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Background: Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Results: Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Conclusions: Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic. [ABSTRACT FROM AUTHOR]

Background: Human genetic factors such as blood group antigens may affect the severity of infectious diseases. Presence of specific ABO and Lewis blood group antigens has been shown previously to be associated with the risk of different enteric infections. The aim of this study was to determine the relationship of the Lewis blood group antigens with susceptibility to cholera, as well as severity of disease and immune responses to infection. Methodology: We determined Lewis and ABO blood groups of a cohort of patients infected by Vibrio cholerae O1, their household contacts, and healthy controls, and analyzed the risk of symptomatic infection, severity of disease if infected and immune response following infection. Principal Findings: We found that more individuals with cholera expressed the Le(a+b-) phenotype than the asymptomatic household contacts (OR 1.91, 95% CI 1.03-3.56) or healthy controls (OR 1.90, 95% CI 1.13-3.21), as has been seen previously for the risk of symptomatic ETEC infection. Le(a-b+) individuals were less susceptible to cholera and if infected, required less intravenous fluid replacement in hospital, suggesting that this blood group may be associated with protection against V. cholerae O1. Individuals with Le(a-b-) blood group phenotype who had symptomatic cholera had a longer duration of diarrhea and required higher volumes of intravenous fluid replacement. In addition, individuals with Le(a-b-) phenotype also had lessened plasma IgA responses to V. cholerae O1 lipopolysaccharide on day 7 after infection compared to individuals in the other two Lewis blood group phenotypes. Conclusion: Individuals with Lewis blood type Le(a+b-) are more susceptible and Le(a-b+) are less susceptible to V. cholerae O1 associated symptomatic disease. Presence of this histo-blood group antigen may be included in evaluating the risk for cholera in a population, as well as in vaccine efficacy studies, as is currently being done for the ABO blood group antigens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

At the International Centre for Diarrhoeal Disease Research. Bangladesh, one-half of the rice-water stool samples that were culture-positive for Vibrio cholerae did not contain motile V. cholerae by standard darkfield microscopy and were defined as darkfield-negative (DF-). We evaluated the host and microbial factors associated with DF status, as well as the impact of DF status on transmission. Viable counts of V. cholerae in DF stools were three logs lower than in DF- stools, although DF and DF- stools had similar direct counts of V. cholerae by microscopy. In DF- samples, non-V. cholerae bacteria outnumbered V. cholerae 10:1. Lytic V. cholerae bacteriophage were present in 90% of DF- samples compared with 35% of DF- samples, suggesting that bacterio-phage may limit culture-positive patients from producing DF- stools. V. cholerae in DF and DF- samples were found both planktonically and in distinct nonplanktonic populations; the distribution of organisms between these compartments did not differ appreciably between DF- and DF4 stools. This biology may impact transmission because epidemiological data suggested that house-hold contacts of a DF- index case were at greater risk of infection with V. cholerae. We propose a model in which V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, viable counts of V. cholerae drop, stools become DF, other microorganisms bloom, and cholera transmission is reduced. [ABSTRACT FROM AUTHOR]