Cholera epidemics have long been known to spread through water contaminated with human fecal material containing the toxigenic bacterium Vibrio cholerae. However, detection of V. cholerae in water is complicated by the existence of a dormant state in which the organism remains viable, but resists cultivation on routine bacteriological media. Growth in the mammalian intestine has been reported to trigger "resuscitation" of such dormant cells, and these studies have prompted the search for resuscitation factors. Although some positive reports have emerged from these investigations, the precise molecular signals that activate dormant V. cholerae have remained elusive. Quorum-sensing autoinducers are small molecules that ordinarily regulate bacterial gene expression in response to cell density or interspecies bacterial interactions. We have found that isolation of pathogenic clones of V. cholerae from surface waters in Bangladesh is dramatically improved by using enrichment media containing autoinducers either expressed from cloned synthase genes or prepared by chemical synthesis. These results may contribute to averting future disasters by providing a strategy for early detection of V. cholerae in surface waters that have been contaminated with the stools of cholera patients or asymptomatic infected human carriers.biofilm formation | CVEC | transmissibility T he natural habitats of the species Vibrio cholerae are estuarine or fresh water aquatic environments (1-3). In cholera endemic areas such as Bangladesh, viable V. cholerae can be readily detected in water during seasonal cholera epidemics; however, as disease incidence decreases, the isolation of viable V. cholerae becomes dramatically more difficult perhaps in part due to the influence of lytic bacteriophages (4, 5). However, during the interepidemic period, the organism can also occasionally be found in a viable but dormant state, which has been alternately referred to as the viable but nonculturable (VBNC) cells (1), conditionally viable environmental cells (CVEC) (6), or active but nonculturable (ABNC) (7). Recently we have documented the existence of CVEC in surface waters of Bangladesh by using fluorescent antibody based microscopy, which revealed clumps of V. cholerae O1 cells in water, which were often negative for V. cholerae O1 by conventional culture. To detect possible presence of relatively small number of culturable cells in such water we also used enrichment and selection approaches that depend on antibiotic resistance profiles displayed by the strains that have caused the preceding cholera epidemic. This approach referred to as antibiotic selection technique (AST) (8) allowed enhanced detection of V. cholerae by suppressing growth of other environmental bacteria that would otherwise mask the small number of V. cholerae colonies.In our previous studies, CVEC were found to be organized as aggregates of cells embedded in extracellular material, presumably Vibrio extracellular polysaccharide (VPS) (6). The genes responsible for VPS production are c...

Viruses are the most abundant microorganisms in the aquatic environment, yet the identification of viruses and assessing their diversity still remains a challenge. Here, we present a robust, routinely usable approach to identify viruses from two freshwater lakes of the lower Great Lakes region, Lake Ontario, and Lake Erie. We collected water samples from six different beaches of these two lakes during the summer period of 2012 and 2013, and separated into three distinct fractions, namely a bacterial fraction, a virus like particle (VLP) fraction, and a fraction of eDNA (environmental DNA). DNA extracted from all three fractions was sequenced and bioinformatic analyses of sequences revealed the presence of viruses from major viral families. The analyzed viral sequences were dominated by bacteriophage sequences, but also contained many plant and animal viruses. Within the context of this study, geographic location does not appear to have a major impact on viral abundance and diversity, since virome composition of both lakes were similar. Comparative analyses between eDNA and viral fractions showed that eDNA can be used in combination with VLP fractions to identify viruses from the environment.

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Helicobacter pylori is a prevalent bacterium that can cause gastric ulcers and cancers. Lactic acid bacteria (LAB) ameliorate treatment outcomes against H. pylori, suggesting that they could be a source of bioactive molecules usable as alternatives to current antibiotics for which resistance is mounting. We developed an in vitro framework to compare the anti-H. pylori properties of 25 LAB and their secretions against H. pylori. All studies were done at acidic and neutralized pH, with or without urea to mimic various gastric compartments. Eighteen LAB strains secreted molecules that curtailed the growth of H. pylori and the activity was urea-resistant in five LAB. Several LAB supernatants also reduced the urease activity of H. pylori. Pre-treatment of H. pylori with acidic LAB supernatants abrogated its flagella-mediated motility and decreased its ability to elicit pro-inflammatory IL-8 cytokine from human gastric cells, without reverting the H. pylori-induced repression of other pro-inflammatory cytokines. This study identified the LAB that have the most anti-H. pylori effects, decreasing its viability, its production of virulence factors, its motility and/or its ability to elicit pro-inflammatory IL-8 from gastric cells. Once identified, these molecules can be used as alternatives or complements to current antibiotics to fight H. pylori infections.

Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has been under the longest active boil water advisory in Canada due to FIB contamination in groundwater wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet's groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources. This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.

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