning immunity, with infection both from human and environmental sources, explains 50 yr of mortality data from 26 districts of Bengal, the pathogen's endemic home. We find that the asymptomatic ratio in cholera is far higher than had been previously supposed and that the immunity derived from mild infections wanes much more rapidly than earlier analyses have indicated. We find, too, that the environmental reservoir(5,6) (free-living pathogen) is directly responsible for relatively few infections but that it may be critical to the disease's endemicity. Our results demonstrate that inapparent infections can hold the key to interpreting the patterns of disease outbreaks. New statistical methods(7), which allow rigorous maximum likelihood inference based on dynamical models incorporating multiple sources and outcomes of infection, seasonality, process noise, hidden variables and measurement error, make it possible to test more precise hypotheses and obtain unexpected results. Our experience suggests that the confrontation of time-series data with mechanistic models is likely to revise our understanding of the ecology of many infectious diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62519/1/nature07084.pd

Transmission of Vibrio cholerae Is Antagonized by Lytic Phage and Entry into the Aquatic Environment

Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water—the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment