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...

The development of the oral biofilm requires a complex series of interactions between host tissues and the colonizing bacteria as well as numerous interspecies interactions between the organisms themselves. Disruption of normal host–microbe homoeostasis in the oral cavity can lead to a dysbiotic microbial community that contributes to caries or periodontal disease. A variety of approaches have been pursued to develop novel potential therapeutics that are active against the oral biofilm and/or target specific oral bacteria. The structure and function of naturally occurring antimicrobial peptides from oral tissues and secretions as well as external sources such as frog skin secretions have been exploited to develop numerous peptide mimetics and small molecule peptidomimetics that show improved antimicrobial activity, increased stability and other desirable characteristics relative to the parent peptides. In addition, a rational and minimalist approach has been developed to design small artificial peptides with amphipathic α‐helical properties that exhibit potent antibacterial activity. Furthermore, with an increased understanding of the molecular mechanisms of beneficial and/or antagonistic interspecies interactions that contribute to the formation of the oral biofilm, new potential targets for therapeutic intervention have been identified and both peptide‐based and small molecule mimetics have been developed that target these key components. Many of these mimetics have shown promising results in in vitro and pre‐clinical testing and the initial clinical evaluation of several novel compounds has demonstrated their utility in humans.

Porphyromonas gingivalis is associated with chronic periodontitis and may initially colonize the oral cavity by adhering to streptococci. Adhesion to streptococci is driven by interaction of the minor fimbrial antigen (Mfa1) with streptococcal antigen I/II. We identified the region of antigen I/II required for this interaction and developed small molecule mimetics that inhibited P. gingivalis adherence. However, the functional motifs of Mfa1 involved in the interaction with antigen I/II remain uncharacterized. A series of N‐ and C‐terminal peptide fragments of Mfa1 were expressed and tested for inhibition of P. gingivalis adherence to S. gordonii. This approach identified residues 225–400 of Mfa1 as essential for P. gingivalis adherence. Using the three‐dimensional structure of Mfa1, a putative binding cleft was identified using SiteMap and five small molecule mimetics could dock in this site. Site‐specific mutation of residues in the predicted cleft, including R240A, W275A, D321A and A357P inhibited the interaction of Mfa1 with streptococci, whereas mutation of residues not in the predicted cleft (V238A, I252F and ΔK253) had no effect. Complementation of an Mfa1‐deficient P. gingivalis strain with wild‐type mfa1 restored adherence to streptococci, whereas complementation with full‐length mfa1 containing the R240A or A357P mutations did not restore adherence. The mutations did not affect polymerization of Mfa1, suggesting that the complemented strains produced intact minor fimbriae. These results identified specific residues and structural motifs required for the Mfa1‐antigen I/II interaction and will facilitate the design of small molecule therapeutics to prevent P. gingivalis colonization of the oral cavity.

Enterococcus faecalis , a leading cause of health care-associated infections, forms biofilms and is resistant to many antimicrobial agents. Planktonic-phase E. faecalis is resistant to high concentrations of the enzyme lysozyme, which catalyzes the hydrolysis of N -acetylmuramic acid and N -acetylglucosamine linkages in peptidoglycan and is also a cationic antimicrobial peptide (CAMP). E. faecalis lysozyme resistance in planktonic cells is stimulated upon activation of the extracytoplasmic function sigma factor SigV via cleavage of the anti-sigma factor RsiV by the transmembrane protease Eep.