Clinical Isolates Of Enterococcus Faecalis Research Articles

A New Kind of Quinonic-Antibiotic Useful Against Multidrug-Resistant S. aureus and E. faecium Infections.

A rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics and reducing the therapeutic arsenal available for treatment of infectious diseases. In the present study, we developed a new class of compounds with antibacterial activity obtained by a simple, two step synthesis and screened the products for in vitro antibacterial activity against ATCC® strains using the broth microdilution method. The compounds exhibited minimum inhibitory concentrations (MIC) of 1–32 μg/mL against Gram-positive ATCC® strains. The structure–activity relationship indicated that the thiophenol ring is essential for antibacterial activity and the substituents on the thiophenol ring module, for antibacterial activity. The most promising compounds detected by screening were tested against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) clinical isolates. We found remarkable activity against VREF for compounds 7 and 16, were the MIC50/90 were 2/4 µg/mL and 4/4 µg/mL, respectively, while for vancomycin the MIC50/90 was 256/512 µg/mL. Neither compound affected cell viability in any of the mammalian cell lines at any of the concentrations tested. These in vitro data show that compounds 7 and 16 have an interesting potential to be developed as new antibacterial drugs against infections caused by VREF.

Bactericidal activity of acetone extract of Alpinia galanga on multidrug resistant clinical isolates of Enterococcus faecalis

Introduction: The objective of the study was to evaluate the inhibitory activity of five traditional Indian medicinal herbs against multidrug resistant clinical isolates of E. faecalis. Methods: Sixty five E. faecalisderived from clinical specimens were tested for their resistance pattern to 7 antibiotics by disc diffusion method. Extracts from 5 medicinal plant parts were obtained and tested for inhibitory activity against multi drug resistant clinical E. faecalisisolates. The minimum bactericidal concentration (MBC) for the active plant extract exhibiting highest activity towards the susceptible isolateswas determined by macro broth dilution method followed by sub culturing on agar plate. Results: All the clinically derived E. faecalis isolates were resistant to two or more antibiotics. Acetone extract of Alpiniagalanga inhibited 67.69% of E. faecalis and concentration of <5.0mg/ml was found to exert bactericidal activity against multi drug resistant isolates. Conclusion: Acetone extract of A. galanga exhibit bactericidal activity against multi-drug resistant clinical isolates of E. faecalis and hence could be potential phytotherapeutic candidate.

Phenotype and genotype alteration during adaptive evolution of Enterococcus faecalis to antimicrobials

The current worldwide emergence of resistance to antibiotics in bacteria constitutes an important growing public health threat. The mechanisms of the emergence and dissemination of resistance remain to be elucidated. Adaptation laboratory evolution provide an approach to investigate the acquisition of de novo mutations that confers drug resistance. In our study, 3 Enterococcus faecalis clinical isolates and E. faecalis ATCC29212 were evolved resistant to 8 kinds of antimicrobials spanning five chemical classes, including ciprofloxacin, levofloxacin, gatifloxacin, penicillin, imipenem, vancomycin, chloramphenicol and gentamicin. After 10 passages for 40 days, strains exhibited high level resistance to selected drugs except for imipenem. The greatest increase was observed in those evolved to quinolones, which caused >256-fold increase in MIC compared to the wild type. Cross-resistance and collateral-sensitivity were widely found after evolution. Through genotypic analysis of quinolones resistance strains, amino acid changes were observed in the QRDR region of GyrA and ParC. Substitutions occurred at GyrA were detected as Ser84Asn/Ser84Ile/Ser84Arg/Gly106Asp. However, Substitutions in ParC were found as Ser82Ile/Glu86Lys/Glu86Gly/His105Tyr. Compared with ancestral strains, the growth rates of evolved resistant strains slowed down and the logarithmic phase was delayed >7 h. While, the biofilm formation capacity of strains was not significantly changed by evolutionary adaptation. Our data verified that long-term exposure to sub-lethal concentrations of antimicrobial agents could selectively enrich drug resistant mutant of E. faecalis, conferring to cross-resistance and collateral-sensitivity towards other antimicrobials and fitness costs. Defining these effects can provide alternative antimicrobial strategies directed to mitigate the selection of antibiotic resistant microbes.

Association between Extracellular Material and Biofilm Formation in Response to Sodium Hypochlorite by Clinical Isolates of Enterococcus faecalis

IntroductionExtracellular material (ECM) surrounding Enterococcus faecalis may play a role in increasing resistance to environmental stresses. Our aim was to determine ECM levels in response to subminimal inhibitory concentrations of sodium hypochlorite (sub-MIC/NaOCl) or anaerobic growth and determine the impact on biofilm development. MethodsFrom 37 E. faecalis clinical strains, 19 were selected according to their biofilm-producing ability by using a crystal violet biofilm assay: 10 strong, 4 intermediate, and 5 non-biofilm producers. Biofilm assays were subsequently performed on all strains when subjected to sub-MIC/NaOCl. All strains were evaluated for ECM production under aerobic and anaerobic conditions and with sub-MIC/NaOCl. ECM production was assessed by using scanning electron microscopy. Double-blinded independent assessors were used to score levels of ECM production. The esp gene was detected by using polymerase chain reaction. Gelatinase activity was determined by using Todd-Hewitt and gelatin agar. ResultsIn aerobic conditions, ECM was expressed in all strains. In the presence of sub-MIC/NaOCl, of the 10 strong biofilm producers, 5 increased their ECM production, and 4 showed increased biofilm growth. Two strains had less ECM production and showed decreased biofilm growth. One isolate demonstrated no observable changes. Most non-biofilm producers demonstrated no observable differences in ECM production, although 1 strain increased biofilm growth. ECM production in anaerobic conditions was highly variable. The esp gene (n = 15) and gelatinase activity (n = 7) were evident among the isolates. ConclusionsClonal diversity among strains of E. faecalis suggests that some strong biofilm producers can upregulate ECM production and increase biofilm growth in response to sub-MIC/NaOCl.

Isolation and structure elucidation of lipopeptide antibiotic taromycin B from the activated taromycin biosynthetic gene cluster.

In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.

Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain)

IntroductionIn order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised. MethodsMolecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records. ResultsA total of 79 vancomycin-resistant E. faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak. ConclusionThe outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones.

Comparison of biofilm-producing Enterococcus faecalis, Enterococcus faecium, and unusual Enterococcus strains

The present study focused on determining the prevalence of biofilm-forming ability in Enterococcus faecalis , E. faecium , and unusual Enterococcus clinical isolates, and comparison of resistance and the prevalence of selected virulence factors among biofilm-positive strains. The ability to form biofilm was detected in 13.3% of E. faecalis , 90% of E. faecium , and 57.1% of unusual Enterococcus strains ( p =0.026). All E. faecalis strains were susceptible to β-lactams, while 37.5% of unusual and all E. faecium isolates were resistant to these antibiotics. Resistance to gentamicin was detected in 75% of E. faecalis , 55.5% of E. faecium , and 25% of other strains; resistance to streptomycin in 25%, 83.3%, and 50%, respectively. Analysis of the virulence revealed that the enterococcal surface protein ( esp ) gene was found in all E. faecium , 75.0% of E. faecalis , and 37.5% of other strains; collagen adhesin gene ( ace ) in 100%, 25.0%, and 37.5%; and hyaluronidase gene ( hyl ) in 83.3%, 0%, and 37.5%, respectively. Analysis of the resistance and virulence patterns showed that E. faecium isolates had the greatest variety of virulence and resistance determinants, while the lowest variety was exhibited by unusual strains. These findings indicate that unusual biofilm-producing Enterococcus strains have lower resistance and virulence potency than E. faecalis and E. faecium .

Detection of aminoglycoside resistance gene aph(2")Ic in clinical isolates of Enterococcus faecalis

Detection of aminoglycoside resistance gene aph(2")Ic in clinical isolates of Enterococcus faecalis

Antibiotic-potentiating activity, phytochemical profile, and cytotoxicity of Acalypha integrifolia Willd. (Euphorbiaceae)

Acalypha integrifolia Willd. (Euphorbiaceae) (AI), an indigenous medicinal plant of the Mascarene Islands is traditionally used to manage infectious diseases. The authors aimed to evaluate the antimicrobial, antibiotic-potentiating activity and cytotoxicity of AI. Decoction as traditionally used and organic extracts (hexane, dichloromethane, ethyl acetate, and methanol) of AI leaves were screened for their antimicrobial activity against nine ATCC strains and 10 clinical isolates. A fractional inhibitory concentration index (FICI) was determined to establish any synergistic interaction between the extracts and antibiotics using the variable ratio analysis method. The phytochemical profile was established using chemical and thin-layer chromatography methods. The decoction and methanolic extracts inhibited the growth of all tested bacteria (minimum inhibitory concentration (MIC) ranged between 0.06–2.00mg/ml). The methanolic extract showed potent antibacterial activity against Escherichia coli (EC) (MIC: 0.06mg/ml), Pseudomonas aeruginosa (MIC: 0.50mg/ml), and Staphyloccocus aureus (SA) (MIC: 0.06mg/ml). The highest antibacterial activity was observed against clinical isolates of Enterococcus faecalis, S. aureus, and Methicillin-Resistant S. aureus (MRSA) (MIC: 0.50mg/ml). Moderate antifungal activity was observed against Candida albicans. Combination profiles of the extract with Ciprofloxacin, Chloramphenicol, and Streptomycin showed synergistic action (FICI≤0.50) against ATCC strains of EC and SA. Decoction and methanolic extracts showed low cytotoxicity against normal adult African green monkey kidney cells. Data obtained tends to corroborate with the reported traditional usage of the plant. Since the leaf decoction is traditionally used against skin infections as well as consumed for its astringent and purgative properties, the reported data tend to correlate with the low cytotoxicity as well as its anti-infective property. There may also be the potential towards the discovery of novel antimicrobial compounds.

Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

Antimicrobial influence of different root canal filling techniques in experimentally infected human root canals.

The aim of the present study was to evaluate the antimicrobial influence of different root canal filling techniques using gutta-percha and an epoxy resin-based sealer in experimentally infected root canals of extracted human teeth. In total, 96 intact sterilized, permanent human anterior teeth and premolars with single patent root canals were prepared and infected with a clinical isolate of Enterococcus faecalis. After 72 hours, all root canals were sampled using three sterile paper points. The tooth specimens were randomly divided into three groups and a control of 24 specimens each, according to the respective obturation techniques: lateral condensation (LC group), ProTaper Thermafil (PT group), and vertical compaction technique (VC group). AH Plus was used as sealer. The control group was left untreated (without root canal filling). After 7 days root canal fillings were removed and collected. The root canals were sampled using three sterile paper points and dentin chips were obtained from the root canal walls. The samples were cultured on blood agar, and colony forming units were counted. All root canal filling techniques significantly reduced bacterial viability, eliminating more than 99.9% of E faecalis. In the LC group, three (13%) root canals were culture negative. In the PT group, 21 (88%) root canals and in the VC group 15 (54%) were culture negative. All root canal filling techniques significantly reduced E faecalis in root canals. In cases where warm filling techniques can be applied, these should be preferred to cold obturation.

Rapid Identification of Vancomycin Resistant Enterococcus Faecalis Clinical Isolates using a Sugar Fermentation Method.

Vancomycin Resistant Enterococci (VRE) can be found all over the world. Thus, rapid detection of the isolates could be of high importance in the treatment or prevention of the associated disease. To measure the turanose fermentation in Enterococcus faecalis clinical isolates for rapid differentiation of VRE and Vancomycin-Susceptible E. faecalis (VSE) isolates. Forty E. faecalis samples were isolated from 200 clinical samples in Tehran Medical Center, Iran, from October 2012 to December 2012. These isolates were detected according to the standard microbial and biochemical tests. Detection of VRE isolates was originally performed by disk diffusion using 1 μg vancomycin disk, followed by Polymerase Chain Reaction (PCR) amplification of the vanA gene. Finally, the turanose consumption in 1%, 0.7% and 0.5% dilutions was detected by a phenotypic method. Among the 40 E. faecalis isolates, 20 vancomycin-susceptible and 20 vancomycin-resistant E. faecalis were isolated according to the disk diffusion and PCR of the vanA gene. There was a considerable difference between VRE and VSE isolates in 0.7% dilution of turanose. However, there was no significant difference between VRE and VSE in 1% and 0.5% dilutions of turanose. Since detection of VRE isolates is of high importance, especially in nosocomial infections, phenotypic methods may be highly useful for this purpose. In conclusion, our data indicate that VRE isolated from clinical samples could be distinguished from VSE isolates by turanose fermentation at dilution 0.7%.

Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances.

Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner.

The increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to the d-Ala-d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variant d-Ala-d-Lac, which is produced by VanA. Here we show that exogenous d-Ala competes with d-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of the d-Ala-d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations of d-Ala led to the production of a significant amount of wild-type cell wall precursors, while vanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organism Streptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates of Enterococcus faecium (VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity of S. coelicolor cells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

Persistencia de un clon ST6 de Enterococcus faecalis con genotipo vanB2 en dos hospitales de Aragón

IntroductionIn order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised. MethodsMolecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records. ResultsA total of 79 vancomycin-resistant E.faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak. ConclusionThe outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones.

Antimicrobial activity of crude leaf extracts from medicinal plants against Enterococcus faecalis

Enterococcus faecalis is a Gram negative, commensal enteric bacterial pathogen usually located in the intestinal tracts of either animals or human beings. The pathogenic members of enteric bacteria’s are usually associated to infections that are characterized by enteric fevers, abdominal pain, and diarrhoea and vomiting. This study aimed at determining the effects of the selected medicinal plants extracts against Clinical isolate of Enterococcus faecalis obtained from Kenyatta University health Centre. Kirby Bauer method was used to determine the antimicrobial activity of the plants extracts against Enterococcus faecalis. All the plant extracts showed antimicrobial activity against Enterococcus faecalis with Tagetes minuta producing the largest average zones of inhibition of 18.67±1.03mm when compared to the other plant extracts. Vernonia lasiopus was more active at low concentrations (MIC 3.9µg/ml; MBC 5.0µg/ml) while Aloe secundiflora was less active (MIC 7.0µg/ml; 9.7µg/ml). Ciprofloxacin (5µg/ml) was used as a positive control producing an average zone of inhibition of 21.67±2.66mm while Methanol (33.50±2.56mm) and 4% Dimethyl sulphoxide (0.00±0.00mm) were used as negative control. Qualitative phytochemical screening showed the presence of four phytochemicals namely; saponins, tannins, alkaloids and flavonoids. The study provides insight about the antimicrobial activity of the plant extracts and if they can be used in treatment of as an antimicrobial agent against infections caused by Enterococcus faecalis.

Phenotypic and genotypic characterization of vancomycin-resistant Enterococcus faecium clinical isolates from two hospitals in Mexico: First detection of VanB phenotype-vanA genotype

IntroductionEnterococcus faecium has emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. Our aim was to determine the antimicrobial susceptibility, biofilm production, and clonal relatedness of vancomycin-resistant E. faecium (VREF) clinical isolates from two hospitals in Mexico. MethodsConsecutive clinical isolates (n=56) were collected in two tertiary care hospitals in Mexico from 2011 to 2014. VREF isolates were characterized by phenotypic and molecular methods including pulsed-field gel electrophoresis (PFGE). ResultsVREF isolates were highly resistant to vancomycin, erythromycin, norfloxacin, high-level streptomycin, and teicoplanin, and showed lower resistance to tetracycline, nitrofurantoin and quinupristin-dalfopristin. None of the isolates were resistant to linezolid. The vanA gene was detected in all isolates. Two VanB phenotype-vanA genotype isolates, highly resistant to vancomycin and susceptible to teicoplanin, were detected. Furthermore, 17.9% of the isolates were classified as biofilm producers, and the espfm gene was found in 98.2% of the isolates. A total of 37 distinct PFGE patterns and 6 clones (25% of the isolates as clone A, 5.4% as clone B, and 3.6% each as clone C, D, E, and F) were detected. Clone A was detected in 5 different wards of the same hospital during 14 months of surveillance. ConclusionThe high resistance to most antimicrobial agents and the moderate cross-transmission of VREF detected accentuates the need for continuous surveillance of E. faecium in the hospital setting. This is also the first reported incidence of the E. faecium VanB phenotype-vanA genotype in the Americas.

Evaluation of DiversiLab®, MLST and PFGE typing for discriminating clinical Enterococcus faecium isolates

We evaluated and critically assessed the performance and discriminatory power of a rep-PCR based commercial test DiversiLab® Enterococcus kit (bioMerieux) for typing a set of 65 representative isolates of Enterococcus faecium/VRE and compared it to state-of-the-art typing techniques such as PFGE and MLST.

The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

The toxin–antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

Increased frequency of linezolid resistance among clinical Enterococcus faecium isolates from German hospital patients.

Linezolid is an antibiotic of last resort for the treatment of infections with vancomycin-resistant enterococci (VRE). Here we report the increasing prevalence of linezolid resistance among clinical Enterococcus faecium strains from German hospital patients. Linezolid minimum inhibitory concentrations (MICs) were determined for 4461 clinical E. faecium strains isolated between 2008 and 2014. Isolates originated from the network of diagnostic laboratories collaborating with the National Reference Centre (NRC) for Staphylococci and Enterococci covering all German federal states. All linezolid-resistant isolates were determined by broth microdilution and confirmed by Etest as well as by analysing the 23S rDNA for putative mutations. Marker genes were determined by PCR. Genotyping was performed by SmaI macrorestriction analysis in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for selected isolates. An increase in linezolid resistance was observed, from <1% in 2008 to >9% in 2014. Occasionally, outbreaks with linezolid-resistant VRE (ST117) were observed. In total, 232 (92.4%) of 251 linezolid-resistant E. faecium isolates (including 61 vanA and 29 vanB) contained the G2576T 23S rDNA mutation and showed a varying mixture of wild-type and mutated alleles per genome sufficient to confer linezolid resistance. In vitro growth experiments revealed a stable linezolid MIC. Of the 251 linezolid-resistant isolates, 5 were cfr-positive. In conclusion, these NRC data identified a country-wide ongoing trend of increasing linezolid resistance among clinical E. faecium isolates within the last 5 years.