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Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients., (Copyright © 2018 American Society for Microbiology.)

Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.

Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfm disruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfm is associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfm mutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfm mutant. These findings indicate that AtlAEfm is important in biofilm and collagen binding in E. faecium, making AtlAEfm a promising target for treatment of E. faecium infections.

Enterococcus faecalis and Enterococcus faecium are among the leading causative agents of nosocomial infections and are infamous for their resistance to many antibiotics. They cause difficult-to-treat infections, often originating from biofilm-mediated infections associated with implanted medical devices or endocarditis. Biofilms protect bacteria against antibiotics and phagocytosis, and physical removal of devices or infected tissue is often needed but is frequently not possible. Currently there are no clinically available compounds that disassemble biofilms. In this review we discuss all known structural and regulatory genes involved in enterococcal biofilm formation, the compounds directed against biofilm formation that have been studied, and potentially useful targets for future drugs to treat enterococcal biofilm-associated infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hospital-acquired clonal complex 17 (CC17) Enterococcus faecium strains are genetically distinct from indigenous strains and are enriched with resistance genes and virulence genes. We identified a genomic island in CC17 E. faecium tentatively encoding a metabolic pathway involved in carbohydrate transport and metabolism, which may provide a competitive advantage over the indigenous E. faecium microbiota.

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 clade-specific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing-based clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment.

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.

To substantiate a common genetic background of ciprofloxacin-resistant Enterococcus faecium, 32 ciprofloxacin-resistant (Cip(r)) and 31 ciprofloxacin-susceptible (Cip(s)) isolates from outbreaks, clinical infections, surveillances, and animals from 10 different countries were genotyped by multilocus sequence typing. Additionally, susceptibilities to ampicillin and vancomycin and the presence of esp were determined and the quinolone resistance-determining regions of parC, gyrA, parB, and gyrE were sequenced. High-level Cip(r) (MIC > or = 64 microg/ml) due to point mutations in the quinolone resistance-determining region was unique to a distinct hospital-adapted genetic complex in E. faecium, previously designated CC17. Low-level Cip(r) (MIC = 4 microg/ml) in non-CC17 strains is not attributable to point mutations in any subunit of the topoisomerase genes, and the mechanism of resistance remains unclear. Acquisition of mutations in parC and gyrA, leading to high-level Cip(r), is, in addition to ampicillin resistance and the presence of a putative pathogenicity island, another cumulative step in hospital adaptation of CC17.

The epidemiology of vancomycin-resistant Entero- coccus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.

Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization. [ABSTRACT FROM AUTHOR]