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.
Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes
(glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.
IMPORTANCE The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
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
Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the “susceptible” cls allele from S447 for the “resistant” one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains.
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.
The genus Enterococcus includes some of the most important nosocomial multidrug-resistant organisms, and these pathogens usually affect patients who are debilitated by other, concurrent illnesses and undergoing prolonged hospitalization. This Review discusses the factors involved in the changing epidemiology of enterococcal infections, with an emphasis on Enterococcus faecium as an emergent and challenging nosocomial problem. The effects of antibiotics on the gut microbiota and on colonization with vancomycin-resistant enterococci are highlighted, including how enterococci benefit from the antibiotic-mediated eradication of Gram-negative members of the gut microbiota. Analyses of enterococcal genomes indicate that there are certain genetic lineages, including an E. faecium clade of ancient origin, with the ability to succeed in the hospital environment, and the possible virulence determinants that are found in these genetic lineages are discussed. Finally, we review the most important mechanisms of resistance to the antibiotics that are used to treat vancomycin-resistant enterococci.
Mutations in liaFSR, a three-component regulatory system controlling cell-envelope stress response, were recently linked with the emergence of daptomycin (DAP) resistance in enterococci. Our previous work showed that a liaF mutation increased the DAP MIC of a vancomycin-resistant Enterococcus faecalis strain from 1 to 3 μg/ml (the DAP breakpoint is 4 μg/ml), suggesting that mutations in the liaFSR system could be a pivotal initial event in the development of DAP resistance. With the hypothesis that clinical enterococcal isolates with DAP MICs between 3 and 4 μg/ml might harbor mutations in liaFSR, we studied 38 Enterococcus faecium bloodstream isolates, of which 8 had DAP MICs between 3 and 4 μg/ml by Etest in Mueller-Hinton agar. Interestingly, 6 of these 8 isolates had predicted amino acid changes in the LiaFSR system. Moreover, we previously showed that among 6 DAP-resistant E. faecium isolates (MICs of >4 μg/ml), 5 had mutations in liaFSR. In contrast, none of 16 E. faecium isolates with a DAP MIC of ≤2 μg/ml harbored mutations in this system (P < 0.0001). All but one isolate with liaFSR changes exhibited DAP MICs of ≥16 μg/ml by Etest using brain heart infusion agar (BHIA), a medium that better supports enterococcal growth. Our findings provide a strong association between DAP MICs within the upper susceptibility range and mutations in the liaFSR system. Concomitant susceptibility testing on BHIA may be useful for identifying these E. faecium first-step mutants. Our results also suggest that the current DAP breakpoint for E. faecium may need to be reevaluated.
We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus.
In this study, we examined the effect of CO2/HCO3-, pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3- increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2- bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested.
This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.
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
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.
We describe a patient who developed Corynebacterium striatum native valve endocarditis after receiving two 6-week courses of daptomycin for the treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. The organism exhibited in vitro heteroresistance to daptomycin, with two subpopulations showing daptomycin susceptibility (MIC of ≤0.094 μg/ml) and high-level resistance to daptomycin (MIC of ≥256 μg/ml). The selection of daptomycin-resistant Gram-positive skin flora with the potential of causing invasive disease may be a concern during prolonged courses of daptomycin.
Daptomycin MICs for enterococci are typically 1- to 2-fold higher than those for Staphylococcus aureus, and there is an imminent need to establish the optimal dose for appropriate treatment of enterococcal infections. We investigated the bactericidal activity of daptomycin at various dose exposures compared to that of linezolid against vancomycin-resistant enterococcus (VRE) in an in vitro pharmacokinetic/pharmacodynamic model utilizing simulated endocardial vegetations over 96 h. Daptomycin at doses of 6, 8, 10, and 12 mg/kg of body weight/day and linezolid at a dose of 600 mg every 12 h were evaluated against two clinical vancomycin-resistant Enterococcus faecium strains (EFm11499 and 09-184D1051), one of which was linezolid resistant (09-184D1051), and one clinical vancomycin-resistant Enterococcus faecalis strain (EFs11496). Daptomycin MICs were 4, 2, and 0.5 μg/ml for EFm11499, 09-184D1051, and EFs11496, respectively. Bactericidal activity, defined as a ≥3 log10 CFU/g reduction from the initial colony count, was demonstrated against all three isolates with all doses of daptomycin; however, bactericidal activity was not sustained with the daptomycin 6- and 8-mg/kg/day regimens. Linezolid was bacteriostatic against EFm11499 and displayed no appreciable activity against 09-184D1051 or EFs11496. Concentration-dependent killing was displayed with more sustained reduction in colony count (3.58 to 6.46 and 5.89 to 6.56 log10 CFU/g) at 96 h for the simulated regimen of daptomycin at doses of 10 and 12 mg/kg/day, respectively (P ≤ 0.012). No E. faecium mutants with reduced susceptibility were recovered at any dosage regimen; however, the E. faecalis strain developed reduced daptomycin susceptibility with daptomycin at 6, 8, and 10 but not at 12 mg/kg/day. Daptomycin displayed a dose-dependent response against three VRE isolates, with high-dose daptomycin producing sustained bactericidal activity. Further research is warranted.
Most previous reports have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen
The influence of various conditions on E. faecalis adherence to ECM proteins was evaluated using a radiolabeled adherence assay.
Among conditions studied, growth in 40% serum, a biological cue with potential clinical relevance, elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen, but not to elastin; the adherence levels were independent of strain source and was eliminated by treating cells with trypsin. As previously reported, serum also elicited collagen adherence. While prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (<5 min) to serum had an immediate, although partial, effect on adherence to fibronectin and collagen. Pre-treatment of bacteria with chloramphenicol did not decrease the enhanced fibronectin and collagen adherence, indicating that protein synthesis is not required for the latter effect.
Together, these data suggest that serum components may serve i) as host environmental stimuli to induce production of extracellular matrix protein binding adhesin(s), as previously seen with collagen adherence, and also ii) as activators of adherence, perhaps by forming bridges between ECM proteins and adhesins.
Enterococcus faecalis; extracellular matrix; adherence; collagen; fibrinogen; fibronectin; elastin; serum induction; MSCRAMMs
We recently showed that the ubiquitous Enterococcus faecalis ebp (endocarditis and biofilm-associated pilus) operon was important for biofilm and experimental endocarditis. Here we assess its role in murine urinary tract infection using wild type E. faecalis OG1RF and its nonpiliated ebpA allelic replacement mutant (TX5475).
OG1RF and TX5475 were administered transurethrally in a 1:1 ratio (competition assay) or individually (monoinfection). Kidneys and bladders were cultured 48 h postinfection. These strains were also tested in a peritonitis model.
No differences were observed in the peritonitis model. In mixed urinary infection, OG1RF significantly outnumbered TX5475 in kidneys (P=0.0033) and bladders (P=<0.0001). More OG1RF CFU were also recovered from kidneys of monoinfected mice at the four inocula tested (P=0.015 to 0.049) and ID50s of OG1RF for kidney and bladder (9.1 × 101 and 3.5 × 103 CFU, respectively) were two-three log10 lower than with TX5475. Increased tropism for the kidney relative to the bladder was observed by both OG1RF and TX5475.
The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.
Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalisis associated with host cell adherence and endocarditis. The Fsr quorum-sensing system of E. faecalis, a two-component signal transduction system, has also been repeatedly linked to virulence in E. faecalis, due in part to the transcriptional induction of an extracellular metalloprotease, gelatinase (GelE). In this study, we discovered that disruption of the Fsr pathway significantly increased the levels of Ace on the cell surface in the latter phases of growth. Furthermore, we observed that, in addition to fsrBmutants, other strains identified as deficient in GelE activity also demonstrated a similar phenotype. Additional experiments demonstrated the GelE-dependent cleavage of Ace from the surface of E. faecalis, confirming that GelE specifically reduces Ace cell surface display. In addition, disruption of the Fsr system or GelE expression significantly improved the ability of E. faecalisto adhere to collagen, which is consistent with higher levels of Ace on the E. faecalissurface. These results demonstrate that the display of Ace is mediated by quorum sensing through the action of GelE, providing insight into the complicated world of Gram-positive pathogen adhesion and colonization.
Recent studies have pointed to the existence of two subpopulations of Enterococcus faecium, one containing primarily commensal/community-associated (CA) strains and one that contains most clinical or hospital-associated (HA) strains, including those classified by multi-locus sequence typing (MLST) as belonging to the CC17 group. The HA subpopulation more frequently has IS16, pathogenicity island(s), and plasmids or genes associated with antibiotic resistance, colonization, and/or virulence. Supporting the two clades concept, we previously found a 3–10% difference between four genes from HA-clade strains vs. CA-clade strains, including 5% difference between pbp5-R of ampicillin-resistant, HA strains and pbp5-S of ampicillin-sensitive, CA strains. To further investigate the core genome of these subpopulations, we studied 100 genes from 21 E. faecium genome sequences; our analyses of concatenated sequences, SNPs, and individual genes all identified two distinct groups. With the concatenated sequence, HA-clade strains differed by 0–1% from one another while CA clade strains differed from each other by 0–1.1%, with 3.5–4.2% difference between the two clades. While many strains had a few genes that grouped in one clade with most of their genes in the other clade, one strain had 28% of its genes in the CA clade and 72% in the HA clade, consistent with the predicted role of recombination in the evolution of E. faecium. Using estimates for Escherichia coli, molecular clock calculations using sSNP analysis indicate that these two clades may have diverged ≥1 million years ago or, using the higher mutation rate for Bacillus anthracis, ∼300,000 years ago. These data confirm the existence of two clades of E. faecium and show that the differences between the HA and CA clades occur at the core genomic level and long preceded the modern antibiotic era.
Recent studies have shown that ampicillin resistance has increased steadily over the past 3 decades within U.S. Enterococcus faecium isolates. Analysis of the predicted PBP5 protein of 41 isolates showed a consensus PBP5 pattern for the 9 isolates with MICs of <4 μg/ml that is distinctly different from the PBP5 consensus of the 32 isolates with MICs of >4 μg/ml with ∼5% difference between these; however, there were no consistent amino acid changes that correlated with specific increases in the MICs of ampicillin within the latter group. Analysis of three other genes encoding cell wall/surface proteins also showed that there are two distinct evolutionary groups for each gene, but with occasional mixing of genes, consistent with a species that evolves by recombination.
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
Most Enterococcus faecalis isolates carry gelE, but many are gelatinase nonproducers due to the lack of fsrC (EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activates gelE expression. Analysis of 22 accessible E. faecalis genomes revealed the identity of the 53-amino-acid propeptide of fsrD across multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen in fsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surrounding fsrC-EF_1841. However, analysis of five sequenced strains carrying the fsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) in gelE and an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conserved gelE SNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kb fsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown by fsr-gelE mutants. In conclusion, we describe the identity of fsrD despite high plasticity within the fsrC-EF_1841 region and the surrounding sequence. However, strains lacking the fsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and in gelE, suggesting that the deletion may result from horizontal transfer and recombination.
Enterococcus faecium; fms genes; pili; hyaluronidase; transferable plasmid