In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecularweight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P < 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P < 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.Enterococci are a leading cause of nosocomial infections in the United States and account for 5 to 15% of cases of infective endocarditis, with most isolates being Enterococcus faecalis (9). Better understanding of the pathogenicity of enterococci may help to develop more effective therapies or preventative modalities for E. faecalis infections.Polysaccharides on bacterial surfaces may interact with the human host and play important roles in bacterial pathogenesis. There is evidence indicating that polysaccharides are involved in hindering leukocyte killing of enterococci. Arduino and colleagues (1, 12) reported that exposure of Enterococcus faecium TX0016 (also called TEX16 as well as DO), a strain resistant to phagocytosis and killing by leukocytes in the presence of normal human sera (NHS); for partial sequence, see http:// www.hgsc.bcm.tmc.edu/microbial/efaecium/), to sodium periodate, but not to trypsin, pronase, or phospholipase C, eliminated its resistance to phagocytosis. Recently, members of our group showed that rabbit antiserum against formalin-killed E. faecium TX0016 promoted opsonization and killing, and this effect was dramatically reduced by adsorption of the antiserum with carbohydrate purified from TX0016, but not by incubation with ...

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of l-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalisinside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

The enterococcal surface protein Esp, specifically linked to nosocomial Enterococcus faecium, is involved in biofilm formation. To assess the role of Esp in endocarditis, a biofilm-associated infection, an Esp-expressing E. faecium strain (E1162) or its Esp-deficient mutant (E1162Δesp) were inoculated through a catheter into the left ventricle of rats. After 24 hours, less E1162Δesp than E1162 were recovered from heart valve vegetations. In addition, anti-Esp antibodies were detected in Esp-positive E. faecium bacteremia and endocarditis patient sera. In conclusion, Esp contributes to colonization of E. faecium at the heart valves. Furthermore, systemic infection elicits an Esp-specific antibody response in humans.

During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%o) of 26 strains of E. faecium tested were susceptible to phagocytosis (.7 organisms per PMN); the other 13 strains showed c3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each ofEnterococcus ralfinosus and Enterococcus solitarius were readily phagocytosed. Exposure ofE. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydratecontaining moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to