Pathogenicity of Enterococci

Pathogenicity of Enterococci

Lynn E. Hancock and Michael S. Gilmore*
Department of Microbiology and Immunology
University of Oklahoma Health Sciences Center
Oklahoma City, OK 73104
Phone: 405-271-1083
Fax: 405-271-8128
Email: michael-gilmore@ouhsc.edu

INTRODUCTION

Enterococcal pathogenicity was initially addressed at the end of the 19th century by MacCallum and Hastings (46), who isolated an organism from a case of acute endocarditis, and designated it Micrococcus zymogenes based on its fermentative properties. The organism was shown to be resistant to dessication, heating to 60°C, and several antiseptics, including carbolic acid and chloroform (46). It was also found to be lethal when injected intraperitoneally in white mice, and capable of producing endocarditis in a canine model (46). A century later, enterococci are prominent among nosocomial pathogens, ranking second only to E. coli in total nosocomial infections, accounting for more than 12% of all cases (56).

Infections caused by the genus Enterococcus (most notably Enterococcus faecalis, which accounts for ~80% of all infections) include urinary tract infections, bacteremia, intra-abdominal infections, and endocarditis (33, 53). The problem of nosocomial enterococcal infection is compounded by multiple antibiotic resistance. A comparison of outcomes for patients with bacteremia due to vancomycin-resistant Enterococcus faecium or vancomycin-susceptible E. faecium found a median length of stay of 46 days after the first episode of bacteremia in the group of patients with vancomycin-resistant E. faecium, as compared to 19 days for patients infected by a susceptible strain (43). The presence of VRE (vancomycin-resistant enterococci) in the bloodstream has also been associated with increased mortality (14). Patients with enterococcal bacteremia were observed to be twice as likely to die (37% vs. 16%) when the infecting isolate was resistant to vancomycin (14). However, more recent studies indicate that VRE-status is not a major predictor for clinical outcome (45, 48). Whether more recent findings describe cases that benefit from further evolution in the treatment of VRE requires further analysis. As vancomycin frequently represents the last available therapeutic for multiple antibiotic resistant enterococci, the rapid increase in vancomycin resistance (33) indicates that enterococcal infection will pose an increasing therapeutic challenge.

Resistance trends among E. faecalis and E. faecium were recently reviewed (33). Data compiled for approximately 15,000 isolates over a 3 year period (1995-1997) showed that while resistance to ampicillin and vancomycin is relatively uncommon among E. faecalis isolates (<2%),>

Biology and Epidemiology

The classification of enterococci as group D streptococci dates back to the scheme established by Rebecca Lancefield in the early 1930's (42). In 1984, enterococci were given formal genus status after DNA-DNA and DNA-RNA hybridization studies demonstrated a more distant relationship with the streptococci (64).

Enterococci are generally considered commensals of the gastrointestinal tract of a variety of organisms including man, and are morphologically indistinguishable from other streptococci. They are found in a number of environments, probably because of dissemination in animal excrement and environmental persistence. Several intrinsic features of Enterococcus may allow members of this genus to survive for extended periods of time, leading to its persistence and nosocomial spread. E. faecalis is able to grow in 6.5% NaCl, at temperatures ranging from 10-45°C, and can survive 30 minutes at 60°C. Additionally, the organism grows in the presence of 40% bile salts and over a broad pH range (53). The earliest descriptions of the organism noted that it was "hardy and tenacious of life" (46).
Several studies have examined the nature of the environmental ruggedness of the organism (16, 18). E. faecalis were observed to adapt to the presence of lethal levels of bile salts and detergents, such as sodium dodecyl sulfate (SDS), when first cultured at sub-lethal levels for as little as five seconds (16). The ability of enterococci to adapt and persist in the presence of detergents may allow them to survive inadequate cleaning regimens, contributing to their persistence in the hospital. Numerous epidemiologic studies have shown that enterococci can be transmitted from person to person in the hospital (8, 38). This transmission typically occurs via the hands of healthcare workers or on clinical instruments, such as ear-probe thermometers (61).

Antibiotic Resistance

The intrinsic ruggedness of enterococci also confers an unusual level of tolerance to several classes of antibiotics including aminoglycosides, beta-lactams and quinolones. For example, the resistance of enterococci to aminoglycosides results from the ability of enterococci to block the uptake of the drug at the cell wall (29, 51). Consequently, aminoglycosides are only effective against enterococci when used in combination with cell wall active antibiotics. This combination treatment modality has been compromised, however, by the rapid spread of high-level aminoglycoside resistance among enterococci (>2000 µg/ml) (30). Although the mechanism of high-level resistance was determined to be the result of a bifunctional enzyme (15), the molecular basis for the intrinsic resistance of enterococci to low-levels of aminoglycosides remains to be determined. Further discussion of the genetic and physiologic basis for antibiotic resistance can be found elsewhere in this volume.

PATHOGENIC MECHANISMS

In order to infect, enterococci must first be able to colonize, primarily at mucosal surfaces. From the site of colonization, the organism must then evade the host clearance, and ultimately produce pathologic changes in the host, either through direct toxic activity, or indirectly by inducing an inflammatory response (40).

Colonization and Translocation

Enterococci normally colonize the gastrointestinal tract of man. They are found in relative abundance in human feces (105-107 organisms per gram) (57). A close association is likely to exist between enterococci and its host, or the organism would be eliminated due to normal intestinal motility (38). Studies in progress are examining the specific binding of enterococci to intestinal epithelium (63). Many infection-derived enterococcal isolates were found to be clonal, indicating nosocomial transmission. Moreover, a number of studies have documented patient colonization following hospital admission, and have shown that colonization with multiply resistant strains is a predisposing factor for subsequent infection (33, 55). To colonize the lower bowel, enterococci must survive transit through the low pH of the stomach. Several studies have examined the acid tolerance of E. faecalis (17, 71). Flahaut et al. (17) demonstrated that exposure of E. faecalis to a sub-lethal pH (pH 4.8) for 15-30 minutes protected the organism from a normally lethal challenge at pH 3.2. Suzuki and colleagues (71) have shown that an E. faecalis mutant defective in F1 -F0 H+-ATPase activity was unable to grow at pH<6. The H+-ATPase is used to regulate the cytoplasmic pH of E. faecalis by proton extrusion. This enzyme has been shown to be activated at low pH (71). From these studies, it is apparent that enterococci possess the ability to withstand the low gastric pH, which would facilitate colonization. This attribute may be critical in the ability of multi-drug resistant enterococcal strains to colonize the intestinal tract and cause hospital ward outbreaks. Whether infection-derived enterococcal isolates show enhanced acid tolerance is yet to be determined.

Therapy with antibiotics possessing little anti-enterococcal activity is a key predisposing factor leading to enterococcal colonization and infection (8). Studies in mice with antibiotic- induced intestinal E. faecalis overgrowth demonstrated that organisms can adhere to epithelial surfaces of the ileum, cecum, and colon (74, 75). These same studies showed that enterococci possess the ability to translocate from the intestinal lumen to the mesenteric lymph nodes, liver, and spleen (74, 75). As prior antibiotic therapy appears to be a predisposing factor for enterococcal infection, antibiotic-induced intestinal overgrowth by E. faecalis, followed by translocation of the organism into the circulation may offer one explanation for bacteremias of unknown etiology (23).

The mechanisms responsible for enterococcal translocation are not clearly defined. One hypothesis is that enterococci are phagocytosed by tissue macrophages or intestinal epithelial cells, and are transported across the intestinal wall to the underlying lymphatic system. Failure to kill the phagocytosed organisms could then lead to systemic spread (76). Olmsted et al. (59) examined the role of the plasmid-encoded surface protein, aggregation substance, in the ability of E. faecalis to be internalized by cultured intestinal epithelial (HT-29) cells. The presence of aggregation substance significantly augmented E. faecalis internalization by HT-29 cells. However, in contrast to the one order of magnitude in enhanced uptake efficiency conferred by aggregation substance, a difference of three orders of magnitude of difference was observed between various enterococcal strains tested. This indicated that additional unknown features of this species play major roles as determinants of internalization efficiency.

The vast majority (87/91) of E. faecalis produce superoxide (O2-), whereas E. faecium isolates (5/13) do so less frequently (32). When clinical and commensal isolates of E. faecalis were compared for O2- production, strains associated with bacteremia produced O2- in vitro at a rate 60% higher than stool isolates. What role, if any, superoxide production plays in enterococcal pathogenesis, however, is not well defined. Perhaps, enterococcal strains capable of producing O2- are better adapted physiologically to utilize limited resources in the intestinal environment, leading to overgrowth of the organism. Alternatively, O2- production may enhance niche control in proximity to the intestinal epithelium. The membrane damaging effects of oxygen radicals may then potentiate the ability of the organism to translocate across a weakened epithelial barrier. The enrichment of this trait among E. faecalis infection derived isolates presents intriguing questions and warrants further study.

One of the enigmas of nosocomial enterococcal infection not easily explained is the ready colonization of an ecology already occupied by members of the same species. As noted, antibiotics lacking substantial anti-enterococcal activity (i.e. antibiotics that do not deleteriously affect indigenous enterococci) are important predisposing factors for infection. These infections are frequently caused by multiply resistant enterococcal isolates that have been exogenously acquired and appear to have out competed indigenous enterococci in the absence of direct selection. The numbers of exogenously acquired, multiply resistant enterococci would be expected to be minuscule compared to the numbers of indigenous enterococci that are present and well positioned to occupy any niche suitable for enterococcal colonization. The fact that exogenous, multiply resistant, nosocomially transmitted enterococci efficiently colonize the gastrointestinal tract suggests that they may not compete directly for the same niche as indigenous strains. A novel surface protein capable of enabling enterococci to colonize a new area of the gastrointestinal tract, an area perhaps less endowed with immune clearance mechanisms, may explain the ability of these outbreak strains to efficiently colonize and cause disease. Another mechanism that could render colonization by exogenous strains noncompetitive would be an ability to utilize nutrients in a new or enhanced way. For instance, an enhanced ability to utilize mammalian intestinal mucin as an energy source may enable outbreak strains to colonize deeper layers of the mucosa in more intimate association with intestinal epithelial cells. Few studies have comprehensively examined and compared the physiology of commensal strains and enterococcal strains that caused multiple infections, and so at present, these prospects remain completely speculative. However, a new surface protein of novel structure has been described, which is enriched among endocarditis (40%) and bacteremia isolates (29%), but is rare among fecal isolates (<3%)>

Bacteremia

Nosocomial surveillance data for the period October 1986-April 1997 list enterococci as the third most common cause of nosocomial bacteremia, accounting for 12.8% of all isolates (56). The translocation of enterococci across an intact intestinal epithelial barrier is thought to lead to many bacteremias with no identifiable source (38, 76). Other identifiable sources for enterococcal bacteremia include intravenous lines, abscesses, and urinary tract infections (38). The risk factors for mortality associated with enterococcal bacteremia include severity of illness, patient age, and use of broad spectrum antibiotics, such as third-generation cephalosporins or metronidazole (68). Huycke et al. (34) showed that patients infected with hemolytic, gentamicin- resistant E . faecalis strains had a fivefold-increased risk for death within three weeks compared to patients infected with nonhemolytic, gentamicin-susceptible strains. Moreover, mode of treatment was not associated with outcome, discounting the contribution of aminoglycoside resistance to this enhanced lethality of infection. In a more recent study, Caballero-Granado et al. (7) analyzed the clinical outcome, including mortality, for bacteremia caused by Enterococcus spp. with and without high-level gentamicin resistance. Mortality associated with high-level gentamicin resistance (29%) was not significantly different from gentamicin-susceptible strains (28%). In addition, these workers found no significant difference in the length of hospitalization after acquisition of enterococcal bacteremia. Taken together, these studies suggest that high-level aminoglycoside resistance does not affect clinical outcome, and that the presence of the E. faecalis cytolysin (hemolysin) may enhance the severity of the infection. A number of well controlled independent animal studies confirm the toxicity of the enterococcal cytolysin. Cytolysin significantly lowers the 50% lethal dose (LD50) of the infecting strain for mice (13, 35, 50). As discussed below, cytolysin also contributes to the acute toxicity of lupine endocarditis and endophthalmitis models (35, 39).

Urinary Tract

Enterococci have been estimated to account for 110,000 urinary tract infections (UTI) annually in the United States (33). A few studies have been aimed at understanding the interaction of enterococci with uroepithelial tissue (25, 41, 73). Kreft et al. (41) showed a potential role for the plasmid-encoded aggregation substance in the adhesion of enterococci to renal epithelial cells. E. faecalis harboring the pheromone responsive plasmid pAD1, or various isogenic derivatives, were better able to bind to the cultured pig renal tubular cell line, LLC-PK, than plasmid free cells. Their findings also showed that a synthetic peptide containing the fibronectin motif, Arg-Gly-Asp-Ser, could inhibit binding. This structural motif mediates the interaction between fibronectin and eucaryotic surface receptors of the integrin family (20).

Guzman and coworkers (25) analyzed strains of E. faecalis isolated from either urinary tract infections or endocarditis, for their ability to adhere to urinary tract (UT) epithelial cells and the Girardi heart cell line. UTI isolates adhered to the UT epithelial cells in vitro, whereas strains from endocarditis adhered efficiently to the Girardi heart cell line. A key observation from these experiments was that growth in pooled human serum enhanced the binding of UTI isolates to the Girardi heart cell line (8-fold increase). The authors noted that the serum-dependent alterations to cell adhesion were lost by several sub-cultures in brain heart infusion broth (25). In a later study, E. faecalis adherence was found to be mediated by carbohydrate antigens present on the cell surface (26). Thus, the nature of the interaction of enterococci with uroepithelial tissue appears to be quite complex, involving surface adhesins of protein and/or carbohydrate nature.

Endocarditis

Of the diverse infections caused by enterococci, infective endocarditis (IE) is one of the most therapeutically challenging (49). Enterococci are the third leading cause of infective endocarditis, accounting for 5-20% of cases of native valve IE, and 6-7% of prosthetic valve endocarditis (49). As noted above, enterococci cultured in serum exhibit enhanced binding to Girardi heart cells. This interaction is inhibited by periodate treatment of the bacterial cell as well as competitive inhibition of binding, by prior incubation of the target cells with specific sugar residues, including D-galactose and L-fucose (26). This suggests that a carbohydrate antigen mediates the adherence of enterococci to cultured heart cells which were derived from the right auricular appendage (Girardi heart).
The presence of the pheromone-responsive plasmid pAD1 enhances vegetation formation in enterococcal endocarditis (9). By comparing endocarditis caused by isogenic mutants in either cytolysin (hemolysin) production or aggregation substance, which are encoded on pAD1, it was observed that the presence of the cytolysin contributed to overall lethality (6/11 animals killed compared to 2/13 in the non-cytolytic mutant, p<0.01), whereas the presence of aggregation substance led to a 2-fold increase in mean vegetation weight. It was noted, however, that all strains tested were able to cause endocarditis, even the plasmid-free controls. This data suggests that the virulence traits encoded by auxiliary genetic elements can enhance the pathogenicity of the organism, but may not be essential in establishing infection.

Serum from a patient with E. faecalis endocarditis was used to identify an E. faecalis antigen selectively expressed in serum but not in broth culture (44). This protein antigen, designated EfaA, had a predicted molecular weight of 34,768. Database homology searches revealed extensive sequence similarity with several streptococcal adhesins. The authors hypothesized that this surface antigen might function as an important adhesin in endocarditis, but there is no published data to support this.

Endophthalmitis

Colonization of host tissue may play a role in the pathogenesis of endophthalmitis. Enterococci are among the the most destructive agents that cause this post-operative complication of cataract surgery (27, 39). Experiments designed to determine whether aggregation substance targeted E. faecalis to alternate anatomical structures within the eye showed that enterococci attach to membranous structures in the vitreous, but that such adherence is not dependent on the presence of aggregation substance (37). In summary, the preponderance of data indicates that E. faecalis adhesion to host tissues is complex and involves multiple adhesins including surface carbohydrates as well as proteins.

Immune Evasion

For enterococci to maintain an infection, they must successfully evade both specific and nonspecific host defense mechanisms. Other gram-positive pathogens possess attributes which allow them to survive in the host in spite of powerful nonspecific host defenses mediated primarily by professional phagocytes, i.e. neutrophils, monocytes, and macrophages. These factors include antiphagocytic polysaccharide capsules, antiphagocytic surface proteins, such as the group A streptococcal M protein, and various secreted toxins with direct toxicity for phagocytic cells.

Studies designed to characterize the host response to enterococcal infection have been conducted (2, 19, 28, 58). Harvey et al. (28) concluded that "neutrophil mediated killing of enterococci was largely a function of complement with antibody playing a less essential but potentially important role." Arduino et al. (2) reached similar conclusions with regard to the role of complement in the clearance of enterococci by polymorphonuclear cells (PMNs). The latter study also tested several defined virulence traits, which included gelatinase, cytolysin, and aggregation substance of E. faecalis and found no significant correlation between a given trait and resistance to phagocytosis. However, these phagocytosis assays were performed under conditions unlikely to support cytolysin expression (72), and production of the other traits in this environment was similarly not controlled. Although cytolysin is not made at appreciable levels in standard laboratory media, it can be detected when organisms are cultured in serum (unpublished data), so it may be of value to reexamine the roles of these factors under more physiologic conditions.

In the course of studying neutrophil-mediated phagocytosis, Arduino et al. (1) identified a strain of E. faecium that exhibited increased resistance to phagocytosis. They were able to detect electron-dense clumps adjacent to the cell wall consistent with the presence of capsular material. It was noted, however, that similar electron-dense clumps surrounded the cell wall of strains of E. faecalis that appeared to be sensitive to PMN-mediated clearance. The nature of the material providing protection to phagocytosis for the E. faecium strain was shown to be protease resistant and periodate sensitive, implicating a carbohydrate. Further characterization of this material has not been reported. In a recent case report, Bottone and colleagues (6) isolated three highly mucoid-encapsulated strains of E. faecalis from patients with urinary tract infections. Whether these encapsulated strains are more resistant to phagocytic killing by neutrophils remains to be demonstrated.

Additional evidence for the importance of anti-enterococcal antibodies in promoting clearance by opsonophagocytic killing was recently reported by Gaglani et al. (19). These authors compared killing efficacy by neutrophils using normal human serum, hypogammaglobulinemic serum, or normal human serum adsorbed with the homologous bacterial strain. Their findings suggest that normal human serum has sufficient anti-enterococcal antibodies to promote > 90% reduction of the bacterial inoculum with serum concentrations as low as 0.5%. Hypogammaglobulinemic serum also promoted > 90% reduction of the bacterial inoculum, but only at serum concentrations above 5%. Adsorption with the homologous strain significantly reduced bactericidal activity of the normal human serum. These findings indicate that the normal human host possesses antibodies to enterococci that aid in opsonophagocytic killing, which might be expected for a normal bowel organism in constant association with the host. The protective role of anti-enterococcal antibodies should be examined to determine whether such protection extends from the strain level to either genus or species. Work in progress is examining these issues (60, 67).

In 1992, Maekawa et al. (47) proposed a new serologic typing scheme for E. faecalis, in which E. faecalis isolates were classified into 21 distinct serotypes, but 4 serotypes accounted for 72% of all typable strains, indicating that certain serotypes are more prevalent among clinical isolates. The exact nature of the serotype antigens is not yet known, and could be protein (such as M protein in the case of group A streptococci), or a polysaccharide (as is the case for group B streptococci and Streptococcus pneumoniae).

More recently it has been observed that enterococci possess the ability to survive within professional phagocytes (21, 70). Preliminary evidence (70), indicates an important role for aggregation substance in the adherence, entry, and survival in macrophages. Further studies into the basis for the survival of enterococci within phagocytes will add to our understanding of how enterococci escape immune surveillance.

Pathologic Tissue Damage

Following adhesion to host cell surfaces, and evasion of the host immune response, the last step in the pathogenesis of infection is the production of pathologic changes in the host. Such changes can be induced by the host inflammatory cascade or by direct tissue damage as a result of secreted toxins or proteases. Both mechanisms have been observed in studies of E. faecalis pathogenesis.

Indirect Tissue Damage

Enterococcal lipoteichoic acid (LTA), also known as the group D streptococcal antigen, has been implicated in a variety of biological processes (24-26, 54). Some properties ascribed to LTA include modulation of the host immune response as well as mediating the adherence of enterococci to host cells. Bhakdi et al. (4) found the LTA from enterococci to be as inflammatory as lipopolysaccharide of gram-negative bacteria. LTA may also contribute to the ability of enterococci to exchange and rapidly disseminate genetic determinants, a subject that has been investigated extensively (12).

The role of LTA and aggregation substance (AS) in cardiac infections was recently examined (65). Strains of E. faecalis defective in AS and the enterococcal binding substance (EBS), which is at least partially derived from LTA, did not induce clinical signs of illness when injected intraventricularly at levels of 108 cfu/ml. However, EBS+AS- or EBS-AS+ strains induced signs of illness and pericardiac inflammation. All rabbits injected with the EBS+AS+ strain developed illness and died. Surprisingly little inflammation was observed in rabbits injected with the EBS+AS+ strain despite the lethality observed. The authors state that such observations are consistent with the presence of a superantigen. The presence of LTA (EBS) and AS together may mediate effects on the host immune response that differ from those seen when either component acts alone. These results warrant further investigation as one of the strains tested possessed multiple transposon insertions, and the culture fluid extract used to demonstrate superantigen behavior was crude and of unknown physiologic relevance in terms of concentration. Nevertheless, the hypothesis that aggregation substance may bind and display LTA in a conformation that promotes an enhanced inflammatory response is intriguing.

Direct Tissue Damage

The enterococcal cytolysin and the zinc metalloprotease (gelatinase) are secreted factors well suited to contribute to disease severity (9, 13, 22, 35, 39). The role of the enterococcal cytolysin in disease pathogenesis has been well established in several independent laboratories and models (9, 35, 39, 50). The cytolysin is enriched in clinical isolates, and occurs at a frequency of 45-60% (31, 34, 36). The cytolysin is a unique bacterial toxin that is distantly related to lantibiotic bacteriocins, a family of small, post-translationally modified antimicrobial peptides (5). The cytolysin possesses both toxin and bacteriocin activities, and may provide several levels of selective advantage for E. faecalis strains expressing this trait. We observed that the presence of the cytolytic phenotype promotes the appearance of enterococci in the blood when compared to a non-cytolytic isogenic mutant in a mouse model of septicemia (10). A substantial body of data supports the role for cytolysin in enterococcal infection, both in humans, (34) and animal models (9, 35, 39).

The most direct and quantitative evidence for pathologic damage attributable to the cytolysin was obtained using a rabbit model of endophthalmitis (39). This model was selected because of the natural aberrations in the intraocular immune response, allowing a robust infection to be established with as few as 10 organisms. This limited response provides the offending bacterium an opportunity to adapt to in vivo growth conditions and environmental cues. Moreover, highly sensitive and quantitative measurements of the evolution of disease can be made. A role for the cytolysin in tissue pathology was unambiguously demonstrated both by a reduction in retinal function as measured by electroretinography (B-wave response), and complete destruction of retinal architecture by 24 hours postinfection. The contribution of cytolysin to the severity of disease has also been observed in animal models of systemic disease (35) and endocarditis (9). These findings conclusively demonstrate the importance of the E. faecalis cytolysin as a major virulence factor in E. faecalis infections. However, the cytolytic phenotype is a variable trait typically encoded on pheromone-responsive plasmids (38). In about half of E. faecalis infections, the cytolytin is absent, emphasizing the importance of other traits in pathogenicity (11,31, 34, 36).

The enterococcal gelatinase may also play a measurable role in systemic disease (13), as well as in a caries model using germ-free rats (22). Dupont et al. (13) showed a reduced LD50 for mice injected with gelatinase producing (Gel+) strains. These studies were limited, however, in that the basis for comparison was a Gel- strain which was generated by chemical mutagenesis, and therefore other uncharacterized traits may have contributed to the observed attenuation.
Using germ-free rats, Gold et al. (22) showed that a proteolytic (Gel+) strain exhibited cariogenic activity, whereas three nonproteolytic strains exhibited little cariogenecity. There is some indication that clinical isolates may be enriched for this trait, as greater than 50% of isolates from both endocarditis and other clinical sources exhibited gelatinase activity, whereas only 27% of community fecal isolates possessed this trait (11). In this study, highly virulent lineages that caused multiple infections were discounted by study design, skewing the correlation between phenotype and disease incidence (11).

Although demonstration of the involvement of gelatinase in tissue pathology and virulence requires the comparison of isogenic mutants, in vitro targets of this enzyme do provide clues for a potential role (69). The gene for gelatinase, gelE, encodes an enzyme that shares significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa (69). The proposed targets of this enzyme include gelatin, casein, hemoglobin, and other bioactive peptides, including the E. faecalis sex pheromones. The observation that enterococcal pheromones are potent chemoattractants (62), and that gelatinase can cleave these and other bioactive peptides, indicates that gelatinase at least has the potential to modulate the host response to enterococcal infection.

CONCLUSIONS AND FUTURE PERSPECTIVES

Enterococci are well adapted for survival and persistence in a variety of adverse environments, including sites of infection and inanimate hospital surfaces. The rapid emergence of antimicrobial resistance among enterococci undoubtedly also contributes to their emergence as prominent nosocomial pathogens, making them among the most difficult to treat. An understanding of the molecular pathogenesis of enterococcal infection, however, is in its infancy, largely because rogue commensals do not fit the mold of what has emerged as the modern conception of "pathogenesis". These organisms are not highly toxigenic, highly invasive, or highly infectious by most measures. They do, nevertheless, cause a substantial amount of human disease, highlighting the inadequacy of existing pathogenesis precepts. Additional study on the pathogenesis of enterococcal infection is urgently needed to reverse the trend toward larger numbers of refractory infections caused by these organisms, for which we have limited understanding.

Acknowledgments

The authors wish to thank Viswanathan Shankar, Mark Huycke, Wolfgang Haas, Pravina Srinivas, Phil Coburn, Brett Shepard, Michelle Callegan, and Mary Booth for helpful discussions and comments. Portions of the work described were supported by PHS grants AI41108 and EY08289.

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