New Insights into the Antimicrobial Effect of Mast Cells against Enterococcus faecalis

Enterococcus faecalis has emerged as an important cause of life-threatening multidrug-resistant bacterial infections in the hospital setting. The pathogenesis of enterococcal infections has remained a relatively neglected field despite their obvious clinical relevance. The objective of this study was to characterize the interactions between mast cells (MCs), an innate immune cell population abundant in the intestinal lamina propria, and E. faecalis. This study was conducted with primary bone marrow-derived murine MCs. The results demonstrated that MCs exerted an antimicrobial effect against E. faecalis that was mediated both by degranulation, with the concomitant discharge of the antimicrobial effectors contained in the granules, and by the release of extracellular traps, in which E. faecalis was snared and killed. In particular, the cathelicidin LL-37 released by the MCs had potent antimicrobial effect against E. faecalis. We also investigated the specific receptors involved in the recognition of E. faecalis by MCs. We found that Toll-like receptors (TLRs) are critically involved in the MC recognition of E. faecalis, since MCs deficient in the expression of MyD88, an adaptor molecule required for signaling by most TLRs, were significantly impaired in their capacity to degranulate, to reduce E. faecalis growth as well as to release tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) after encountering this pathogen. Furthermore, TLR2 was identified as the most prominent TLR involved in the recognition of E. faecalis by MCs. The results of this study indicate that MCs may be important contributors to the host innate immune defenses against E. faecalis.     

Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC)

Initial Enterococcus faecalis–endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria–cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium–bacterium aggregation and bacterium–host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.     

Zebrafish as a Novel Vertebrate Model To Dissect Enterococcal Pathogenesis

Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence.     

Independent risk factors for the co-colonization of vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus in the region most endemic for vancomycin-resistant Staphylococcus aureus isolation

In the majority of cases of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococcus faecalis (VR E. faecalis) served as the vanA donor to S. aureus. Previous studies that evaluated the risk factors for co-colonization with VRE and MRSA did not differentiate between VR E. faecalis and VR E. faecium. This study aimed to identify variables associated with VR E. faecalis and MRSA co-colonization. A retrospective case–control study from January 2008 to December 2009 was conducted at the Detroit Medical Center. Data were extracted from charts and pharmacy records. Unique patients co-colonized with VR E. faecalis and MRSA (defined as isolation of MRSA within 7 days of VR E. faecalis isolation) were compared with patients with VR E. faecalis who were not co-colonized with MRSA. A total of 546 patients with VR E. faecalis isolation were identified. 85 (15.6 %) VR E. faecalis patients were co-colonized with MRSA and 461 (84.4 %) VR E. faecalis patients were not co-colonized with MRSA. The mean age of the study cohort was 65.9?±?16.4 years, 424 (77.7 %) were African–American, and 270 (49.5 %) were residing in long-term care institutions. Independent predictors of co-colonization of VR E. faecalis and MRSA were male gender, impaired consciousness, ICU stay prior to VR E. faecalis isolation, indwelling devices, and isolation of VR E. faecalis from wounds. MRSA was frequently isolated from the same culture specimen as VR E. faecalis (n?=?39, 45.9 %), most commonly from wounds. This large study of patients with VR E. faecalis identified the severity of illness, indwelling devices, and chronic wounds as independent predictors of co-colonization with VR E. faecalis and MRSA     

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2^−/−) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2^−/− mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2^−/− mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2^−/− mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.     

Characterization and genome analysis of novel phage vB_EfaP_IME195 infecting <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

Enterococcus faecalis is one of the main bacteria in the human and animal intestine but is also classed as an opportunistic pathogen. During normal growth, E. faecalis produces natural antibiotics and is conducive to human health. As ectopic parasites, E. faecalis is capable of causing infective endocarditis, neonatal sepsis, bloodstream infections, bacteremia, and intraabdominal infections. With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control is a promising treatment option to combat bacterial resistance. In this study, a lytic phage, designated vB_EfaP_IME195, was isolated from hospital sewage using a clinical multidrug-resistant Enterococcus faecalis strain as an indicator. The one-step growth curve with the optimal multiplicity of infection of (MOI) 0.01 revealed a latent period of ~?30 min and a burst size of ~?120 plaque-forming units (pfu) per cell. Transmission electron microscopy showed that the phage belongs to the family Podoviridae. Phage vB_EfaP_IME195 has a linear, double-stranded DNA genome of 18,607 bp with a G?+?C content of 33% and 27 coding sequences (GenBank accession no. KT932700). Run-off sequencing experiments showed that the phage has a unique 59-bp inverted repeat sequences at the terminal ends. BLASTn analysis revealed that vB_EfaP_IME195 shares 92% identity (93% genome coverage) with unpublished E. faecalis phage Idefix. This study reported a novel E. faecalis phage with unique genome termini containing inverted repeats. The isolation and characterization of this novel lytic E. faecalis phage provides the basis for the development of new therapeutic agents like phage cocktails for multidrug-resistant E. faecalis infection, and its unique genomic feature would also provide valuable knowledge and insight for further phage genome analysis.     

Enterococcus faecalis Infection Activates Phosphatidylinositol 3-Kinase Signaling To Block Apoptotic Cell Death in Macrophages

Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.     

Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections.     

Virulence factors in isolates of Enterococcus faecalis from infective endocarditis and from the normal flora

Infective endocarditis (IE) is a severe infection associated with significant morbidity and mortality. Enterococcus faecalis is among the most common causes of this disease, and yet little is known about the pathogenesis of E. faecalis IE. We screened 21 E. faecalis isolates from IE and 21 isolates from normal flora for the putative virulence factors ace, asa1, gelE, and esp with PCR. In addition, we determined the ability of the isolates to form biofilm and to aggregate platelets. With the exception of biofilm formation, which was more pronounced in the normal flora group, there was no difference between the groups, indicating that many isolates have virulence properties and that host factors might determine if E. faecalis causes IE.     

Infection-Derived Enterococcus faecalis Strains Are Enriched in esp, a Gene Encoding a Novel Surface Protein

We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of ~202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.     

Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans

The Gram-positive bacterium Enterococcus faecalis and the fungus Candida albicans are both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using a Caenorhabditis elegans model of polymicrobial infection, we discovered that E. faecalis and C. albicans negatively impact each other''s virulence. Much of the negative effect of E. faecalis on C. albicans was due to the inhibition of C. albicans hyphal morphogenesis, a developmental program crucial to C. albicans pathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence in E. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibit C. albicans filamentation in the context of an in vitro biofilm. Finally, a screen of an E. faecalis transposon mutant library identified other genes required for suppression of C. albicans hyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.     

Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals

The high prevalence of Enterococcus faecalis in root canal treated teeth with post-treatment disease, as evidenced by both molecular and traditional culturing methods, suggests that this species may be a key player in endodontic treatment failure. This study aimed to detect virulence factors by phenotypic and western blotting tests, and virulence genes by PCR from 20 clinical strains of E. faecalis isolated from treated root canals of teeth with (10) or without (10) apical periodontitis. Moreover, genomic diversity of these strains was assessed by pulsed-field gel electrophoresis (PFGE) and rep-PCR. All 20 strains presented the gelE gene (gelatinase), but 10 of them did not hydrolyze gelatin. Seven of the 10 gelatinase-producing isolates were recovered from root canals with lesions, which suggests a role for this virulence factor in the pathogenesis of post-treatment disease. The esp gene was expressed only in cases where gelatinase production was negative. The other virulence genes were found in 90% (efaA and ace genes), 45% (agg gene) and 95% (cpd gene) of the E. faecalis isolates. As for PFGE and rep-PCR, no specific clonal type of E. faecalis was found in association with teeth with or without disease, revealing the interindividual clonal diversity of endodontic infections.     

Prevalence,outcome and risk factor associated with vancomycin-resistant Enterococcus faecalis and Enterococcus faecium at a Tertiary Care Hospital in Northern India

Purpose: To determine the prevalence, genotype, risk factors and mortality in patients having vancomycin-resistant Enterococcus faecalis (VR E. faecalis) and Enterococcus faecium (VR E. faecium) infection or colonisation. Materials and Methods: A total of 1488 clinical isolates of E. faecalis and E. faecium were tested for vancomycin resistance by phenotypic (disk diffusion, E-test and broth micro-dilution test) and genotypic polymerase chain reaction methods. Records of all 1488 patients who had E. faecalis or E. faecium infection or colonisation were reviewed for the identification of host, hospital and medication related risk factors associated with VR E. faecalis and VR E. faecium. Results: Of 1488 isolates, 118 (7.9%) were vancomycin-resistant and their distributions were as follows: E. faecalis =72 (61%) and E. faecium =46 (39%). All 118 vancomycin-resistant isolates were vanA genotype (minimum inhibitory concentration [MIC] to vancomycin ≥64 μg/ml and MIC to teicoplanin ≥32 μg/ml) and none of the isolates was vanB genotype. Multivariate logistic regression analysis identified ventilator support and hospital stay for ≥48 h as independent risk factors associated with VR E. faecalis and VR E. faecium infection or colonisation. Hospital stay ≥48 h was the only independent risk factor for mortality in patients infected with vancomycin-resistant enterococci. Conclusions: Strategies to limit the nosocomial infection especially in patients on ventilator support can reduce VRE incidence and related mortality.     

EfaR Is a Major Regulator of Enterococcus faecalis Manganese Transporters and Influences Processes Involved in Host Colonization and Infection

Metal ions, in particular manganese, are important modulators of bacterial pathogenicity. However, little is known about the role of manganese-dependent proteins in the nosocomial pathogen Enterococcus faecalis, a major cause of bacterial endocarditis. The present study demonstrates that the DtxR/MntR family metalloregulator EfaR of E. faecalis controls the expression of several of its regulon members in a manganese-dependent way. We also show that efaR inactivation impairs the ability of E. faecalis to form biofilms, to survive inside macrophages, and to tolerate oxidative stress. Our results reveal that EfaR is an important modulator of E. faecalis virulence and link manganese homeostasis to enterococcal pathogenicity.     

Influence ofEscherichia coli onStreptococcus faecalis in mixed cultures and experimental animal infections

Strains ofEscherichia coli (four strains) andStreptococcus faecalis (three strains) randomly isolated from faecal samples were cultured together in vitro and in experimental animal infections in order to evaluate the interaction of these two species.Streptococcus faecalis was eliminated from mixed cultures and infections at population ratios ofEscherichia coli toStreptococcus faecalis of 10–100∶1. The mechanism involved was probably an antibiotic substance produced byEscherichia coli which was antagonistic toStreptococcus faecalis. The substance appeared to be diffusible, water-soluble, of low molecular weight and insensitive to the protease trypsin. It was produced and remained active in vitro and in vivo and can probably be classified as a microcin.     

A Genomic Virulence Reference Map of Enterococcus faecalis Reveals an Important Contribution of Phage03-Like Elements in Nosocomial Genetic Lineages to Pathogenicity in a Caenorhabditis elegans Infection Model

In the present study, the commensal and pathogenic host-microbe interaction of Enterococcus faecalis was explored using a Caenorhabditis elegans model system. The virulence of 28 E. faecalis isolates representing 24 multilocus sequence types (MLSTs), including human commensal and clinical isolates as well as isolates from animals and of insect origin, was investigated using C. elegans strain glp-4 (bn2ts); sek-1 (km4). This revealed that 6 E. faecalis isolates behaved in a commensal manner with no nematocidal effect, while the remaining strains showed a time to 50% lethality ranging from 47 to 120 h. Principal component analysis showed that the difference in nematocidal activity explained 94% of the variance in the data. Assessment of known virulence traits revealed that gelatinase and cytolysin production accounted for 40.8% and 36.5% of the observed pathogenicity, respectively. However, coproduction of gelatinase and cytolysin did not increase virulence additively, accounting for 50.6% of the pathogenicity and therefore indicating a significant (26.7%) saturation effect. We employed a comparative genomic analysis approach using the 28 isolates comprising a collection of 82,356 annotated coding sequences (CDS) to identify 2,325 patterns of presence or absence among the investigated strains. Univariate statistical analysis of variance (ANOVA) established that individual patterns positively correlated (n = 61) with virulence. The patterns were investigated to identify potential new virulence traits, among which we found five patterns consisting of the phage03-like gene clusters. Strains harboring phage03 showed, on average, 17% higher killing of C. elegans (P = 4.4e⁻⁶). The phage03 gene cluster was also present in gelatinase-and-cytolysin-negative strain E. faecalis JH2-2. Deletion of this phage element from the JH2-2 clinical strain rendered the mutant apathogenic in C. elegans, and a similar mutant of the nosocomial V583 isolate showed significantly attenuated virulence. Bioinformatics investigation indicated that, unlike other E. faecalis virulence traits, phage03-like elements were found at a higher frequency among nosocomial isolates. In conclusion, our report provides a valuable virulence map that explains enhancement in E. faecalis virulence and contributes to a deeper comprehension of the genetic mechanism leading to the transition from commensalism to a pathogenic lifestyle.     

Longer Intestinal Persistence of Enterococcus faecalis Compared to Enterococcus faecium Clones in Intensive-Care-Unit Patients

The dynamics of intestinal colonization with enterococcal clones in intensive-care-unit (ICU) patients was evaluated. Eight patients admitted directly to the neurosurgical ICU at the Ramón y Cajal University Hospital (Madrid, Spain) from the community and with no overlapping stay during a 10-month period in 2006 were studied. Rectal swab specimens were collected on admission and daily until the patients were discharged. Clonality was determined by pulsed-field gel electrophoresis and multilocus sequence typing. Clonal colonization dynamics were estimated by using two new parameters: the clonal diversity per patient per day (CDPD) and the clonal persistence ratio (CPR). Enterococcus faecalis isolates (n = 123) and Enterococcus faecium isolates (n = 66) were resolved into 13 and 15 clones, respectively. The CDPD of E. faecalis steadily increased during admission, and E. faecalis showed a higher (P = 0.001) CPR value than E. faecium (0.86 and 0.42, respectively). E. faecium, with the exception of an ampicillin-resistant clone belonging to clonal complex 17, frequently appeared as a short-term colonizer, even though the E. faecalis clones had significantly (P = 0.03) more days under antibiotic exposure than E. faecium (77.5 and 65 days/100 colonization days, respectively). E. faecalis had a longer persistence than E. faecium, except for the CC17 ampicillin-resistant clone, and E. faecalis showed a cumulative increase in CDPD, whereas E. faecium did not. CDPD and CPR were useful for measuring the dynamics of intestinal colonization with enterococcal clones.     

Molecular Epidemiology of Enterococcal Bacteremia in Australia

Enterococci are a major cause of health care-associated infections and account for approximately 10% of all bacteremias globally. The aim of this study was to determine the proportion of enterococcal bacteremia isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to ampicillin and the glycopeptides, and to characterize the molecular epidemiology of the Enterococcus faecalis and Enterococcus faecium isolates. From 1 January to 31 December 2011, 1,079 unique episodes of bacteremia were investigated, of which 95.8% were caused by either E. faecalis (61.0%) or E. faecium (34.8%). The majority of bacteremias were health care associated, and approximately one-third were polymicrobial. Ampicillin resistance was detected in 90.4% of E. faecium isolates but was not detected in E. faecalis isolates. Vancomycin nonsusceptibility was reported in 0.6% and 36.5% of E. faecalis and E. faecium isolates, respectively. Unlike Europe and the United States, where vancomycin resistance in E. faecium is predominately due to the acquisition of the vanA operon, 98.4% of E. faecium isolates harboring van genes carried the vanB operon, and 16.1% of the vanB E. faecium isolates had vancomycin MICs at or below the susceptible breakpoint of the CLSI. Although molecular typing identified 126 E. faecalis pulsed-field gel electrophoresis pulsotypes, >50% belonged to two pulsotypes that were isolated across Australia. E. faecium consisted of 73 pulsotypes from which 43 multilocus sequence types were identified. Almost 90% of the E. faecium isolates were identified as CC17 clones, of which approximately half were characterized as ST203, which was isolated Australia-wide. In conclusion, the Australian Enterococcal Sepsis Outcome Programme (AESOP) study has shown that although they are polyclonal, enterococcal bacteremias in Australia are frequently caused by ampicillin-resistant vanB E. faecium.     

Incidence,clinical characteristics and 30-day mortality of enterococcal bacteraemia in Denmark 2006–2009: a population-based cohort study

Enterococci currently account for approximately 10% of all bacteraemias, reflecting remarkable changes in their epidemiology. However, population-based data of enterococcal bacteraemia are scarce. A population-based cohort study comprised all patients with a first episode of Enterococcus faecalis or Enterococcus faecium bacteraemia in two Danish regions during 2006–2009. We used data collected prospectively during clinical microbiological counselling and hospital registry data. We determined the incidence of mono- and polymicrobial bacteraemia and assessed clinical and microbiological characteristics as predictors of 30-day mortality in monomicrobial bacteraemia by logistic regression analysis. We identified 1145 bacteraemic patients, 700 (61%) of whom had monomicrobial bacteraemia. The incidence was 19.6/100 000 person-years (13.0/100 000 person-years for E. faecalis and 6.6/100 000 person-years for E. faecium). The majority of bacteraemias were hospital-acquired (E. faecalis, 45.7%; E. faecium, 85.2%). Urinary tract and intra-abdominal infections were the predominant foci for the two species, respectively. Infective endocarditis (IE) accounted for 25% of patients with community-acquired E. faecalis bacteraemia. Thirty-day mortality was 21.4% in patients with E. faecalis and 34.6% in patients with E. faecium. Predictors of 30-day mortality included age, co-morbidity and hospital-acquired bacteraemia. In addition, intra-abdominal infection, unknown focus and high-level gentamicin resistance were predictors of mortality in E. faecalis patients. E. faecium was associated with increased risk of mortality compared with E. faecalis. The study emphasizes the importance of enterococci both in terms of incidence and prognosis. The frequency of IE in patients with E. faecalis bacteraemia emphasizes the importance of echocardiography, especially in community-acquired cases.