Opportunistic Pathogen Enterococcus Faecalis Research Articles

Epidemiology and genetic diversity of linezolid resistant enterococci clinical isolates in Belgium from 2013 to 2021

ObjectivesLinezolid resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are an emerging health threat as limited therapeutic options remain. The aim was to investigate the epidemiology, resistance mechanisms and genetic diversity of Belgian linezolid resistant enterococci (LRE) isolated between 2013 and 2021 received at the Belgian National Reference Centre (NRC) for Enterococci. MethodsLinezolid susceptibility testing was performed upon request on 2458 submitted Enterococci strains. Whole genome sequencing was performed on all LRE strains. ResultsSeventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid resistant E. faecalis strains, 97% harboured the optrA (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA and 23% the optrA gene. Furthermore two E. faecium strains were identified with a combination of two resistance mechanisms ((i) optrA and poxtA and (ii) cfr(B) and G2576T point mutation respectively). Vancomycin resistance was observed in 15% (n=12) of the LRE. ST480 (n=42/63 typed strains, 67%) was the most frequently detected sequence type (STs) in linezolid resistant E. faecalis strains, while ST203 (n=5/15 typed strains, 33%) was the most frequently detected STs in linezolid resistant E. faecium strains. ConclusionsE. faecalis isolates harbouring optrA were the predominant LRE in Belgium with ST480 as the most prominent MLST. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.

A moonlighting role for LysM peptidoglycan binding domains underpins Enterococcus faecalis daughter cell separation

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.

Disruption of the tagF Orthologue in the epa Locus Variable Region of Enterococcus faecalis Causes Cell Surface Changes and Suppresses an eep-Dependent Lysozyme Resistance Phenotype.

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.

A Carotenoid- and Nuclease-Producing Bacterium Can Mitigate Enterococcus faecalis Transformation by Antibiotic Resistance Genes.

Dissemination of antibiotic resistance genes (ARGs) through natural transformation is facilitated by factors that stabilize extracellular DNA (eDNA) and that induce reactive oxygen species (ROS) that permeabilize receptor cells and upregulate transformation competence genes. In this study, we demonstrate that Deinococcus radiodurans can mitigate this ARG dissemination pathway by removing both eDNA and ROS that make recipient cells more vulnerable to transformation. We used plasmid RP4 as source of extracellular ARGs (tetA, aphA, and blaTEM-2) and the opportunistic pathogen Enterococcus faecalis as receptor. The presence of D. radiodurans significantly reduced the transformation frequency from 2.5 ± 0.7 × 10-6 to 7.4 ± 1.4 × 10-7 (p < 0.05). Based on quantification of intracellular ROS accumulation and superoxide dismutase (SOD) activity, and quantitative polymerase chain reaction (qPCR) and transcriptomic analyses, we propose two mechanisms by which D. radiodurans mitigates E. faecalis transformation by ARGs: (a) residual antibiotics induce D. radiodurans to synthesize liposoluble carotenoids that scavenge ROS and thus mitigate the susceptibility of E. faecalis for eDNA uptake, and (b) eDNA induces D. radiodurans to synthesize extracellular nucleases that degrade eARGs. This mechanistic insight informs biological strategies (including bioaugmentation) to curtail the spread of ARGs through transformation.

Antimicrobial tolerance and its role in the development of resistance: Lessons from enterococci.

Bacteria have developed resistance against every antimicrobial in clinical use at an alarming rate. There is a critical need for more effective use of antimicrobials to both extend their shelf life and prevent resistance from arising. Significantly, antimicrobial tolerance, i.e., the ability to survive but not proliferate during antimicrobial exposure, has been shown to precede the development of bona fide antimicrobial resistance (AMR), sparking a renewed and rapidly increasing interest in this field. As a consequence, problematic infections for the first time are now being investigated for antimicrobial tolerance, with increasing reports demonstrating in-host evolution of antimicrobial tolerance. Tolerance has been identified in a wide array of bacterial species to all bactericidal antimicrobials. Of particular interest are enterococci, which contain the opportunistic bacterial pathogens Enterococcus faecalis and Enterococcus faecium. Enterococci are one of the leading causes of hospital-acquired infection and possess intrinsic tolerance to a number of antimicrobial classes. Persistence of these infections in the clinic is of growing concern, particularly for the immunocompromised. Here, we review current known mechanisms of antimicrobial tolerance, and include an in-depth analysis of those identified in enterococci with implications for both the development and prevention of AMR.

Spatial segregation and cooperation in radially expanding microbial colonies under antibiotic stress

Antibiotic resistance in microbial communities reflects a combination of processes operating at different scales. In this work, we investigate the spatiotemporal dynamics of bacterial colonies comprised of drug-resistant and drug-sensitive cells undergoing range expansion under antibiotic stress. Using the opportunistic pathogen Enterococcus faecalis with plasmid-encoded β-lactamase, we track colony expansion dynamics and visualize spatial patterns in fluorescently labeled populations exposed to antibiotics. We find that the radial expansion rate of mixed communities is approximately constant over a wide range of drug concentrations and initial population compositions. Imaging of the final populations shows that resistance to ampicillin is cooperative, with sensitive cells surviving in the presence of resistant cells at otherwise lethal concentrations. The populations exhibit a diverse range of spatial segregation patterns that depend on drug concentration and initial conditions. Mathematical models indicate that the observed dynamics are consistent with global cooperation, despite the fact that β-lactamase remains cell-associated. Experiments confirm that resistant colonies provide a protective effect to sensitive cells on length scales multiple times the size of a single colony, and populations seeded with (on average) no more than a single resistant cell can produce mixed communities in the presence of the drug. While biophysical models of drug degradation suggest that individual resistant cells offer only short-range protection to neighboring cells, we show that long-range protection may arise from synergistic effects of multiple resistant cells, providing surprisingly large protection zones even at small population fractions.

Enterococcus phage Nonaheksakonda infecting clinical isolates of Enterococcus faecalis represents a new lineage in the family Siphoviridae.

Enterococcus phage Nonaheksakonda was isolated from wastewater, using a vancomycin-resistant strain of the opportunistic pathogen Enterococcus faecalis (VRE) as a host. Nonaheksakonda is a lytic phage infecting E. faecalis V583 and clinical isolates with at least four different multi-locus sequence types (MLSTs). The genome is a 41.9-kb double-stranded DNA molecule (34.6% GC) with 74 coding sequences. Comparative analysis revealed only one close relative, Enterococcus phage heks. All other phages had low protein similarity and shared less than 54% nucleotide sequence identity with phage Nonaheksakonda. The most similar phages were all classified and unclassified efquatroviruses. We propose that the phages Nonaheksakonda and heks represent a novel genus within the family Siphoviridae, order Caudovirales, for which we propose the name "Nonaheksakondavirus".

Bacterial lyso-form lipoproteins are synthesized via an intramolecular acyl chain migration

All bacterial lipoproteins share a variably acylated N-terminal cysteine residue. Gram-negative bacterial lipoproteins are triacylated with a thioether-linked diacylglycerol moiety and an N-acyl chain. The latter is transferred from a membrane phospholipid donor to the α-amino terminus by the enzyme lipoprotein N-acyltransferase (Lnt), using an active-site cysteine thioester covalent intermediate. Many Gram-positive Firmicutes also have N-acylated lipoproteins, but the enzymes catalyzing N-acylation remain uncharacterized. The integral membrane protein Lit (lipoprotein intramolecular transacylase) from the opportunistic nosocomial pathogen Enterococcus faecalis synthesizes a specific lysoform lipoprotein (N-acyl S-monoacylglycerol) chemotype by an unknown mechanism that helps this bacterium evade immune recognition by the Toll-like receptor 2 family complex. Here, we used a deuterium-labeled lipoprotein substrate with reconstituted Lit to investigate intramolecular acyl chain transfer. We observed that Lit transfers the sn-2 ester-linked lipid from the diacylglycerol moiety to the α-amino terminus without forming a covalent thioester intermediate. Utilizing Mut-Seq to analyze an alanine scan library of Lit alleles, we identified two stretches of functionally important amino acid residues containing two conserved histidines. Topology maps based on reporter fusion assays and cysteine accessibility placed both histidines in the extracellular half of the cytoplasmic membrane. We propose a general acid base-promoted catalytic mechanism, invoking direct nucleophilic attack by the substrate α-amino group on the sn-2 ester to form a cyclic tetrahedral intermediate that then collapses to produce lyso-lipoprotein. Lit is a unique example of an intramolecular transacylase differentiated from that catalyzed by Lnt, and provides insight into the heterogeneity of bacterial lipoprotein biosynthetic systems.

Safety and Quality of Fish and Game Meats Prepared by First Nations Communities in British Columbia, Canada

Canadian First Nations communities rely on traditional preservation methods such as the smoking, drying, and canning of fish and game meats to ensure long-term food security. Unlike commercial food production, there are no recognized official standards for these methods, rendering their efficacy in producing microbiologically safe foods relatively unknown. In this study, 81 fresh or processed fish and game samples obtained from four British Columbia First Nations communities were analyzed for quality indicator microbes, foodborne pathogens, and mineral levels. Aerobic counts, coliforms (CC), Escherichia coli (EC), lactic acid bacteria, Staphylococcus aureus (STA), and yeast and molds (YM) were enumerated using the TEMPO, whereas the presence of E. coli O157:H7, Listeria monocytogenes, and Salmonella were detected using the VIDAS immunoassay system. The opportunistic pathogens Enterococcus faecalis and Enterococcus faecium were additionally detected using culture methods with subsequent PCR confirmation, and minerals (Ca, Cu, K, Mg, Mn, Na, and Zn) were detected using mass spectrometry. With the exception of STA, microbial loads were significantly (P < 0.05) higher in processed fish and meat samples compared with unprocessed samples, and game samples contained higher microbial levels than fish; however, differences were only significant (P < 0.05) for coliforms, E. coli, and STA. E. coli O157:H7 was detected in one smoked moose sample, and E. faecalis and E. faecium were isolated from 21 and 2 samples, respectively. Although smoked samples contained significantly higher Na levels, they were effective in reducing microbial levels. These results indicate that current food preservation methods practiced by British Columbia First Nations communities are infrequently effective at reducing microbial populations, and in many cases, resulted in increased microbial loads. More efforts should be made to improve the dissemination of safe food handling and processing knowledge to ensure long-term food security and well-being.

Identification of Novel Bacteriophages with Therapeutic Potential That Target Enterococcus faecalis.

The Gram-positive opportunistic pathogen Enterococcus faecalis is frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections. E. faecalis is intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance of E. faecalis to several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that target E. faecalis strains isolated from the oral cavity of patients suffering root canal infections. All phages isolated were Siphoviridae with similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicate E. faecalis biofilms formed in vitro on a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistant E. faecalis infections.

Characterization of the gen locus involved in β-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-β-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β-1,6- rather than β-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

Activation of the extracytoplasmic function σ factor σV by lysozyme.

σV is an extracytoplasmic function (ECF) σ factor that is found exclusively in Firmicutes including Bacillus subtilis and the opportunistic pathogens Clostridioides difficile and Enterococcus faecalis. σV is activated by lysozyme and is required for lysozyme resistance. The activity of σV is normally inhibited by the anti-σ factor RsiV, a transmembrane protein. RsiV acts as a receptor for lysozyme. The binding of lysozyme to RsiV triggers a signal transduction cascade which results in degradation of RsiV and activation of σV . Like the anti-σ factors for several other ECF σ factors, RsiV is degraded by a multistep proteolytic cascade that is regulated at the step of site-1 cleavage. Unlike other anti-σ factors, site-1 cleavage of RsiV is not dependent upon a site-1 protease whose activity is regulated. Instead constitutively active signal peptidase cleaves RsiV at site-1 in a lysozyme-dependent manner. The activation of σV leads to the transcription of genes, which encode proteins required for lysozyme resistance.

Using Nanoscopy To Probe the Biological Activity of Antimicrobial Leads That Display Potent Activity against Pathogenic, Multidrug Resistant, Gram-Negative Bacteria.

Medicinal leads that are also compatible with imaging technologies are attractive, as they facilitate the development of therapeutics through direct mechanistic observations at the molecular level. In this context, the uptake and antimicrobial activities of several luminescent dinuclear RuII complexes against E.coli were assessed and compared to results obtained for another ESKAPE pathogen, the Gram-positive major opportunistic pathogen Enterococcus faecalis, V583. The most promising lead displays potent activity, particularly against the Gram-negative bacteria, and potency is retained in the uropathogenic multidrug resistant EC958 ST131 strain. Exploiting the inherent luminescent properties of this complex, super-resolution STED nanoscopy was used to image its initial localization at/in cellular membranes and its subsequent transfer to the cell poles. Membrane damage assays confirm that the complex disrupts the bacterial membrane structure before internalization. Mammalian cell culture and animal model studies indicate that the complex is not toxic to eukaryotes, even at concentrations that are several orders of magnitude higher than its minimum inhibitory concentration (MIC). Taken together, these results have identified a lead molecular architecture for hard-to-treat, multiresistant, Gram-negative bacteria, which displays activities that are already comparable to optimized natural product-based leads.

Fecal Bacterial Communities in Insectivorous Bats from the Netherlands and Their Role as a Possible Vector for Foodborne Diseases

Bats are commonly regarded as vectors for viruses, but little is known about bacterial communities in bats and the possible role of bats in the transmission cycle of foodborne diseases. To gain more insight, microbial communities in fecal samples from 37 insectivorous bats of different species from the Netherlands were investigated by polymerase chain reaction and denaturant gradient gel electrophoresis (PCR-DGGE). Subsequently, 10 samples from the following bat species: common pipistrelle (Pipistrellus pipistrellus; n = 3), Daubenton's bat (Myotis daubentonii; n = 3), serotine bat (Eptesicus serotinus; n = 1), whiskered bat (Myotis mystacinus; n = 1), Geoffroy's bat (Myotis emarginatus; n = 1) and Natterer's bat (Myotis nattereri; n = 1) were selected and used in bacterial 16S rDNA cloning and sequencing. The fecal microbiota in bats was found to be diverse with predominant bacterial genera Carnobacterium, Serratia, Pseudomonas, Enterococcus and Yersinia. The presence of opportunistic pathogens Citrobacter freundii, Escherichia coli, Enterococcus faecalis, Serratia fonticola and Rahnella aquatilis was also recorded. Based on cloning results, we found no proof that bats in the Netherlands are a major vector for the transmission of bacterial zoonotic diseases, although previous findings in literature reported isolation of foodborne pathogens from bats.

Comparative evaluation of disinfection mechanism of sodium hypochlorite, chlorine dioxide and electroactivated water on Enterococcus faecalis

The aim of this study is to compare the efficiency of chlorine-based disinfectants, namely, sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), and electroactivated water (EAW), and to investigate their inactivation mechanisms on a Gram positive, opportunistic pathogen Enterococcus faecalis. Cell viability, damages on membrane integrity and cellular components were examined to reveal underlying mechanisms of the disinfection process. Among three disinfectants, EAW was the most effective one to inactivate E. faecalis in less time with more damage. According to flow cytometry results, EAW destroyed 95.4% of bacteria within 1 min whereas NaOCl and ClO2 provided 73.9% and 72.6% bacterial inactivation after 20 min of treatment. The rapid increment at conductivity and lipid peroxidation within 30 s of treatment indicates that EAW causes membrane damage much more expeditiously compared to other disinfectants. DNA and protein leakage increased gradually during the treatment with EAW and reached 44.9 and 5.8 μg/ml, respectively. All disinfectants were found as inactivating the bacteria by disrupting cell membrane integrity, unbalancing the ion concentration and damaging the cellular components such as DNA, protein, lipid molecules. It is considered that EAW is more effective due to its superior content including chlorine dioxide, free radicals and ozone besides hypochlorous acid.

An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition.

ABSTRACTAntibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this “CRISPR tolerance.” We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.

Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections.

Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In E. faecalis and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from E. faecalis, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive E. faecalis strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of E. faecalis in vitro. We also show that two daptomycin-resistant (DapR) strains of E. faecalis have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen E. faecalis, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked.

Detection of Esp gene for the presence of extracellular surface protein among clinical isolates of Enterococcus species

Esp gene is a high molecular weight surface protein whose frequency is significantly increased among infection derived Enterococcus faecalis isolates. Esp gene is an Enterococcal surface protein gene which is present in the opportunistic pathogens Enterococcus faecalis and Enterococcus faecium. It has been proposed that esp gene can be used as a marker of human pollution in environmental waters. This gene has been shown to be a part of a 150-in putative pathogenicity island. A sum of 20 isolates of Enterococci were assessed for the presence of esp gene that encodes for extracellular surface protein. We have observed 5% positivity in our study. This indicates that, the pathogenicity of urinary tract infections by these pathogens might be crucially played by this gene encoding for extra cellular surface protein.