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Enterococci, common hospital-acquired infections in immunocompromised patients, have garnered attention in clinical microbiology. To determine the clinical relevance of enterococci as food-borne pathogens, 116 fish, 90 vegetables, and 120 human diarrheal samples were tested for E. faecalis and E. faecium pathogenicity. Conventionally, 69 of 326 (21.17%) samples were positive for Enterococcus species, 52 (15.95%) of which were molecularly classified as E. faecalis and 13 (3.99%) as E. faecium. The E. faecalis contamination percentage of fresh fish (19.70%) was higher than frozen fish (4%). Cauliflower had the highest E. faecalis percentage (16.67%) when fish and vegetable samples didn't harbor the E. faecium atpA gene. 23.33% and 10.83% of participants' samples were molecularly confirmed as E. faecalis and E. faecium positive, respectively. E. faecalis isolates had all virulence genes, with gels being the most common (65.38%), while cylA and asa1 genes couldn't be detected in E. faecium isolates. E. faecalis showed the highest resistance against vancomycin and tetracycline (69.23%), whereas E. faecium extremely resisted tetracycline (76.92%) and erythromycin (69.23%) with the recognition of MDR among 44.2% of E. faecalis and 38.5% of E. faecium isolates. The great similarity of our isolates showed the clinical importance of food-borne antibiotic-resistant enterococci., (© 2024. The Author(s).)

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

This study presents the empirical findings of an in-depth genomic analysis of Enterococcus faecalis and Enterococcus lactis isolates from South Africa. It offers valuable insights into their genetic characteristics and their significant implications for public health. The study uncovers nuanced variations in the gene content of these isolates, despite their similar GC contents, providing a comprehensive view of the evolutionary diversity within the species. Genomic islands are identified, particularly in E. faecalis, emphasizing its propensity for horizontal gene transfer and genetic diversity, especially in terms of antibiotic resistance genes. Pangenome analysis reveals the existence of a core genome, accounting for a modest proportion of the total genes, with 2157 core genes, 1164 shell genes, and 4638 cloud genes out of 7959 genes in 52 South African E. faecalis genomes (2 from this study, 49 south Africa genomes downloaded from NCBI, and E. faecalis reference genome). Detecting large-scale genomic rearrangements, including chromosomal inversions, underscores the dynamic nature of bacterial genomes and their role in generating genetic diversity. The study uncovers an array of antibiotic resistance genes, with trimethoprim, tetracycline, glycopeptide, and multidrug resistance genes prevalent, raising concerns about the effectiveness of antibiotic treatment. Virulence gene profiling unveils a diverse repertoire of factors contributing to pathogenicity, encompassing adhesion, biofilm formation, stress resistance, and tissue damage. These empirical findings provide indispensable insights into these bacteria's genomic dynamics, antibiotic resistance mechanisms, and virulence potential, underlining the pressing need to address antibiotic resistance and implement robust control measures., (© 2024. The Author(s).)

Aim: Previous endodontic research has provided limited understanding of the prevalence and roles of haemolytic and non-haemolytic Enterococcus faecalis strains in root filled teeth. This study aimed to determine the prevalence of these strains in root filled teeth with periradicular lesions and investigate their associated virulence factors., Methodology: A total of 36 root canal samples were collected from 36 subjects. The prevalence of E. faecalis was determined using culture and PCR methods. Antibiotic susceptibility of haemolytic and non-haemolytic E. faecalis strains was assessed using the broth dilution assay. The cytokine stimulation in periodontal ligament (PDL) cells and neutrophil migration were evaluated using real-time PCR and migration assay, respectively. Cell invasion ability of the strains was assessed using a cell culture model. Additionally, the virulence gene expression of the haemolytic and non-haemolytic strains was investigated using real-time PCR. The Mann-Whitney U and Spearman's ρ tests were used to examine the significant difference between the two strains and to analyse the correlation between phenotype and gene expression, respectively., Results: Enterococcus faecalis was detected in 33.3% and 88.9% of samples by culture and real-time PCR, respectively. Haemolytic strains were found in 36.4% of subjects. Non-haemolytic strains exhibited susceptibility to erythromycin and varying susceptibility to tetracycline, while all haemolytic strains were resistant to both antibiotics. Haemolytic strains significantly upregulated the expression of IL-8, OPG and RANKL in PDL cells (p < .05). Notably, the fold increases in these genes were higher: IL-8 (556.1 ± 82.9 vs. 249.6 ± 81.8), OPG (2.2 ± 0.5 vs. 1.3 ± 0.2) and RANKL (1.8 ± 0.3 vs. 1.2 ± 0.1). Furthermore, haemolytic strains had a greater effect on neutrophil migration (68.7 ± 15.2% vs. 46.9 ± 11.4%) and demonstrated a higher level of internalization into oral keratinocyte cells (68.6 ± 0.4% vs. 33.8 ± 0.5%) (p < .05). They also showed enhanced expression of virulence genes associated with haemolysin, surface proteins, collagen-binding and aggregation substances. Gelatinase activity was only detectable in non-haemolytic strains., Conclusions: This study revealed that haemolytic strains E. faecalis possessed enhanced abilities in host invasion and a higher abundance of virulence factors, suggesting their potential contribution to more severe disease manifestations., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues, with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this study, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems (Δ5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron deficiency. Virulence of the Δ5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of Δ5Fe under iron depletion and virulence in an invertebrate infection model. This study revealed that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factor V and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 μM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factor V after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factor V reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The Enterococci, mainly Enterococcus faecalis and E. faecium, are ubiquitous members of the human gastrointestinal tract consortia but also a leading cause of opportunistic infections. The global rise in human-associated enterococcal infections, often caused by multidrug resistant strains, highlights an urgent need to identify the bacterial factors contributing to its pathogenicity such that new therapies can be devised. The use of the Galleria mellonella (greater wax moth) larvae, commonly known as wax worm, as a model to study host-pathogen interactions has allowed the identification and characterization of numerous bacterial factors that contribute to disease in humans, serving both as an alternative and complementary approach to mammalian models. Here, we describe the methods for using G. mellonella to characterize the virulence factors of E. faecalis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) ( ef0114 , ef0361 , and ef2863 ), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or β-1,4 endo-β- N -acetylglucosaminidases (ENGases) based on their β-1,4 endo- N -acetyl-β-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro . In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis ( cdaA ) and degradation ( dhhP and gdpP ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ cdaA strain) or c-di-AMP accumulation (Δ dhhP , Δ gdpP , and Δ dhhP Δ gdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the Δ cdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo , and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the Δ cdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

The purpose of this study was to design silver nanoparticles (AgNPs) poloxamer thermoreversible gel (AgNPs-PL) and investigate whether this gel could provide sustained antibacterial activity against Enterococcus faecalis (E. faecalis) in the root canal. The gels fabricated were characterized in terms of gelatin temperature, particle size, in-vitro Ag + release, and elemental content. Cytotoxicity of AgNPs-PL on primary human periodontal ligament fibroblasts (HPDLFs) was examined by CCK-8 assay. Characterization of AgNPs-PL gel revealed that it contained particles existing as large clumps/fused aggregates of different shapes, with a mean diameter of 21.624 ± 14.689 nm, exhibited sustained release of Ag + for 9 days, and non-toxic to HPDLFs at a low dose (4-32 μg/mL) through 24, 48, and 72 h exposures. The antibacterial effect of 16 and 32 μg/mL concentrations of AgNPs-PL was compared with blank poloxamer gel (PL) and calcium hydroxide (CH) using three methods: (I) agar counting plate, (II) scanning electron microscope (SEM) observations, and (III) confocal laser scanning microscope (CLSM) analysis. AgNPs-PL at the two doses above was more effective than PL and CH in removing E. faecalis biofilm at 1, 3, 9 days. Thus, AgNPs-PL exhibits strong activity against E. faecalis and is easy to produce, with a continuous release profile of Ag + . AgNPs-PL gel may be a candidate for a new root canal disinfection.

Upon exposure to harmful microorganisms, hosts engage in protective molecular and behavioral immune responses, both of which are ultimately regulated by the nervous system. Using the nematode Caenorhabditis elegans , we show that ingestion of Enterococcus faecalis leads to a fast pathogen avoidance behavior that results in aversive learning. We have identified multiple sensory mechanisms involved in the regulation of avoidance of E. faecalis. The G-protein coupled receptor NPR-1-dependent oxygen-sensing pathway opposes this avoidance behavior, while an ASE neuron-dependent pathway and an AWB and AWC neuron-dependent pathway are directly required for avoidance. Colonization of the anterior part of the intestine by E. faecalis leads to AWB and AWC mediated olfactory aversive learning. Finally, two transient receptor potential melastatin (TRPM) channels, GON-2 and GTL-2, mediate this newly described rapid pathogen avoidance. These results suggest a mechanism by which TRPM channels may sense the intestinal distension caused by bacterial colonization to elicit pathogen avoidance and aversive learning by detecting changes in host physiology., Competing Interests: AF, JL, AA No competing interests declared, (© 2021, Filipowicz et al.)

Artificial sweeteners (AS) are synthetic sugar substitutes that are commonly consumed in the diet. Recent studies have indicated considerable health risks which links the consumption of AS with metabolic derangements and gut microbiota perturbations. Despite these studies, there is still limited data on how AS impacts the commensal microbiota to cause pathogenicity. The present study sought to investigate the role of commonly consumed AS on gut bacterial pathogenicity and gut epithelium-microbiota interactions, using models of microbiota ( Escherichia coli NCTC10418 and Enterococcus faecalis ATCC19433) and the intestinal epithelium (Caco-2 cells). Model gut bacteria were exposed to different concentrations of the AS saccharin, sucralose, and aspartame, and their pathogenicity and changes in interactions with Caco-2 cells were measured using in vitro studies. Findings show that sweeteners differentially increase the ability of bacteria to form a biofilm. Co-culture with human intestinal epithelial cells shows an increase in the ability of model gut bacteria to adhere to, invade and kill the host epithelium. The pan-sweet taste inhibitor, zinc sulphate, effectively blocked these negative impacts. Since AS consumption in the diet continues to increase, understanding how this food additive affects gut microbiota and how these damaging effects can be ameliorated is vital.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

Enterococcus  faecalis is a significant cause of hospital-acquired bacteremia. Herein, the discovery is reported that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those formed by Streptococcus pneumoniae during invasive pneumococcal disease. However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation. Rather, disulfide bond forming protein A (DsbA) was found to be required for E. faecalis virulence in a Caenorhabditis elegans model and was necessary for efficient cardiac microlesion formation. Furthermore, E. faecalis promoted cardiomyocyte apoptotic and necroptotic cell death at sites of microlesion formation. Additionally, loss of DsbA caused an increase in proinflammatory cytokines, unlike the wild-type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterium and the host response that are mechanistically involved., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The nematode Caenorhabditis elegans is commonly used as a model organism in studies of the host immune response. The worm encodes twelve peroxidase-cyclooxygenase superfamily members, making it an attractive model in which to study the functions of heme peroxidases. In previous work, loss of one of these peroxidases, SKPO-1 (ShkT-containing peroxidase), rendered C. elegans more sensitive to the human, Gram-positive pathogen Enterococcus faecalis. SKPO-1 was localized to the hypodermis of the animals where it also affected cuticle development as indicated by a morphological phenotype called "dumpy." In this work, a better understanding of how loss of skpo-1 impacts both sensitivity to pathogen as well as cuticle development was sought by subjecting a deletion mutant of skpo-1 to transcriptome analysis using RNA sequencing following exposure to control (Escherichia coli) and pathogenic (E. faecalis) feeding conditions. Loss of skpo-1 caused a general upregulation of genes encoding collagens and other proteins related to cuticle development. On E. faecalis, these animals also failed to upregulate guanylyl cyclases that are often involved in environmental sensing. Hoechst straining revealed increased permeability of the cuticle and atomic force microscopy exposed the misalignment of the cuticular annuli and furrows. These findings provide a basis for better understanding of the morphological as well as the pathogen sensitivity phenotypes associated with loss of SKPO-1 function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America.)

Aim: Enterococcus faecalis is a leading nosocomial pathogen in biofilm-associated polymicrobial infections. The study aims to understand pathogenicity and biofilm determinants of the pathogen by genome analysis. Methodology: Genome sequencing of a strong biofilm forming clinical isolate Enterococcus faecalis SK460 devoid of Fsr quorum-signaling system, was performed and comparative genomics was carried out among a set of pathogenic biofilm formers and nonpathogenic weak biofilm formers. Results: Analysis revealed a pool of virulence and adhesion related factors associated with pathogenicity. Absence of CRISPR-Cas system facilitated acquisition of pheromone responsive plasmid, pathogenicity island and phages. Comprehensive analysis identified a subset of accessory genes encoding polysaccharide lyase, sugar phosphotransferase system, phage proteins and transcriptional regulators exclusively in pathogenic biofilm formers. Conclusion: The study identified a set of genes specific to pathogenic biofilm formers and these can act as targets which in turn help to develop future treatment endeavors against enterococcal infections.

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I-saline; group II-propolis 100 µg/mL; group III-propolis 300 µg/mL; group IV-propolis nanoparticle 100 µg/mL; group V-propolis nanoparticle 300µg/mL; group VI-6% sodium hypochlorite; group VII-2% chlorhexidine. Dentin shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal-Wallis and Mann-Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and β-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups ( p < 0.05) except 6% NaOCl and 2% CHX ( p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs ( p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min ( p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm -1 band and an increase in the 870 cm -1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF&#95;11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities., Competing Interests: The authors have declared that no competing interests exist.

Traditionally, treatments for bacterial infection have focused on killing the microbe or preventing its growth. As antimicrobial resistance becomes more ubiquitous, the feasibility of this approach is beginning to wane and attention has begun to shift toward disrupting the host-pathogen interaction by improving the host defense. Using a high-throughput, fragment-based screen to identify compounds that alleviate Pseudomonas aeruginosa -mediated killing of Caenorhabditis elegans , we identified over 20 compounds that stimulated host defense gene expression. Five of these molecules were selected for further characterization. Four of five compounds showed little toxicity against mammalian cells or worms, consistent with their identification in a phenotypic, high-content screen. Each of the compounds activated several host defense pathways, but the pathways were generally dispensable for compound-mediated rescue in liquid killing, suggesting redundancy or that the activation of unknown pathway(s) may be driving compound effects. A genetic mechanism was identified for LK56, which required the Mediator subunit MDT-15/MED15 and NHR-49/HNF4 for its function. Interestingly, LK32, LK34, LK38, and LK56 also rescued C. elegans from P. aeruginosa in an agar-based assay, which uses different virulence factors and defense mechanisms. Rescue in an agar-based assay for LK38 entirely depended upon the PMK-1/p38 MAPK pathway. Three compounds-LK32, LK34, and LK56-also conferred resistance to Enterococcus faecalis , and the two lattermost, LK34 and LK56, also reduced pathogenesis from Staphylococcus aureus This study supports a growing role for MDT-15 and NHR-49 in immune response and identifies five molecules that have significant potential for use as tools in the investigation of innate immunity. IMPORTANCE Trends moving in opposite directions (increasing antimicrobial resistance and declining novel antimicrobial development) have precipitated a looming crisis: the nearly complete inability to safely and effectively treat bacterial infections. To avert this, new approaches are needed. One idea is to stimulate host defense pathways to improve the clearance of bacterial infection. Here, we describe five small molecules that promote resistance to infectious bacteria by activating C. elegans ' innate immune pathways. Several are effective against both Gram-positive and Gram-negative pathogens. One of the compounds was mapped to the action of MDT-15/MED15 and NHR-49/HNF4, a pair of transcriptional regulators more generally associated with fatty acid metabolism, potentially highlighting a new link between these biological functions. These studies pave the way for future characterization of the anti-infective activity of the molecules in higher organisms and highlight the compounds' potential utility for further investigation of immune modulation as a novel therapeutic approach., (Copyright © 2021 Hummell et al.)

Here, we have analysed and explored the genome sequences of three newly isolated bacteria that were recently characterised for their probiotic activities and ability to produce bacteriocins. These strains, isolated from faeces of animals living in captivity at the zoological garden of Lille (France), are Escherichia coli ICVB443, Enterococcus faecalis ICVB501 and Pediococcus pentosaceus ICVB491. Their genomes have been analysed and compared to those of their pathogenic or probiotic counterparts. The genome analyses of E. coli ICVB443 and Ent. faecalis ICVB501 displayed similarities to those of probiotics E. coli 1917 Nissle, and Ent. faecalis Symbioflor 1, respectively. Furthermore, E. coli ICVB443 shares at least 89 genes with the enteroaggregative E. coli 55989 (EAEC), and Ent. faecalis ICVB501 shares at least 315 genes with the pathogenic Ent. faecalis V583 strain. Unlike Ped. pentosaceus ICVB491, which is devoid of virulence genes, E. coli ICVB443 and Ent. faecalis ICVB501 both carry genes encoding virulence factors on their genomes. Of note, the bioinformatics analysis of these two genomes located the bsh gene, which codes for bile salt hydrolase (BSH). The presence of BSH is of major importance, as it can help to increase the viability of these two strains in the gastrointestinal tract (GIT). The genome analysis of Ped. pentosaceus ICVB491 confirmed its GRAS status (Generally Recognised As Safe), as no genomic virulence factor determinant was found.

Infections are the leading cause of failure of osteogenic material implantation. Antibiotic treatment, treatment with bone cement, or collagen sponge placement can result in drug resistance and difficulties in operation. To address this, gellan gum (GG) was selected in this study and prepared as an injectable hydrogel containing chlorhexidine (CHX) and nanohydroxyapatite (nHA) that overcomes these intractable problems. Scanning electron microscopy and micro-computed tomography revealed a three-dimensional polymeric network of the hydrogel. The hydrogel had excellent biocompatibility, as detected by cell counting kit-8 and Live/Dead assay. Bone marrow mesenchymal stem cells could be encapsulated into the network, showing that the structure was suitable for cell growth. Additionally, loading the hydrogel with nHA improved its mechanical, biodegradable, and osteogenic properties. Quantitative alkaline phosphatase and Alizarin Red S staining validated its osteogenic ability. Furthermore, antibacterial activity assessment showed that the hydrogel loaded with 50 μg/mL CHX inhibited Enterococcus faecalis in a concentration-dependent manner. Thus, we report an injectable GG-based hydrogel with superior antibacterial effect against E. faecalis and osteogenesis, which holds promise for treating infectious bone defects caused by refractory periradicular periodontitis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate how carbohydrates (glucose or sucrose) affect the characteristics of Enterococcus faecalis (E. faecalis) planktonic and biofilm in vitro. For this study, E. faecalis was cultured in tryptone-yeast extract broth with 0% glucose + 0% sucrose, 0.5% glucose, 1% glucose, 0.5% sucrose, or 1% sucrose. Viability of E. faecalis was examined by colony forming unit counting assays. Biofilm formation was assessed by measuring extracellular DNA (eDNA), a component of the biofilm matrix. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression of virulence-associated genes. Field emission scanning electron microscopy analysis, confocal laser scanning microscopy analysis, and crystal violet colorimetric assay were conducted to study E. faecalis biofilms. E. faecalis showed the highest viability and eDNA levels in 1% sucrose medium in biofilms. The result of qRT-PCR showed that the virulence-associated genes expressed highest in 1% sucrose-grown biofilms and in 1% glucose-grown planktonic cultures. E. faecalis showed highly aggregated biofilms and higher bacteria and exopolysaccharide (EPS) bio-volume in sucrose than in 0% glucose + 0% sucrose or glucose. The results indicate that the production of eDNA and EPS and expression of virulence-associated genes in E. faecalis are affected by the concentration of carbohydrates in biofilm or planktonic culture.

Enterococcus faecalis is one of the important causative agents of nosocomial and life-threatening infections in human. Several studies have demonstrated that the presence of CRISPR- cas  is associated with antibiotic susceptibility and lack of virulence traits. In this study, we aimed to assess the phenotypic and genotypic virulence determinants in relation to CRISPR elements from the dental-root canals and hospital-acquired isolates of  E. faecalis . Eighty-eight hospital-acquired and 73 dental-root canal isolates of  E. faecalis  were assessed in this study. Phenotypic screening of the isolates included biofilm formation, and gelatinase and hemolysis activities. Genotypical screening using PCR was further used to evaluate the presence of CRISPR elements and different virulence-associated genes such as  efaA, esp, cylA, hyl, gelE, ace, ebpR , and  asa1 . Biofilm formation, gelatinase, and hemolysis activities were detected in 93.8%, 29.2%, and 19.2% of the isolates, respectively. The most prevalent virulence-associated gene was  ace , which was followed by  efaA , whereas  cylA  was the least identified. The presence of CRISPR1- cas , orphan CRISPR2, and CRISPR3- cas  was determined in 13%, 55.3%, and 17.4% of the isolates, respectively. CRISPR elements were significantly more prevalent in the dental-root canal isolates. An inverse significant correlation was found between CRISPR- cas  loci,  esp,  and  gelE , while direct correlations were observed in the case of  cylA, hyl, gelE  (among CRISPR-loci 1 and 3),  asa1, ace , biofilm formation, and hemolysis activity. Findings, therefore, indicate that CRISPR- cas  might prevent the acquisition of some respective pathogenicity factors in some isolates, though not all; so selective forces could not influence pathogenic traits. Abbreviations: BHI: brain-heart infusion agar; CRISPRs: Clustered regularly interspaced short palindromic repeats; Esp: Cell wall-associated protein; ENT: ear-nose-throat; ICU: intensive care units; OD: optical densities; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; UTI: urinary tract infection.

Bacterial prostatitis affects 1% of men, with increased incidence in the elderly. Acute bacterial prostatitis frequently progresses to chronicity, marked by recurrent episodes interspersed with asymptomatic periods of variable duration. Antibiotic treatment is standard of care; however, dissemination of antimicrobially resistant uropathogens threatens therapy efficacy. Thus, development of nonantibiotic-based approaches to treat chronic disease is a priority. Currently, why chronic prostatitis arises is unclear, as the immune response to prostate infection is incompletely understood. As 80% of prostatitis cases are caused by Gram-negative uropathogenic Escherichia coli (UPEC) or Gram-positive Enterococcus faecalis , we used a mouse transurethral instillation model to address the hypothesis that an innate immune response fails to develop following prostate infection with these uropathogens, leading to chronic disease. Surprisingly, infection induced robust proinflammatory cytokine expression and myeloid cell infiltration. Following a second infection, cytokine responses and innate cell infiltration were largely comparable to primary infection. Characteristic of memory responses, more lymphoid cells infiltrated the prostate in a second infection compared with a first, suggesting that adaptive immunity develops to eliminate the pathogens. Unexpectedly, bacterial burden in prostates challenged with either UPEC or E. faecalis was equal or greater than primary infection despite that a protective adaptive response to UPEC infection was evident in the bladder of the same animals. Our findings support that chronic or recurrent prostatitis develops despite strong innate immune responses and may be the result of a failure to develop immune memory to infection, pointing to actionable targets for immunotherapy., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Biogenesis of iron-sulfur (FeS) clusters in an essential process in living organisms due to the critical role of FeS cluster proteins in myriad cell functions. During biogenesis of FeS clusters, multi-protein complexes are used to drive the mobilization and protection of reactive sulfur and iron intermediates, regulate assembly of various FeS clusters on an ATPase-dependent, multi-protein scaffold, and target nascent clusters to their downstream protein targets. The evolutionarily ancient sulfur formation (Suf) pathway for FeS cluster assembly is found in bacteria and archaea. In Escherichia coli, the Suf pathway functions as an emergency pathway under conditions of iron limitation or oxidative stress. In other pathogenic bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for FeS clusters and therefore is a potential target for the development of novel antibacterial compounds. Here we summarize the considerable progress that has been made in characterizing the first step of mobilization and protection of reactive sulfur carried out by the SufS-SufE or SufS-SufU complex, FeS cluster assembly on SufBC 2 D scaffold complexes, and the downstream trafficking of nascent FeS clusters to A-type carrier (ATC) proteins. Cell Biology of Metals III edited by Roland Lill and Mick Petris., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Continued mining of natural products from the strain Streptomyces sp. MA37 in our laboratory led to the discovery of a minor specialized metabolite (SM) called accramycin A. Owing to its low yield (0.2 mg/L) in the wild type strain, we investigated the roles of regulatory genes in the corresponding biosynthetic gene cluster ( acc BGC) through gene inactivation with the aim of improving the titer of this compound. One of the resulting mutants (∆ accJ ) dramatically upregulated the production of accramycin A 1 by 330-fold (66 mg/L). Furthermore, ten new metabolites, accramycins B-K 2 - 11 , were discovered, together with two known compounds, naphthacemycin B 1 12 and fasamycin C 13 from the mutant extract. This suggested that accJ , annotated as multiple antibiotic resistance regulator (MarR), is a negative regulator gene in the accramycin biosynthesis. Compounds 1 - 13 inhibited the Gram-positive pathogens ( Staphylococcus aureus, Enterococcus faecalis ) and clinical isolates Enterococcus faecium (K59-68 and K60-39) and Staphylococcus haemolyticus with minimal inhibitory concentration (MIC) values in the range of 1.5-12.5 µg/mL. Remarkably, compounds 1 - 13 displayed superior activity against K60-39 (MIC = 3.1-6.3 µg/mL) compared to ampicillin (MIC = 25 µg/mL), and offered promising potential for the development of accramycin-based antibiotics that target multidrug-resistant Enterococcus clinical isolates. Our results highlight the importance of identifying the roles of regulatory genes in natural product discovery.

Introduction: Enterococcus faecalis ( E. faecalis ) is the most important species in dentistry and plays a significant role in the etiology of persistent apical lesions after root canal treatment. Up to date, the intracanal application of 2% chlorhexidine for 7 days is the best way to eliminate E. faecalis. However, due to the ability of this bacterium to persist and survive in harsh environments, many studies have been directed towards finding an alternative strategy for prevention or eradication of it. This study was conducted to investigate the effect of bismuth nanoparticles on E. faecalis , as an etiologic factor in recurrent root canal infections., Methods: Forty patients, referred to Endodontic Ward of Shiraz University of Medical Science for endodontic pretreatment, provided root canal samples. First, all samples were transferred in Enterococcosel broth and incubated. Then, samples which showed growth were plated on blood agar plates and incubated for further PCR procedure. Nanoparticle powder was dissolved in high-purity water, and the final concentration of bismuth nanoparticles (BiNPs) was measured by the spectrophotometer. Minimum inhibitory concentration (MIC) of BiNPs against E. faecalis was determined by microbroth dilution method according to methods for antimicrobial susceptibility tests. Also, bactericidal assays were conducted in Mueller-Hinton broth medium and reported as the concentration of BiNPs that reduced the viable bacterial count by 99.9%., Results: Of all samples, 77.5% revealed the presence of E. faecalis by PCR. Also, E. faecalis growth inhibition was observed at concentrations ranging from 0.625  μ g/ml to 20  μ g/ml (geometric mean: 2.337  μ g/ml), and the MBC values were between 1.25  μ g/ml and 40  μ g/ml (geometric mean: 4.781  μ g/ml), which in comparison with chlorhexidine, these values were about one-eighth of chlorhexidine., Conclusion: The experimental data suggest that bismuth nanoparticles could be an interesting alternative to combat E. faecalis , which, in view of the advantages mentioned for bismuth nanoparticle like inhibiting Streptococcus mutans biofilm formation and higher antibacterial activity compared to chlorhexidine, can be suggested to be used in different fields of dentistry., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Azita Azad et al.)

Enterococci are commensals of human and other animals' gastrointestinal tracts. Only making up a small part of the microbiota, they have not played a significant role in research, until the 1980s. Although the exact year is variable according to different geographical areas, this was the decade when vancomycin-resistant enterococci (VRE) were discovered and since then their role as causative agents of human infections has increased. Enterococcus faecium is on the WHO's list of "bacteria for which new antibiotics are urgently needed," and with no new antibiotics in development, the situation is desperate. In this review, different aspects of VRE are outlined, including the mortality caused by VRE, antibiotic resistance profiles, animal-modeling efforts, and virulence. In addition, the limitations of current antibiotic treatments for VRE and prospective new treatments, such as bacteriocins, are reviewed.

Enterococcus faecalis infective endocarditis (EFIE) is a severe disease of increasing incidence. The objective was to analyze whether the outcome of patients with native valve EFIE (NVEFIE) treated with a short course of ampicillin plus ceftriaxone (4wAC) was similar to patients treated according to international guidelines (6wAC). Between January 2008 and June 2018, 1,978 consecutive patients with definite native valve IE were prospectively included in a national registry. Outcomes of patients with NVEFIE treated with 4wAC were compared to those of patients who received 6wAC. Three hundred and twenty-two patients (16.3%) had NVEFIE. One hundred and eighty-three (56.8%) received AC. Thirty-nine patients (21.3%) were treated with 4wAC for four weeks and 70 patients (38.3%) with 6wAC. There were no differences in age or comorbidity. Patients treated 6wAC presented a longer duration of symptoms before diagnosis (21 days, IQR 7-60 days vs. 7 days, IQR 1-22 days; p = 0.002). Six patients presented perivalvular abscess and all of these received 6wAC. Surgery was performed on 14 patients (35.9%) 4wAC and 34 patients (48.6%) 6wAC (p = 0.201). In-hospital mortality, one-year mortality and relapses among 4wAC and 6wAC patients were 10.3% vs. 11.4% (p = 0.851); 17.9% vs. 21.4% (p = 0.682) and 5.1% vs. 4.3% (p = 0.833), respectively. In conclusion, a four-week course of AC may be considered as an alternative regimen in NVEFIE, notably in patients with shorter duration of symptoms and those without perivalvular abscess. These results support the performance of a randomized clinical trial to evaluate the efficacy of this short regimen., Competing Interests: Dr. Ramos-Martínez declares personal fees from Merck, Astellas and Pfizer, outside the submitted work. Dr. Ojeda-Burgos reports personal fees from Merck, personal fees from Janssen Cilag, personal fees from Gilead, outside the submitted work. Dr. Miro reports grants and personal fees from AbbVie, Bristol-Myers Squibb, Contrafect, Genentech, Jansen, Medtronic, Merck, Novartis, Gilead Sciences, and ViiV Healthcare, outside the submitted work. Dr. Juan M Pericas. Dr. Ana Fernández-Cruz, Dr. Patricia Muñoz, Dr. Maricela Valerio, Dr. Martha Kestler, Dr. Miguel Montejo, Dr. Mª Carmen Fariñas, Dr. Dolores Sousa, Dr. Fernando Domínguez, Dr. Antonio Plata and Dr. Laura Vidal have nothing to disclouse. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like Enterococcus faecalis . This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated in vitro , and the results are in accordance with a degradosome structure close to the one of Bacillus subtilis . At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the Galleria mellonella infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the Δ cshA mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5'Unstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.

The bacterium-bacterium interaction between pathogenic and probiotic Enterococcus as well as the bacterium-host interaction between Enterococcus and intestinal epithelium has drawn increasing attentions, but the influence of those interactions on host pregnancy remains largely unexplored. In the present study, we evaluated the effects of probiotic E. faecalis Symbioflor 1 or/and pathogenic E. faecalis OG1RF on the miscarriage of pregnant mice. Using in vitro assays of competition and exclusion and displacement, antagonistic property of E. faecalis Symbioflor 1 against E. faecalis OG1RF was observed, and the former inhibited the translocation of the later in vivo. The rate of miscarriage induced by E. faecalis OG1RF challenge was significantly reduced by 28% with E. faecalis Symbioflor 1 intervention; and the tissue integrity of ileum, colon, uterus, and placenta and placental blood cell density in pregnant mice were drastically improved by such probiotic intervention. Compared with the controls, probiotic intervention significantly upregulated the level of IL-10 and TGF-β, downregulated levels of IFN-γ, and increased progesterone level that reversed the trend of being Th1 predominance state reported for adverse pregnancy outcome at early pregnancy stage. In conclusion, E. faecalis Symbioflor 1 decreased the translocation of E. faecalis OG1RF, prevented pathogen-induced tissue damage, and changed Th1-Th2 homeostasis toward Th2 predominance during early pregnancy resulting in decreased miscarriage. KEY POINTS: •The mechanism of how probiotic E. faecalis Symbioflor 1 improves pregnancy of mice • Influence of interactions of pathogenic and probiotic Enterococcus on host pregnancy • E. faecalis Symbioflor 1 change Th1-Th2 homeostasis toward Th2 predominance.

This research was conducted using 50 samples of popular traditional cheeses and 160 enterococcal clinical isolates. Phenotypic and genotypic methods used for identification of enterococci. Then, the incidences of antibacterial resistance and virulence traits were investigated. In total, 165 E. faecalis and 43 E. faecium obtained from traditional cheeses and different clinical isolates were analyzed in the study. Antibiotic susceptibility testing revealed 175(84.1%) isolates with multi-drug resistance (MDR) patterns, which was more common among clinical sources. The predominant virulence profile, including gelE, asa1 and cpd was detected within 47 (22.6%) of the MDR isolates. Our results showed that traditional cheeses and clinical E. faecalis isolates have distinct patterns of virulence traits. The identified enterococci with antibiotic resistance and associated virulence factors, could provide a potential risk to the public health.

Purpose: Enterococci express high degree of resistance towards wide range of antibiotics. Production of biofilm and many virulence factors along with drug resistance makes it difficult to eradicate the infection from urinary tract. The present study detected the expression of such factors including biofilm production by multidrug-resistant (MDR) enterococci., Materials and Methods: Drug susceptibility of 103 uropathogenic enterococci was performed followed by estimation of minimum inhibitory concentration of high-level gentamicin and vancomycin by microbroth dilution method. Vancomycin-resistant genes were detected by multiplex polymerase chain reaction. Production of virulence factors such as haemagglutination, caseinase, lipase, gelatinase, haemolysin and β-lactamase was detected by phenotypic methods in MDR strains. Biofilm production was detected by calcofluor-white fluorescence staining and semi-quantitative adherence assay., Results: 45% and 18.4% of the isolates were high-level gentamicin-resistant and vancomycin-resistant enterococci (VRE), respectively. vanA gene was detected in 14 and vanB gene in 5 strains. Biofilm, caseinase and gelatinase were the most expressed virulence factor. Expression of caseinase, gelatinase and lipase was significantly higher in Enterococcus faecalis (P < 0.05). Expression of haemagglutination, gelatinase and haemolysin among the vancomycin-resistant isolates was significantly higher (P < 0.05)., Conclusion: VanA and vanB are the prevalent genotypes responsible for vancomycin resistance. The high prevalence of MDR enterococcal strains producing biofilm and virulence determinants raises concern. asa1, hyl, esp, gelE, cyl and other genes are known to express these factors and contribute to biofilm formation. Most uropathogenic enterococci expressed biofilm at moderate level and can be detected effectively by calcofluor-white staining. No correlation was noted between vancomycin resistance and biofilm production., Competing Interests: None

Objective: Virulence factors (VFs) among the clinical strains of enterococci play a vital role in pathogenesis. This study was aimed to screen for cylA, asa1, gelE, esp and hyl among Enterococcus faecalis (n = 89) and E. faecium (n = 51) by multiplex PCR. The previously reported multiplex PCR was modified to 2 duplex (asa1 and gelE, cylA and esp) PCRs and 1 simplex (hyl) PCR. The idea of the modification of the multiplex PCR proposed here emerged in the course of the research study when majority of the isolates which phenotypically exhibited virulence traits were found to be negative for the respective gene., Results: cylA, gelE and asa1 were significantly predominant in E. faecalis (59.55%, 85.39%, 86.51%) than E. faecium (1.96%, 60.78%, 9.80%) (p < 0.0001, p = 0.001967, p < 0.0001). hyl was detected in E. faecium (5.9%) only. The number of VFs detected in each isolate was recorded as the VF score. E. faecalis isolates had a VF score pattern of score 4 (34.83%), score 3 (26.96%), score 2 (28.08%) and score 1 (8.98%) while E. faecium had score 4 (1.96%), score 3 (7.84%), score 2 (25.49%) and score 1 (41.18%). This modification of the PCR protocol could resolve the problem of decreased detection of virulence determinants in enterococci.

For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to β-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all β-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Background: Enterococcus faecalis, as a major microorganism in persistent/secondary infections of endodontically treated teeth, is less likely to be eliminated during endodontic therapy. In this study, the effect of root canal disinfection and anti-virulence activities of photodynamic inactivation (PDI) of E. faecalis utilizing reduced graphene oxide-curcumin (rGO-Cur) as a photosensitizing agent following irradiation with light-emitting diode (LED), as well as intracellular ROS production were evaluated on ex vivo biofilms of E. faecalis in comparison with sodium hypochlorite (NaOCl) as the traditional endodontic irrigation solution., Materials and Methods: After formulation and confirmation of synthesized rGO-Cur using scanning electron microscopy (SEM), Fourier transformation infrared (FT-IR), UV-Vis spectra, dynamic light scattering (DLS), and Zeta potential, the minimum biofilm inhibitory concentrations (MBICs) and in vitro anti-biofilm activity of rGO-Cur, light-emitting diode (LED) at the wavelength of 435 ± 20 nm, and rGO-Cur-PDI were determined against 4-week-old pre-formed biofilms of E. faecalis. After preparation of ex vivo biofilm model in root canals, the ex-vivo anti-biofilm potential of rGO-Cur, LED, and rGO-Cur-PDI against E. faecalis were analyzed using the XTT assay and scanning electron microscopy (SEM) in comparison with NaOCl. The effects of sub-MBIC of rGO-Cur and NaOCl, sub-lethal dose of LED, and sub-significant inhibitory (SSI) potential of rGO-Cur-PDI for E. faecalis biofilms on virulence genes (efa, esp, gel, and fsr) expression of E. faecalis were analyzed using real-time polymerase chain reaction (qRT-PCR) assay. Intracellular reactive oxygen species (ROS) level was measured in rGO-Cur-PDI-treated bacterial cells compared to control cells with 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent probe., Results: The FTIR, DLS, Zeta potential, SEM, and UV-Vis spectra analysis indicated the successful synthesis of rGO-Cur. The MBIC of rGO-Cur was 250 μg/ml, which inhibited the growth ofE. faecalis. LED showed insignificant anti-biofilm activity against E. faecalis even after treating for a long irradiation time (300 s). According to checkerboard assay, the MBIC value of rGO-Cur-PDI was reduced noticeably compared to the individual MBIC values of rGO-Cur and LED for E. faecalis. The expression levels of efa, esp, gel, and fsr genes in pre-formed E. faecalis biofilms were markedly reduced after rGO-Cur, rGO-Cur-PDI, and NaOCl treatment in comparison with the control group. Conversely, LED revealed no significant change in the expression of the virulence genes. The intracellular ROS assay showed a significant increase (8.3-fold) in rGO-Cur-PDI when compared to the control., Conclusion: Our data support that rGO-Cur-PDI showed dual inhibitory effects on biofilm formation ability and virulence activity of E. faecalis with potential clinical applications for infection control in endodontics., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dissecting the genetic basis of natural variation in disease response in hosts provides insights into the coevolutionary dynamics of host-pathogen interactions. Here, a genome-wide association study of Drosophila melanogaster survival after infection with the Gram-positive entomopathogenic bacterium Enterococcus faecalis is reported. There was considerable variation in defense against E. faecalis infection among inbred lines of the Drosophila Genetics Reference Panel. We identified single nucleotide polymorphisms associated with six genes with a significant ( p < 10 -08 , corresponding to a false discovery rate of 2.4%) association with survival, none of which were canonical immune genes. To validate the role of these genes in immune defense, their expression was knocked-down using RNAi and survival of infected hosts was followed, which confirmed a role for the genes krishah and S6k in immune defense. We further identified a putative role for the Bomanin gene BomBc1 (also known as IM23 ), in E. faecalis infection response. This study adds to the growing set of association studies for infection in Drosophila melanogaster and suggests that the genetic causes of variation in immune defense differ for different pathogens.

Background: ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△clpP) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling., Results: The ΔclpP mutant strain showed impaired growth at 20 °C or 45 °C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving ΔclpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the ΔclpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE), and orotidine-5'-phosphate decarboxylase (pyrF) all decreased in the ΔclpP mutant strain., Conclusion: The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis -mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa -mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing. NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

Enterococci, which are commonly found in the environment, cause serious infections despite the absence of well-defined virulence factors and toxins. Knowing the virulence properties of enterococci is important to understand the complex pathogenic structures. In this study, we aimed to investigate the virulence factors (asa1, hyl, cylA, efa, ebp, ace, esp, gelE, sprE, fsrA, fsrB, fsrC genes, gelatinase activity, hemolysin, hydrogen peroxide and biofilm production) and antibiotic resistance of Enterococcus faecium and Enterococcus faecalis strains isolated from clinical specimens. A total of 110 enterococcus isolates which were accepted as infectious agents were included in the study. The polymerase chain reaction method was used to identify the isolates and to detect virulence genes. Characteristics of hemolysis, biofilm formation, hydrogen peroxide production and gelatinase activity were investigated by phenotypic methods. The antibiotic susceptibility test was performed with VITEK 2 automated system. E.faecalis ATCC 29212 standard strain was used as a quality control in all tests. Of the 110 enterococci isolates included in the study, 61 were identified as E.faecium and 49 as E.faecalis. The efa gene was the most frequently detected virulence gene (92.7%), followed by ace (83.6%), esp (66.4%), ebp (60.0%), cylA (50.9%), hyl (46.4%), asa1 (45.5%), gelE, sprE, fsrC (33.6%), fsrA (12.7%) and fsrB (11.8%). All genes except hyl were higher in E.faecalis isolates and the difference was statistically significant (p<0.05). Twenty-five (51%) E.faecalis and 1 (1.6%) E.faecium isolates had beta-hemolysis and the difference was statistically significant (p= 0.000). Seven (11.5%) E.faecium and 4 (8.2%) E.faecalis isolates formed biofilm, but the difference was not statistically significant (p> 0.05). Two (3.3%) E.faecium and 14 (28.6%) E.faecalis isolates exhibited gelatinase activity and the difference between the two species was statistically significant (p= 0.000). Hydrogen peroxide production was not detected in any of the isolates. The highest resistance rate was determined against ciprofloxacin (70.9%). The resistance to ampicillin was 69.1%, high level streptomycin 65.1%, high level gentamicin 39.4%, vancomycin and teicoplanin 4.5%, and linezolid 1.8%. In conclusion, our data indicated that virulence factors except hyl gene and biofilm production were higher in E.faecalis isolates but E.faecium isolates were more resistant to antibiotics. In order to prevent infection of such virulent or resistant isolates in the hospital setting, infection control measures must be followed. In vivo studies are needed for the better understanding of the virulence of enterococci.

Background: Recent metagenomic analyses have revealed dysbiosis of the gut microbiota of ulcerative colitis (UC) patients. However, the impacts of this dysbiosis are not fully understood, particularly at the strain level., Results: We perform whole-genome shotgun sequencing of fecal DNA extracts from 13 healthy donors and 16 UC and 8 Crohn's disease (CD) patients. The microbiota of UC and CD patients is taxonomically and functionally divergent from that of healthy donors, with E. faecium being the most differentially abundant species between the two microbial communities. Transplantation of feces from UC or CD patients into Il10 -/- mice promotes pathological inflammation and cytokine expression in the mouse colon, although distinct cytokine expression profiles are observed between UC and CD. Unlike isolates derived from healthy donors, E. faecium isolates from the feces of UC patients, along with E. faecium strain ATCC 19434, promotes colitis and colonic cytokine expression. Inflammatory E. faecium strains, including ATCC 19434 and a UC-derived strain, cluster separately from commercially available probiotic strains based on whole-genome shotgun sequencing analysis. The presence of E. faecium in fecal samples is associated with large disease extent and the need for multiple medications in UC patients., Conclusions: E. faecium strains derived from UC patients display an inflammatory genotype that causes colitis.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality 1-3 . Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice 4 , but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis 5,6 -as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Enterococcus faecalis is a ubiquitous intestinal symbiont and common early colonizer of the neonatal gut. Although colonization with E. faecalis has been previously associated with decreased pathology of necrotizing enterocolitis (NEC), these bacteria have been also implicated as opportunistic pathogens. Here we characterized 21 strains of E. faecalis, naturally occurring in 4-day-old rats, for potentially pathogenic properties and ability to colonize the neonatal gut. The strains differed in hemolysis, gelatin liquefaction, antibiotic resistance, biofilm formation, and ability to activate the pro-inflammatory transcription factor NF-κB in cultured enterocytes. Only 3 strains, BB70, 224, and BB24 appreciably colonized the neonatal intestine on day 4 after artificial introduction with the first feeding. The best colonizer, strain BB70, effectively displaced E. faecalis of maternal origin. Whereas BB70 and BB24 significantly increased NEC pathology, strain 224 significantly protected from NEC. Our results show that different strains of E. faecalis may be pathogenic or protective in experimental NEC., Competing Interests: The authors have declared that no competing interests exist.

Orthopaedic implant-associated infections are a devastating complication of orthopaedic surgery with a significant impact on patients and healthcare systems. The aims of this work were to describe the patterns of antimicrobial resistance, pathogenicity and virulence of clinical bacterial isolates from orthopaedic implant-associated infections and to further isolate and characterise bacteriophages that are efficient in controlling these bacteria. Staphylococcus aureus, Enterococcus faecalis and Escherichia coli isolated from orthopaedic infections showed multiresistance patterns to the most frequently used antibiotics in clinical settings. The presence of mobile genetic elements (mecA, Tn916/Tn1545 and intl1) and virulence determinants (icaB, cna, hlb, cylLs, cylM, agg, gelE, fsr and fimA) highlighted the pathogenicity of these isolates. Moreover, the isolates belonged to clonal complexes associated with the acquisition of pathogenicity islands and antimicrobial resistance genes by recombination and horizontal gene transfer. Bacteriophages vB&#95;SauM&#95;LM12, vB&#95;EfaS&#95;LM99 and vB&#95;EcoM&#95;JB75 were characterised and their ability to infect clinical isolates of S. aureus, E. faecalis and E. coli, respectively, was assessed. Morphological and genomic analyses revealed that vB&#95;EfaS&#95;LM99 and vB&#95;EcoM&#95;JB75 belong to the Siphoviridae and Myoviridae families, respectively, and no genes associated with lysogeny were found. The bacteriophages showed low latent periods, high burst sizes, broad host ranges and tolerance to several environmental conditions. Moreover, they showed high efficiency and specificity to infect and reduce clinical bacteria, including methicillin-resistant S. aureus and vancomycin-resistant enterococci. Therefore, the results obtained suggest that the bacteriophages used in this work are a promising approach to control these pathogens involved in orthopaedic implant-associated infections., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Enterococcus faecalis, a ubiquitous member of the healthy human gut microbiota, is also a common opportunistic pathogen and leading cause of nosocomial infections. This tenacious microbe is well adapted to infect and persist in multiple niches within the mammalian host and can rapidly tune its metabolism to respond to new environments, enabling infection in sites including the gastrointestinal tract, urinary tract, wounded epithelium, heart, and blood. In order to withstand and persist in the face of host immune responses, E. faecalis has an arsenal of strategies to suppress, evade, or inactivate innate and adaptive immune mechanisms. In this review, we present the variety of ways E. faecalis modulates the immune response, enabling this otherwise innocuous gut commensal to transition and persist as a pathogen., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In Firmicutes , the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp 0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp 0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis , a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice., (Copyright © 2019 Colomer-Winter et al.)

Background: Linezolid-resistant enterococci pose great challenges in clinical practice. The aim of this study is to study the mechanisms underlying the resistance and genetic environment of antimicrobial resistance gene of linezolid-resistant enterococci., Results: The linezolid MICs of 16 enterococci were 4 mg/L to 16 mg/L. Four strains belonged to multi-drug resistant (MDR) bacteria. The sequence types (STs) of 13 enterococci strains performed WGS were diverse: 3 ST476, 1 ST86, ST116, ST480, ST59, ST416, ST21, ST67, ST16, ST585 and ST18. None of them carried multi-drug resistance gene cfr. Only one strain had the G2658 T mutation of target 23S rRNA gene. Thirteen (13/16, 81.3%) strains harbored the novel oxazolidinone resistance gene optrA. WGS analysis showed that the optrA gene was flanked by sequence IS1216E insertion in 13 strains, and optrA was adjacent to transposons Tn558 in two strains and Tn554 in one strain. The optrA gene was identified to be co-localized with fexA, the resistance genes mediated florfenicol resistance in 13 strains, and ermA1, the resistance genes mediated erythromycin resistance in 9 strains, indicating that linezolid-resistant strains may be selected due to non-oxazolidinone antibiotics (i.e. macrolides and florfenicol) usage., Conclusion: Our findings demonstrate the high diversity of optrA-carrying genetic platforms. The mobile genetic elements (MGEs) may play an important role in the dissemination of optrA into the enterococci isolates of human origin. The genetic evidence of transferable feature and co-selection of optrA should be gave more attention in clinical practice.

Commensal and generally harmless in healthy individuals, Enterococcus faecalis causes opportunistic infections in immunocompromised patients. Plasmid-cured E. faecalis strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in Galleria mellonella and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the epa locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific epa -variable regions in host adaptation independently of other genetic variations. IMPORTANCE E. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle., (Copyright © 2019 Furlan et al.)

Background: Enterococcus is an important component of normal flora in human and animals, but in recent years, the pathogenicity of Enterococcus has been confirmed in clinical medicine. More and more animal infections have been reported in veterinary clinics. For the last decades, outbreaks of encephalitis in lambs have become much more common in Northern Xinjiang, China. Consequent studies have confirmed that these affected lambs had been commonly infected with E. faecalis. More than 60 E. faecalis were isolated from the brain of infected lambs, A highly virulent strain entitled E. faecalis 2A (XJ05) were selected, sequenced and analyzed., Result: Using whole genome sequence and de novo assembly, 18 contigs with NGS and annotation were obtained. It is confirmed that the genome has a size of 2.9 Mb containing 2783 protein-coding genes, as well as 54 tRNA genes and 4 rRNA genes. Some key features of this strain were identified, which included 7 predicted antibiotic resistance genes and 18 candidate virulence factor genes., Conclusion: The E. faecalis 2A (XJ05) genome is conspicuous smaller than E.faecalis V583, but not significantly different from other non-pathogenic E. faecalis. It carried 7 resistance genes including 4 kind of antibiotics which were consistent with the results of extensive drug resistance phenotypic, including aminoglycoside, macrolide, phenicol, and tetracycline. 2A (XJ05) also carried 18 new virulence factor genes related to virulence, hemolysin genes (cylA, cylB, cylM, cylL) may play an important role in lamb encephalitis by E. faecalis 2A (XJ05).