Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.

A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.Although classically considered a commensal of the gastrointestinal tracts of humans and animals rather than a specialized human pathogen, enterococci have become extremely relevant in hospital-acquired infections. Their ability to acquire specific genetic traits, such as virulence and antibiotic resistance determinants that could increase their fitness in such a complex ecosystem, has been recognized (18). The paradigm of this evolutionary development is the emergence and spread of vancomycin-resistant enterococci (VRE) (20).Among enterococcal species, Enterococcus faecalis is responsible for most human infections in both community and hospital settings. Though resistance to vancomycin and penicillins is very rare, E. faecalis seems to harbor a broader repertoire of potential virulence traits than E. faecium (34). However, little is known about the relationship between the population structure and global epidemiology of E. faecalis. Different molecular typing methods have been developed to analyze E. faecalis epidemiology (3,11,19,36,37,40). Pulsed-field gel electrophoresis (PFGE) is considered a practical "gold standard" due to its high discriminatory abilities (3, 37), but the most important limitation of PFGE is its low interlaboratory reproducibility and its unsuitability for both global and long-term epidemiology studies or for phylogenetic or population structure studies.For many different bacterial species, the most appropriate technique for global and long-term epidemiology studies is multilocus sequence typing (MLST) (38). MLST provides an unambiguous nomenclature for genotypes, and clones and data are easily stored in databases that can be exchanged between different laboratories via the Internet (1). For E. faecium, the development of an MLST scheme has been critical in the understanding of global epidemiology, genetic evolution, and population structure (14,41). A previous ML...

Background: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ϳ105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF ace mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 g/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.