BackgroundDiarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.ResultsWe use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.ConclusionsOur findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

An epidemic of cholera infections was documented in Haiti for the first time in more than 100 years during October 2010. Cases have continued to occur, raising the question of whether the microorganism has established environmental reservoirs in Haiti. We monitored 14 environmental sites near the towns of Gressier and Leogane during April 2012–March 2013. Toxigenic Vibrio cholerae O1 El Tor biotype strains were isolated from 3 (1.7%) of 179 water samples; nontoxigenic O1 V. cholerae was isolated from an additional 3 samples. All samples containing V. cholerae O1 also contained non-O1 V. cholerae. V. cholerae O1 was isolated only when water temperatures were ≥31°C. Our data substantiate the presence of toxigenic V. cholerae O1 in the aquatic environment in Haiti. These isolations may reflect establishment of long-term environmental reservoirs in Haiti, which may complicate eradication of cholera from this coastal country.

The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized thatVibrio choleraeis also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmentalV. choleraecontributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenicV. choleraeO1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenicV. choleraeO1 isolates and show robust evidence of the establishment of aquatic reservoirs as well as ongoing evolution ofV. choleraeisolates from aquatic sites. Novel environmental lineages emerged from sequential population bottlenecks, carrying mutations potentially involved in adaptation to the aquatic ecosystem. Based on such empirical data, we developed a mixed-transmission dynamic model ofV. cholerae, where aquatic reservoirs actively contribute to genetic diversification and epidemic emergence, which underscores the complexity of transmission pathways in epidemics and endemic settings and the need for long-term investments in cholera control at both human and environmental levels.

Since the identification of the first cholera case in 2010, the disease has spread in epidemic form throughout the island nation of Haiti; as of 2014, about 700,000 cholera cases have been reported, with over 8,000 deaths. While case numbers have declined, the more fundamental question of whether the causative bacterium, Vibrio cholerae has established an environmental reservoir in the surface waters of Haiti remains to be elucidated. In a previous study conducted between April 2012 and March 2013, we reported the isolation of toxigenic V. cholerae O1 from surface waters in the Ouest Department. After a second year of surveillance (April 2013 to March 2014) using identical methodology, we observed a more than five-fold increase in the number of water samples containing culturable V. cholerae O1 compared to the previous year (1.7% vs 8.6%), with double the number of sites having at least one positive sample (58% vs 20%). Both seasonal water temperatures and precipitation were significantly related to the frequency of isolation. Our data suggest that toxigenic V. cholerae O1 are becoming more common in surface waters in Haiti; while the basis for this increase is uncertain, our findings raise concerns that environmental reservoirs are being established.

Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of the phylogeny showed a steady accumulation of synonymous substitutions and a progressive increase of nonsynonymous substitutions over time, suggesting diversification likely was driven by positive selection. Short-term accumulation of nonsynonymous substitutions driven by selection may have significant implications for virulence, transmission dynamics, and even vaccine efficacy.