We evaluated ceftobiprole against the well-characterized Enterococcus faecalis strain OG1RF (with and without the β-lactamase [Bla] plasmid pBEM10) in a murine urinary tract infection (UTI) model. Ceftobiprole was equally effective for Bla+ and Bla− OG1 strains, while ampicillin was moderately to markedly (depending on the inoculum) less effective against Bla+ than Bla− OG1 strains. These data illustrate an in vivo effect on ampicillin of Bla production by E. faecalis and the stability and efficacy of ceftobiprole in experimental UTI.
The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.
Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.
Enterococcus faecalis; genomic integration; complementation; green fluorescent protein
Previously, TX5179, a disruption mutant of the enterococcal polysaccharide antigen (epa) gene cluster of Enterococcus faecalis strain OG1RF was shown to be attenuated in translocation, biofilm mouse peritonitis and was more susceptible to polymorphonuclear leukocyte phagocytic killing. Here, wild-type E. faecalis OG1RF and TX5179 strains were tested in a mixed-infection (inoculum, ~1:1) mouse urinary tract infection model. Wild-type OG1RF outnumbered TX5179 in the kidneys (P < .001) and bladder (P < .001). In conclusion, the epa locus of E. faecalis OG1RF contributes to murine urinary tract infection and is the firs such enterococcal polysaccharide locus shown to be important in this site.
Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ∼97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate 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.
Enterococcus faecium; Enterococcal surface protein Esp; endocarditis; pathogenesis; immunity
General stress proteins, Gls24 and GlsB, were previously shown to be involved in bile salts resistance of Enterococcus faecalis and in virulence. Here, we identified 2 gene clusters in Enterococcus faecium each encoding a homolog of Gls24 (Gls33 and Gls20; designated on the basis of their predicted sizes) and of GlsB (GlsB and GlsB1). The sequences of the gls33 and gls20 gene clusters from available genomes indicate distinct lineages, with those of hospital-associated CC17 isolates differing from non-CC17 by ∼7% and ∼3.5%, respectively. Deletion of an individual locus did not have a significant effect on virulence in a mouse peritonitis model, whereas a double-deletion mutant was highly attenuated (P < .004) versus wild-type. However, mutants lacking either gls33-glsB, gls20-glsB1, or both all exhibited increased sensitivity to bile salts. These results suggest that gls-encoded loci may be important for adaptation to the intestinal environment, in addition to being important for virulence functions.
Ebp are endocarditis- and biofilm-associated pili of Enterococcus faecalis that are also important in experimental urinary tract infections (UTIs). Our analyses, using available genomes, found that the ebp locus is unique to enterococci. In E. faecalis, the ebp locus is very highly conserved and only 1/473 E. faecalis isolates tested lacked ebpABC, while only 1.2% had the bee pilus locus. No other pilus-encoding operon was identified in 55 available genomes, indicating that the vast majority of E. faecalis strains (unlike Enterococcus faecium and streptococci) have a single pilus locus. Surface expression studies showed that Ebp pili were produced in vitro by 91/91 brain heart infusion (BHI) plus serum-grown E. faecalis isolates and that strain OG1RF expressed pili at even higher levels in rat endocarditis vegetations. However, Ebp expression was restricted to 30 to 72% of E. faecalis cells, consistent with a bistability mode of expression. We also evaluated E. faecalis interactions with human platelets and found that growth of E. faecalis in BHI plus serum significantly enhanced adherence to human platelets and that sortase deletion mutants (the ΔsrtA, Δbps, and ΔbpsΔsrtA mutants) were markedly defective. Further studies identified that Ebp pili, but not the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) Ace and Fss2, mediate adherence of E. faecalis to platelets. Taken together, our data show that the immunogenic (in human endocarditis patients) and commonly expressed Ebp pili, which are known to be important for experimental endocarditis, are highly conserved and mediate adherence to platelets, suggesting that Ebp pili may be a reasonable immunotherapeutic target for prevention or possibly treatment of endocarditis caused by this species.
Previous studies have demonstrated that the ebp operon and the ace gene of Enterococcus faecalis, encoding endocarditis- and biofilm-associated pili and an adhesin to collagen of E. faecalis, respectively, are both important in experimental urinary tract infections (UTI) and endocarditis. We have also shown that growth of E. faecalis in brain heart infusion (BHI) serum enhances Ebp pilus and Ace production and increases adherence to several host extracellular matrix proteins. Here, we report that deletion of ebpABC almost eliminated serum-elicited adherence to fibrinogen (P < 0.0001), resulted in moderate reduction in adherence to collagen (P < 0.05), and had no effect on fibronectin adherence relative to that of wild-type OG1RF. An OG1RFΔaceΔebpABC double mutant showed further reduced collagen adherence versus that of the OG1RFΔace or OG1RFΔebpABC mutants (P < 0.001). These results were corroborated by complementation and/or studies with native pilus-enriched surface extracts and a collagen-secreting 3T6 fibroblast cell line, as well as antibody inhibition. In the UTI model, both the OG1RFΔace and OG1RFΔaceΔebpABC mutants were found to be significantly attenuated compared to the wild type; however, no significant differences were observed between individual ace or ebp mutants and the OG1RFΔaceΔebpABC mutant. In summary, these data implicate the Ebp pili as having some role in collagen adherence, albeit less than that of Ace, and a very major role in fibrinogen adherence, which may explain in part the importance of these pili in experimental endocarditis. The OG1RFΔaceΔebpABC mutant was attenuated in the UTI model, although not significantly more so than the Δace or ΔebpABC mutants, suggesting involvement of other E. faecalis factors in urinary tract colonization or infection.
Keywords: Enterococcus faecalis; endocarditis; disease reservoirs; swine; multilocus sequence typing; electrophoresis; gel; pulsed-field; biofilms; virulence factors; drug resistance; bacteria
We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX16 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABCfm, is transcribed as an operon, that its putative major pilus subunit, EbpCfm (also called PilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABCfm operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpCfm expression and eliminated EbpCfm-containing pili from the cell surface. However, transcription of the downstream sortase, bpsfm, was not affected. Importantly, the ebpABCfm deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABCfm in trans, which also restored cell surface expression of EbpCfm and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and <0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABCfm locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.
Enterococcus faecium; ebp; pilus; biofilm; virulence; pathogenesis; UTI
Enterococcus faecium; fms genes; pili; hyaluronidase; transferable plasmid
Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model.
Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region
The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.
We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX16 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABCfm, is transcribed as an operon, that its putative major pilus subunit, EbpCfm (also called PilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABCfm operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpCfm expression and eliminated EbpCfm-containing pili from the cell surface. However, transcription of the downstream sortase, bpsfm, was not affected. Importantly, the ebpABCfm deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABCfm in trans, which also restored cell surface expression of EbpCfm and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABCfm locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.
Enterococcus faecium; ebp; pilus; biofilm; virulence; pathogenesis; UTI
Using 98 clinical methicillin-susceptible Staphylococcus aureus isolates of known β-lactamase (Bla) type, we found a pronounced inoculum effect for cephalexin (mostly Bla type A and C strains), a mild inoculum effect for cephalothin (especially types B and C), and no inoculum effects for ceftriaxone and cefuroxime. Ceftobiprole showed the lowest MICs at a high inoculum but with a slight increase for Bla-positive versus Bla-negative strains. Since a potential therapeutic effect associated with a cephalosporin inoculum effect has been described, further studies are warranted.
We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for E. faecalis surface protein) of the recently predicted MSCRAMMs in Enterococcus faecalis strain V583 bind fibrinogen. Despite an absence of extensive primary sequence homology, the three proteins appear to be structurally related. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the fibrinogen-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are mainly composed of β-sheets with only a minor proportion of α-helices, which is characteristic of immunoglobulin folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to fibrinogen. However, the specificity of the fibrinogen-binding MSCRAMMs differs as indicated by far-Western blots which showed that recombinant segments of the MSCRAMMs bound different fibrinogen polypeptide chains. Enterococci, grown in serum-supplemented broth, adhere to fibrinogen-coated surfaces and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, while complementation of the mutant restored full fibrinogen adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resembles the fibrinogen-binding MSCRAMMs of staphylococci.
Enterococcus faecalis; MSCRAMM; adhesin; immunoglobulin fold; fibrinogen
We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of β-sheets with only a minor proportion of α-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.
Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb-sbs7-srtC1, are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis.
The hylEfm gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hylEfm-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hylEfm gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hylEfm, whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hylEfm-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hylEfm plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.
We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.
Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A β-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ. The cefazolin MIC90 was 2 μg/ml for a standard inoculum and 32 μg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of ≥16 μg/ml with 107 CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure (P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.
Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.
Enterococcus faecalis was recognized as a common cause of infective endocarditis (IE) by the early 1900s. It is still third in community-onset IE, but is the second most common cause of hospital-associated IE. Complications due to E. faecalis IE include congestive heart failure, septic emboli and death and current management involves a combination of antimicrobials, often with surgery. Emergence of antimicrobial resistance has created the need for alternative strategies (such as immunoprophylaxis) that target in vivo expressed virulence-associated surface proteins. One such E. faecalis protein is Ace, which is antigenic during human IE and mediates attachment of E. faecalis cells to host extracellular matrix proteins collagen and laminin. Using a rat model, we now show that ace contributes to E. faecalis IE pathogenesis and demonstrate that Ace is expressed at high levels during IE even though produced at low levels under laboratory conditions; both active and passive immunization based on the collagen-binding domain of Ace conferred significant protection against IE. These observations, along with data that human antibodies against Ace inhibit collagen adherence of E. faecalis, indicate that Ace is an important virulence-associated factor and a promising target for prophylactic and possibly therapeutic strategies against E. faecalis IE.