Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Risk factors and outcome associated with the acquisition of MDR linezolid-resistant Enterococcus faecium: a report from tertiary care centre.

OBJECTIVE: The aim of the study was to determine the resistance profile of linezolid-resistant Enterococcus faecium (LREfm) and to investigate risk factors and outcomes associated with LREfm infections. MATERIAL AND METHODS: A prospective case-control study was undertaken (2019 to 2022) and included 202 patients with LREfm infections (cases) and 200 controls with LSEfm infections. Clinical data was prospectively collected and analysed for risk factors and outcomes. Antimicrobial susceptibility was performed, and resistance profile was studied using WHOnet. RESULTS: Risk factors associated with LREfm infection were site of infection UTI (OR 5.87, 95% CI 2.59-13.29, p ≤ 0.001), prior use of carbapenem (OR 2.85 95% CI 1.62-5.02, p ≤ 0.001) and linezolid (OR 10.13, 95% CI 4.13-24.82, p ≤ 0.001), use of central line (OR 5.54, 95% CI 2.35-13.09, p ≤ 0.001), urinary catheter (OR 0.29, 95% CI 0.12-0.70, p ≤ 0.001) and ventilation (OR 14.87, 95% CI 7.86-28.11, p ≤ 0.007). The hospital stay 8-14 days (< 0.001) prior to infection and the mortality rate (p = 0.003) were also significantly high among patients with LREfm infections. Linezolid and vancomycin resistance coexisted; further, MDR, XDR and PDR phenotypes were significantly higher among LREfm. CONCLUSION: This study provided insight into epidemiology of MDR LREfm in a setting where linezolid use is high. The main drivers of infections with LREfm are multiple, including use of carbapenems and linezolid. Invasive procedures and increased hospital stay facilitate spread through breach in infection control practises. As therapeutic options are limited, ongoing surveillance of LREfm and VRE is critical to guide appropriate use of linezolid and infection control policies.

Synergistic properties of linezolid against Enterococcus spp. isolates: a systematic review from in vitro studies.

PURPOSE: Vancomycin-resistant enterococci (VRE) are a leading cause of hospital-acquired infections with limited therapeutic options. Combination of at least two antimicrobials is a possible strategy to obtain rapid and sustained bactericidal effects and overcome the emergence of resistance. We revised the literature on linezolid synergistic properties from in vitro studies to assess its activity in combination with molecules belonging to other antibiotic classes against Enterococcus spp. METHODS: We performed a systematic review of the literature from three peer-reviewed databases including papers evaluating linezolid synergistic properties in vitro against Enterococcus spp. isolates. RESULTS: We included 206 Enterococcus spp. isolates (92 E. faecalis, 90 E. faecium, 2 E. gallinarum, 3 E. casseliflavus, 19 Enterococcus spp.) from 24 studies. When an isolate was tested with different combinations, each combination was considered independently for further analysis. The most frequent interaction was indifferent effect (247/343, 72% of total interactions). The highest synergism rates were observed when linezolid was tested in combination with rifampin (10/49, 20.4% of interactions) and fosfomycin (16/84, 19.0%, of interactions). Antagonistic effect accounted for 7/343 (2.0%) of total interactions. CONCLUSION: Our study reported overall limited synergistic in vitro properties of linezolid with other antibiotics when tested against Enterococcus spp. The clinical choice of linezolid in combination with other antibiotics should be guided by reasoned empiric therapy in the suspicion of a polymicrobial infection or targeted therapy on microbiological results, rather than on an intended synergistic effect of the linezolid-based combination.

Epidemiological trends and susceptibility patterns of bloodstream infections caused by Enterococcus spp. in six German university hospitals: a prospectively evaluated multicentre cohort study from 2016 to 2020 of the R-Net study group.

PURPOSE: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. METHODS: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. RESULTS: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). CONCLUSION: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern.

Performance responses of lactating Rahmani ewes fed diet supplemented with Enterococcus faecium NRC-3 or Lactobacillus rhamnosus.

Production of new types of probiotics for animal nutrition mainly depends on the appropriate bacterial strain and efficient substrate. Therefore, this study aimed to evaluate the impact of two probiotic strains containing 1.2 × 108 (CFU/g), produced on permeate media on performance responses of Rahmani ewes. Thirty early lactating ewes (about 2-3 years old and weighting on average 43.2 ± 0.3 kg) were randomly divided into three groups of 10 animals each using a completely randomized design. The 1st group was fed the basal diet (60% concentrate feed mixture (CFM) + 30% Egyptian clover + 10% bean straw). While the ewes in 2nd and 3rd groups were fed the basal diet + 2 g of Enterococcus faecium NRC-3(EF) and Lactobacillus rhamnosus (LR), respectively for 9 weeks. Ewes' diet supplementation with EF or LR increased (p < 0.05) dry matter, organic matter, crude protein, neutral detergent fiber, acid detergent fiber, and non-structural carbohydrates digestibility compared to ewes of the control group. Glucose, total protein, and albumin concentrations significantly increased in the blood of EF ewes than those of LR and control. Probiotics increased ewes' milk yield as well as milk protein, fat, and lactose yields, but no differences were observed between treatments when milk components were expressed as percentage. Milk fatty acids profile not changed due to EF or LR supplementation. Probiotics (E. faecium and L. rhamnosus) produced on cheese industry waste (permeate) have proven their ability to improve the productive performance of the lactating Rahmani ewes.

Enterococcus faecium Clade Competition in the Presence of ß-Lactam Antibiotics in a Mouse GI Tract Colonization Model.

Previously, we showed that Enterococcus faecium clade B strains outcompeted health care-associated clade A1 strains in murine gastrointestinal colonization. Here, parenterally administered piperacillin-tazobactam and ceftriaxone significantly promoted colonization by clade A1 over clade B strains except that ceftriaxone, at the dose used, did not favor the least ß-lactam-resistant A1 strain. The advantage that ß-lactam administration gives to more highly ampicillin-resistant E. faecium over ampicillin-susceptible strains mirrors what occurs in hospitalized patients administered these antibiotics.

Activity of Oritavancin against Gram-Positive Pathogens Causing Bloodstream Infections in the United States over 10 Years: Focus on Drug-Resistant Enterococcal Subsets (2010-2019).

Oritavancin displayed potent and stable activity (MIC90 range of 0.06 to 0.5 mg/L) over a 10-year period (2010 to 2019) against Gram-positive pathogens that cause bloodstream infections (BSI), including methicillin-resistant Staphylococcus aureus (MRSA) and resistant subsets of Enterococcus spp. Daptomycin and linezolid were also active against methicillin-resistant S. aureus and vancomycin-resistant Enterococcus (VRE). Only oritavancin and linezolid remained active against Enterococcus faecium isolates displaying an elevated daptomycin MIC (i.e., 2 to 4 mg/L). Proportions of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus within the respective S. aureus and enterococcal populations decreased over this period.

Enterococcal contamination of hospital environments in KwaZulu-Natal, South Africa.

AIMS: Enterococci are implicated in hospital-acquired infections and show high tenacity on inanimate objects in the hospital environment. This study investigated the prevalence of Enterococcus spp. in selected wards in public hospitals at four levels of healthcare from a district in KwaZulu-Natal, South Africa. METHODS AND RESULTS: Swabs were collected from frequently touched areas in the paediatric wards and intensive care units (ICUs). Presumptive Enterococcus spp. were isolated and confirmed to genus and species levels, followed by Kirby-Bauer disk diffusion against 14 antibiotics. The results showed that enterococci were recovered from all 11 surfaces tested with the highest contamination rate observed on occupied beds and mops used to clean floors. A total number of 295 Enterococcus was identified. Polymerase chain reaction identified Enterococcus faecalis 83.1% (245/295) and Enterococcus faecium 12.9% (38/295), while whole genome sequencing identified Enterococcus gallinarum 2.0% (6/295) and Enterococcus casseliflavus 2.0% (6/295). Significant prevalence was observed in paediatric wards 64.1% (189/295) compared with the ICUs 35.9% (106/295), p < 0.05, in central, regional and district hospitals. Collectively, 82.0% (242/295) of enterococcal isolates were multidrug resistant, and 80 different antibiograms were observed. The most prominent antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for E. faecalis CIP-TET-ERY. CONCLUSION: E. faecalis was the most frequent enterococcal species isolated in all the hospitals investigated and correlates with studies conducted elsewhere. A substantially greater number of isolates were recovered from the paediatric wards compared with ICUs, and thus improved standards should be developed for infection control practices. It is suggested that the elevated use of antibiotics contributed to the increased nonsusceptible isolates observed from ICUs. This study highlighted the high recovery rate of enterococci in the hospital environment even in a nonoutbreak setting. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterocci had a high prevalence rate on the surfaces within the hospitals studied. This study gives an insight into the possible roles all healthcare staff may play in infection control intervention, including proper handling of hospital cleaning equipment and lack of knowledge about the potential for bacteria dissemination.

Modulation of lymphocyte subpopulations in the small intestine of mice treated with probiotic bacterial strains and infected with Trichinella spiralis.

AIMS: To study the local intestinal lymphocyte immunity in mice with trichinellosis affected by probiotic bacteria. METHODS AND RESULTS: Enterococcus faecium CCM8558, Enterococcus durans ED26E/7, Limosilactobacillus fermentum CCM7421 and Lactiplantibacillus plantarum 17 L/1 were administered daily (109  CFU ml-1 ) and mice were infected with Trichinella spiralis (400 larvae) on the 7th day of treatment. T. spiralis infection significantly inhibited lymphocyte subpopulations from 5 to 25 days postinfection (dpi). L. fermentum CCM7421 and L. plantarum 17 L/1 restored the CD4+ T cell numbers in the epithelium and lamina propria at the control level from 11 dpi. All strains stimulated the CD8+ T cells numbers in infected mice, which were restored in the lamina propria on 11 dpi and in the epithelium only on 32 dpi. B cells (CD19+ ) inhibition after T. spiralis infection was not affected by treatment till 25 dpi. CONCLUSIONS: The strain-specific immunomodulatory effect of tested bacteria was confirmed. L. fermentum CCM7421 and L. plantarum 17 L/1 showed the greatest immunomodulatory potential on CD4+ and CD8+ T lymphocytes in trichinellosis. E. faecium CCM8558 and E. durans ED26E/7 activated only CD8+ T cells in the lamina propria. SIGNIFICANCE AND IMPACT OF THE STUDY: Positive modulation of the gut lymphocyte immunity in T. spiralis infection with bacterial strains showed their beneficial effect with the host's antiparasitic defence.

Insight into the pilot-scale fed-batch fermentation for production of Enterococcus faecium CW3801 using molasses-based medium.

Enterococcus sp. has been used as starters in food fermentation due to their probiotic and antimicrobial properties in food biopreservation. The antimicrobial properties were mainly contributed by the bacteriocin called enterocin. Hence, the availability of a cost-effective pilot-scale cultivation conditions is a necessity for the production of probiotic bacteria. This study aims to investigate optimization of medium composition using sugarcane molasses as a carbon source using response surface methodology and the potential use of fed-batch cultivation for improvement of the cell viability of Enterococcus faecium CW3801 for the use as a probiotic starter culture. Two feeding strategies (ramp and constant) were applied in fed-batch cultivation for enhancement of the production of E. faecium in a 2-L stirred tank bioreactor using the optimized medium and scaled up to a 15-L bioreactor. Optimized fermentation medium which comprised of 10% (v/v) of molasses and 10 g/L of yeast extract at pH 7 yielded maximum cell viability of 29.4 × 1011 CFU/mL with 3900 AU/mL of bacteriocin-like inhibitory substances (BLIS) activity. In the fed-batch, the cell viability (8.4 × 1013) and dry cell weight (6.34 g/L) reached the highest in optimized medium when the ramp (stepwise) feeding was applied. In scaling up to 15-L bioreactor, the growth of E. faecium was achieved at 2.3 × 1013 CFU/mL with the dry cell weight of 5.28 g/L under the same condition. The BLIS in 15-L bioreactor was 6% higher than the 2-L bioreactor. This study demonstrated that molasses and yeast extract are good feedstock for the growth of E. faecium. The E. faecium, a non-vancomycin resistant enterococcus (VRE) was successfully produced by a fed-batch cultivation approach and scaled up to a 15-L bioreactor using a ramp feeding strategy. Results from this study revealed that the fed-batch cultivation using molasses-based medium has industrial potential for the production of probiotics.

Efficacy of Omadacycline against Multidrug-Resistant Enterococcus faecium Strains in a Mouse Peritonitis Model.

Omadacycline (OMC) showed better in vitro potency than daptomycin (DAP) or vancomycin (VAN) against Vanr, Ampr, DAP-nonsusceptible, linezolid-resistant, cfr(B)+ Enterococcus faecium strains. In a mouse peritonitis model, OMC also showed significantly better animal survival during the study and at its end than DAP or VAN with these E. faecium strains. However, OMC, DAP, and VAN showed comparable in vitro and in vivo efficacies against a non-vancomycin-resistant, tetracycline-resistant, DAP-susceptible E. faecium strain.

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

Enterococcus faecium PNC01 isolated from the intestinal mucosa of chicken as an alternative for antibiotics to reduce feed conversion rate in broiler chickens.

BACKGROUND: The development and utilization of probiotics had many environmental benefits for replacing antibiotics in animal production. Bacteria in the intestinal mucosa have better adhesion to the host intestinal epithelial cells compared to bacteria in the intestinal contents. In this study, lactic acid bacteria were isolated from the intestinal mucosa of broiler chickens and investigated as the substitution to antibiotic in broiler production. RESULTS: In addition to acid resistance, high temperature resistance, antimicrobial sensitivity tests, and intestinal epithelial cell adhesion, Enterococcus faecium PNC01 (E. faecium PNC01) was showed to be non-cytotoxic to epithelial cells. Draft genome sequence of E. faecium PNC01 predicted that it synthesized bacteriocin to perform probiotic functions and bacteriocin activity assay showed it inhibited Salmonella typhimurium from invading intestinal epithelial cells. Diet supplemented with E. faecium PNC01 increased the ileal villus height and crypt depth in broiler chickens, reduced the relative length of the cecum at day 21, and reduced the relative length of jejunum and ileum at day 42. Diet supplemented with E. faecium PNC01 increased the relative abundance of Firmicutes and Lactobacillus, decreased the relative abundance of Bacteroides in the cecal microbiota. CONCLUSION: E. faecium PNC01 replaced antibiotics to reduce the feed conversion rate. Furthermore, E. faecium PNC01 improved intestinal morphology and altered the composition of microbiota in the cecum to reduce feed conversion rate. Thus, it can be used as an alternative for antibiotics in broiler production to avoid the adverse impact of antibiotics by altering the gut microbiota.

Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium bacteremia from an Indian tertiary hospital.

We determined the clinical and molecular epidemiology of emerging nosocomial vancomycin-resistant Enterococcus faecium (VREfm)-causing serious bloodstream infections (BSIs) and the correlations between antibiotic resistance and virulence determinants among isolates. All isolates were confirmed by molecular methods (16SrRNA and E. faecium ddl genes) and tested for disk diffusion. PCR was used to detect aac(6')-aph(2″), vanA and vanB resistance genes, and asa1, cylA, ace, esp, gelE and hyl virulence genes. VREfm and high-level gentamicin-resistant (HLGR) representative isolates were selected to characterize by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Of 173 isolates, 73 (42.2%), 146 (84.4%), and 0 (0.0%) were vanA-containing VREfm, aac(6')-aph(2″)-positive HLGR, and vanB-positive. Independent predictors of VREfm infection were hematological malignancies (P = 0.001) and previous hospitalizations (P = 0.007). Observed mortality rate was 34.7%. Independent predictors of BSI-related mortality were endotracheal intubations (P < 0.001), gastrointestinal diseases (P = 0.002), and pulmonary disease (P < 0.001). All VREfm were resistant to vancomycin, teicoplanin, ciprofloxacin, and erythromycin. The esp, hyl, ace, asa1, cylA, and gelE genes were detected at 55.9, 22.5, 2.9, 2.3, 1.7, and 1.2%, respectively. The esp gene was significantly associated with VREfm compared to VSEfm (P = 0.001). PFGE analysis revealed 23 clones, with 7 major clones. The MLST analysis revealed the following five sequence types: ST80, ST17, ST117, ST132, and ST280, all belonging to CC17. The emergence and expansion of VREfm CC17 with limited antibiotic options in our hospital present a serious public health menace and represent challenges to infection control.

Protection of surface layer protein from Enterococcus faecium WEFA23 against Listeria monocytogenes CMCC54007 infection by modulating intestinal permeability and immunity.

Enterococcus faecium WEFA23 was previously found effectively against adherence and colonization of Listeria monocytogenes CMCC54007, which might be closely related to its surface layer protein (SLP). In this study, the protective of SLP of E. faecium WEFA23 against infection of L. monocytogenes CMCC54007 was systemically investigated. In vitro assay showed that SLP actively inhibited L. monocytogenes internalization into Caco-2 cell line, with decreasing mRNA level of pro-inflammation cytokines and virulence factors and restoring destroyed intestinal barrier. In vivo assay through excluding SLP of E. faecium WEFA23 by 5 M LiCl represented that SLP increased body weight, reduced mortality and cell counts of L. monocytogenes CMCC54007 in tissues of mice. Further researches showed that SLP protected against L. monocytogenes CMCC54007 infection by modulation of intestinal permeability and immunity, namely, it decreased fluorescein isothiocyanate (FITC)-Dextran in serum, ameliorated destroyed colon structure, and increased number of goblet cells and protein level of TJ protein (Claudin-1, Occludin, and ZO-1) in colon. For immunity, SLP decreased number of CD4+ and CD8+ T cells in liver, mRNA level, and content of pro-inflammatory factors IL-6, IL-1ß, IFN-γ ,TNF-α, and NO, and restored the structure of liver and spleen. Key Points•SLP of E. faecium inhibited L. monocytogenes internalization and colonization•SLP of E. faecium ameliorated host intestinal barrier dysfunction•SLP of E. faecium decreased pro-inflammatory cytokines and cells.

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridiumscidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus ß-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

BACKGROUND: The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown. METHODS: We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and ß-lactam binding affinity in HOU503 versus R497. RESULTS: DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased ß-lactam binding affinity of PBP5 of HOU503 compared with that of R497. CONCLUSIONS: Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus ß-lactam therapy.

Unexpected Cell Wall Alteration-Mediated Bactericidal Activity of the Antifungal Caspofungin against Vancomycin-Resistant Enterococcus faecium.

Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses, including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most frequently prescribed xenobiotics for ICU patients on fitness, pathogenicity, and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out, and we observed that caspofungin, an antifungal agent belonging to the family of echinocandins, had an important effect on E. faecium growth in vitro We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (1/4× MIC, 8 mg/liter), caspofungin had an impact on cell wall organization, especially with respect to the abundance of some muropeptide precursors. By transcriptome sequencing (RNA-seq), it was also shown that around 20% of the transcriptome was altered in the presence of caspofungin, with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., ß-1,3-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the metabolism of pyruvate seemed to have a drastic impact on bacterial cell viability, while a decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of nonantibiotic xenobiotics against bacterial pathogens.

Tedizolid as Step-Down Therapy following Daptomycin versus Continuation of Daptomycin against Enterococci and Methicillin- and Vancomycin-Resistant Staphylococcus aureus in a Rat Endocarditis Model.

Tedizolid (TZD) and daptomycin (DAP) were assessed in a rat endocarditis model against Enterococcus faecalis, Enterococcus faecium (resistant to vancomycin and ampicillin), and Staphylococcus aureus As a monotherapy, TZD for 5 days was not effective in a comparison with no-treatment controls, while DAP for 5 days was significantly effective against these bacteria. Step-down therapy (DAP for 3 days followed by TZD for 2 days) was as effective as DAP for 5 days and was comparable to 3 days of DAP plus ceftriaxone against all bacteria and to 3 days of DAP plus gentamicin against E. faecalis OG1RF.

Comparative analysis of Bacterial Vaginosis microbiota among pregnant and non-pregnant females and isolation of phages against Enterococcus faecalis, Enterococcus faecium, and Shigella flexneri strains.

BACKGROUND AND OBJECTIVES: Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age. It shifts the paradigms of the vagina from healthy, beneficial microbiota to facultative and strict anaerobes. BV remains one of the most arduous and controversial challenges in modern-day clinical microbiology because of its high prevalence and relapse rates. A lot of research has been carried out on it. Still, its etiology is unknown, which gave this infection global importance. The current study was designed to investigate and compare the microbiota of pregnant and non-pregnant females suffering from BV, and phages were isolated against BV microbiota. MATERIAL AND METHODS: The samples were collected from the vagina by using a speculum, and swabs were streaked on different media to isolate bacteria. The microbiological analysis was performed by microscopy, biochemical testing, and antibiotic susceptibility was determined by using Metronidazole and Clindamycin. Furthermore, the phages were isolated and characterized against BV strains. RESULTS AND CONCLUSION: The Gram staining showed high prevalence of Staphylococcus (36% vs. 33%), followed by Streptococcus (31% vs. 14%) and Enterococcus (7% vs. 14%) in non-pregnant and pregnant females' respectively. However, the exception was observed in non-pregnant BV positive females, who had Shigella flexneri in their samples. The antibiotic sensitivity showed Metronidazole was resistant against all BV microbiota, and Clindamycin showed susceptibility against 3 strains. Phages were isolated against three bacterial strains, i.e. E. faecalis, E. faecium, and S. flexneri. Bacterial reduction assay showed bacterial growth decreases in the presence of phage suspension, pH stability showed phages' maximum lytic activity at pH 7 for E. faecalis and E. faecium and pH 9 for S. flexneri. However, the thermal stability showed phages' highest lytic activity at 55 °C for E. faecalis, 70 °C for E. faecium, and 40 °C for S. flexneri. Phage genome isolation showed that all phages nucleic acid was DNA in nature and between 15 and 20kbp. SEM analysis showed they were circular in shape and might belong to the Podoviridae family. This study provides an understanding of pathogens involved in BV and helps the doctors to treat the patients accordingly. Furthermore, this study showed that Bacterial Vaginosis and BV secondary bacteria have associations. BV secondary microbiota is also involved in the pathogenesis of this infection, whereas bacteriophage therapy has the potential to be used as an alternative treatment to antibiotics.