Abstract
In October, 2010, epidemic cholera was reported for the first time in Haiti in over 100 years. Establishment of cholera endemicity in Haiti will be dependent in large part on the continued presence of toxigenic V. cholerae O1 in aquatic reservoirs. The rugose phenotype of V. cholerae, characterized by exopolysaccharide production that confers resistance to environmental stress, is a potential contributor to environmental persistence. Using a microbiologic medium promoting high-frequency conversion of smooth to rugose (S–R) phenotype, 80 (46.5%) of 172 V. cholerae strains isolated from clinical and environmental sources in Haiti were able to convert to a rugose phenotype. Toxigenic V. cholerae O1 strains isolated at the beginning of the epidemic (2010) were significantly less likely to shift to a rugose phenotype than clinical strains isolated in 2012/2013, or environmental strains. Frequency of rugose conversion was influenced by incubation temperature and time. Appearance of the biofilm produced by a Haitian clinical rugose strain (altered biotype El Tor HC16R) differed from that of a typical El Tor rugose strain (N16961R) by confocal microscopy. On whole-genome SNP analysis, there was no phylogenetic clustering of strains showing an ability to shift to a rugose phenotype. Our data confirm the ability of Haitian clinical (and environmental) strains to shift to a protective rugose phenotype, and suggest that factors such as temperature influence the frequency of transition to this phenotype.
Highlights
Cholera continues to be a major public health threat globally in countries where safe drinking water, adequate sanitation and hygiene are suboptimal [1]
We provide evidence that: (a) unlike typical V. cholerae O1 El Tor strains, the majority of the smooth phenotype of altered V. cholerae O1 strains isolated from Haiti switched to a rugose colony phenotype (S–R conversion) after at least 48 h of growth on L-agar following their growth in APW # 3 for 72 h; (b) in contrast to 2010 clinical O1 isolates, 2012 and 2013 clinical isolates from Haiti exhibited significantly increased smooth to rugose (S–R) conversion; (c) S–R conversion was greatly influenced by incubation temperatures with cultures incubated at 37uC having significantly enhanced rugose production compared to those incubated at 25uC, and (d) the Haitian V. cholerae rugose colony phenotype was influenced by incubation temperatures
A total of 172 V. cholerae O1 and non-O1/non-O139 strains from clinical and environmental sources in Haiti were examined to evaluate their ability to shift to a rugose colony phenotype (Table S1 and Table 1)
Summary
Cholera continues to be a major public health threat globally in countries where safe drinking water, adequate sanitation and hygiene are suboptimal [1]. Toxigenic V. cholerae strains within serogroups O1 and O139 are almost exclusively responsible for epidemic cholera; non-toxigenic strains outside of these O groups (non-O1/O139 V. cholerae) are widely distributed in the environment, but generally are not associated with human illness. The microorganism can survive either in planktonic (free-living) form or in biofilms [2,3]. Suggestions have been made that the bacteria survive between epidemics in these aquatic reservoirs, with environmental triggers causing seasonal increases in counts, followed by ‘‘spill-over’’ into human populations [1]. The genetic and physiologic basis of the persistence of V. cholerae in the environment, during inter-epidemic period, is poorly understood
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