Diverse Enterococcus faecalis strains show heterogeneity in biofilm properties.

Biofilm formation is important for Enterococcus faecalis to cause healthcare-associated infections. It is unclear how E. faecalis biofilms vary in parameters such as development and composition. To test the hypothesis that differences in biofilms exist among E. faecalis strains, we evaluated in vitro biofilm formation and matrix characteristics of five genetically diverse E. faecalis lab-adapted strains and clinical isolates (OG1RF, V583, DS16, MMH594, and VA1128). Biofilm formation of all strains was repressed in TSB+10% FBS. However, DMEM+10% FBS enhanced biofilm formation of clinical isolate VA1128. Crystal violet staining and fluorescence microscopy of biofilms grown on Aclar membranes demonstrated differences between OG1RF and VA1128 in biofilm development over a 48-h time course. None of the biofilms were dispersed by single treatments of sodium (meta)periodate, DNase, or Proteinase K alone, but the biofilm biomass of both OG1RF and DS16 was partially removed by a sequential treatment of sodium (meta)periodate and DNase. Reversing the treatment order was not effective, suggesting that the extracellular DNA targeted by DNase was obscured by carbohydrates that are susceptible to sodium (meta)periodate degradation. Fluorescent staining of biofilm matrix components further demonstrated that more carbohydrates bound by wheat germ agglutinin comprise OG1RF biofilms compared to VA1128 biofilms. This study highlights the existence of heterogeneity in biofilm properties among diverse E. faecalis strains, which may have implications for the design of novel anti-biofilm treatment strategies.

Disruption of the tagF Orthologue in the epa Locus Variable Region of Enterococcus faecalis Causes Cell Surface Changes and Suppresses an eep-Dependent Lysozyme Resistance Phenotype.

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.

The Cationic Antimicrobial Peptide Activity of Lysozyme Reduces Viable Enterococcus faecalis Cells in Biofilms.

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. Planktonically grown E. faecalis lacking eep is more sensitive than wild-type strains to growth inhibition by lysozyme. This study was initiated to determine whether E. faecalis OG1RFΔeep biofilms would be protected from lysozyme. Serendipitously, we discovered that exposure of both E. faecalis OG1RF and OG1RFΔeep biofilms to chicken egg white lysozyme resulted in decreases in biofilm cell viability of 3.7 and 3.8 log10 CFU/mL, respectively. Treatment of biofilms of both strains with recombinant purified human lysozyme was associated with reductions in cell viability of >99.9% for both strains. Lysozyme-treated OG1RF and OG1RFΔeep biofilms contained a higher percentage of dead cells by Live/Dead staining and were associated with more extracellular DNA. Heat-inactivated human lysozyme, which was devoid of muramidase activity, as well as the lysozyme-derived CAMP LP9 and the CAMP polymyxin B, decreased biofilm cell viability. These results are consistent with a model in which the CAMP activity, rather than the muramidase activity, of lysozyme causes lysis of E. faecalis biofilm cells despite them having an intact lysozyme resistance-inducing signaling pathway. Finally, lysozyme was also effective in reducing viable biofilm cells of several other E. faecalis strains, including the vancomycin-resistant strain V583 and multidrug-resistant strain MMH594. This study demonstrates the potential for lysozyme to be developed as a novel antibiofilm therapeutic.