Enterococci are members of the healthy human intestinal flora, but are also leading causes of highly antibiotic-resistant, hospital-acquired infection. We examined the genomes of a strain of Enterococcus faecalis that caused an infectious outbreak in a hospital ward in the mid-1980s (ref. 2), and a strain that was identified as the first vancomycin-resistant isolate in the United States, and found that virulence determinants were clustered on a large pathogenicity island, a genetic element previously unknown in this genus. The pathogenicity island, which varies only subtly between strains, is approximately 150 kilobases in size, has a lower G + C content than the rest of the genome, and is flanked by terminal repeats. Here we show that subtle variations within the structure of the pathogenicity island enable strains harbouring the element to modulate virulence, and that these variations occur at high frequency. Moreover, the enterococcal pathogenicity island, in addition to coding for most known auxiliary traits that enhance virulence of the organism, includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates, identifying a class of new targets associated with disease which are not essential for the commensal behaviour of the organism.

Enterococcus faecalis bacteria isolated from patients with bacteremia, endocarditis, and urinary tract infections more frequently express the surface protein Esp than do fecal isolates. To assess the role of Esp in colonization and persistence of E. faecalis in an animal model of ascending urinary tract infection, we compared an Esp ؉ strain of E. faecalis to its isogenic Esp-deficient mutant. Groups of CBA/J mice were challenged transurethrally with 10 8 CFU of either the parent or mutant strain, and bacteria in the urine, bladder, and kidneys were enumerated 5 days postinfection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the parent strain than from the bladder and urine of mice challenged with the mutant. Colonization of the kidney, however, was not significantly different between the parent and mutant strains. Histopathological evaluations of kidney and bladder tissue done at 5 days postinfection did not show marked histopathological changes consistent with inflammation, mucosal hyperplasia, or apoptosis, and there was no observable difference between the mice challenged with the parent and those challenged with the mutant. We conclude that, while Esp does not influence histopathological changes associated with acute urinary tract infections, it contributes to colonization and persistence of E. faecalis at this site.

Enterococci play a dual role in human ecology. They serve as commensal organisms of the gastrointestinal tract and are also leading causes of multiple antibiotic-resistant hospital-acquired infection. Many nosocomial infections result from the ability of microorganisms to form biofilms. The molecular mechanisms involved in enterococcal biofilm formation are only now beginning to be understood. Enterococcal surface protein, Esp, has been reported to contribute to biofilm formation by Enterococcus faecalis. Recent studies have shown that enterococci form biofilms independently of Esp expression. To precisely determine what role Esp plays in E. faecalis biofilm formation, Esp was expressed on the cell surface of genetically well-defined, natively Espdeficient strains, and isogenic Esp-positive and Esp-deficient strains were compared for their biofilm-forming ability. The results show that Esp expression leads to a significant increase in biofilm formation, irrespective of the strain tested. The contribution of Esp to biofilm formation was found to be most pronounced in the presence of 0.5% (wt/vol) or greater glucose. These results unambiguously define Esp as a key contributor to the ability of E. faecalis to form biofilms.

Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N-and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G؉C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.Enterococci are common inhabitants of the gastrointestinal tracts of humans and animals, and although they have been recognized as pathogens able to cause endocarditis, they were generally considered second-rate pathogens. Recent estimates, however, indicate that enterococci are now among the leading causes of nosocomial infections (57). Of all enterococcal species, Enterococcus faecalis accounted for the most infections in humans (26). However, during the past decade, the incidence of bloodstream infections caused by Enterococcus faecium increased, an increase which has been linked to the emergence of antibiotic resistance in this species (26,40).Little is known about virulence determinants in E. faecium (20). Recently, however, three potential virulence genes, esp, hyl, and acm, have been described for E. faecium. They were all found more frequently in clinical isolates than in fecal isolates or nonhuman isolates (13,41,44,65).Of these three putative virulence genes, only the esp gene is also found in E. faecalis (51). The Esp protein in E. faecalis is expressed as a large surface-exposed protein with a molecular mass of approximately 202 kDa. In E. faecalis, Esp is thought to be an adhesin contributing to colonization of urinary tract epithelial cells and biofilm formation (50, 59). Although detailed experimental evidence is not yet available, the higher prevalence of the E. faecium esp gene in clinical isolates suggests a role of Esp in the pathogenesis of E. faeci...

Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.