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...

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.

The spread of antibiotic resistance among bacterial pathogens is inciting a global public health crisis. Drug-resistant Staphylococcus species, especially S. aureus and S. epidermidis, have emerged in both hospital and community settings, underscoring the urgent need for new strategies to combat staphylococcal infections. Bacterial viruses (phages) and the enzymes that they use to degrade bacterial cell walls (lysins) show promise as alternative antimicrobials; however, only a limited variety of staphylococcal phages and their lysins have yet been identified. Here, we report the discovery and characterization of a novel staphylococcal phage, Andhra. We show that Andhra encodes two lysins (Andhra_gp10 and Andhra_gp14) that inhibit growth and degrade the cell walls of diverse staphylococci, including S. aureus and S. epidermidis strains. Andhra and its unique lysins add to the arsenal of antimicrobials with potential for therapeutic use.

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

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