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Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra., Competing Interests: The authors declare no conflict of interest.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Dual resistance to linezolid and glycopeptides is a milestone reached by certain extensively drug-resistant (XDR) enterococci. This paper describes the molecular and epidemiological investigations of a linezolid-resistant and vancomycin-resistant Enterococcus faecium (E. faecium) (LVREf) outbreak in the French overseas territory of Reunion Island (Indian Ocean)., Methods: All vancomycin-resistant Enterococcus (VRE) isolates detected on Reunion Island between 2015 and 2019 were included in the study. The VRE isolates were phenotypically characterised and genetically explored by whole-genome sequencing (WGS)., Results: Sixteen vancomycin-resistant E. faecium (VREf) isolates were retrieved between 2015 and 2019. Seven isolates obtained in 2019 were involved in the outbreak. These seven LVREf isolates from the 2019 outbreak at the University Hospital of Reunion Island (UHRI) were suspected to be related to a linezolid-susceptible VREf strain imported from India. An epidemiological link was highlighted for six of the seven outbreak cases. All the LVREf outbreak isolates were obtained from rectal swabs (colonisation) and resistant to vancomycin (MIC > 128 mg/L) and linezolid (MIC 8-32 mg/L); one isolate was also resistant to daptomycin (MIC 8 mg/L). The seven outbreak isolates were positive for the vanA and optrA genes and belonged to ST761., Conclusions: These results argue for the strict application of control and prevention measures for VRE clones at high risk of spread, particularly in areas such as Reunion Island where the risk of importation from the Indian subcontinent is high. The regional spread of optrA linezolid-resistance genes in VRE isolates is a matter of concern, due to possibility of treatment failure., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5'untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) ribosome-binding site (RBS) was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objectives: To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments., Methods: All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments., Results: Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids., Conclusions: The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The aim of the study was to characterize the antimicrobial activity of clinically-relevant biocides (chlorhexidine digluconate, benzalkonium chloride, PVP-iodine and triclosan) and to determine the sRNA expression profiles under biocide exposure in two major bacterial opportunistic pathogens, Enterococcus faecium and Staphylococcus aureus. In vitro activities were evaluated against S. aureus HG003 and E. faecium Aus0004. We determined MIC, MBC, sub-inhibitory concentrations (SIC) and growth curves under SIC conditions. sRNA expression study under SIC exposure of biocides was performed by RT-qPCR on 3 sRNAs expressed in S. aureus (RNAIII, SprD and SprX) and the first 9 sRNAs identified as expressed in E. faecium. MICs were higher against E. faecium than for S. aureus. Growth curves under increasing biocide concentrations highlighted two types of bactericidal activity: "on/off" effect for chlorhexidine, benzalkonium chloride, PVP-iodine and a "concentration-dependent" activity for triclosan. Exposure to biocide SICs led to an alteration of several sRNA expression profiles, mostly repressed. The distinct biocide activity profiles must be evaluated with other compounds and bacterial species to enrich the prediction of resistance risks associated with biocide usage. Biocide exposure induces various sRNA-mediated responses in both S. aureus and E. faecium, and further investigations are needed to decipher sRNA-driven regulatory networks.

Eravacycline (ERC), the first fluorocycline, is a new tetracycline with superior activity to tigecycline (TGC) against many bacterial species. This work aimed to determine the in vitro activity of ERC compared with other tetracyclines against enterococcal clinical isolates and to analyse corresponding resistance mechanisms. A collection of 60 enterococcal strains was studied: 54 epidemiologically unrelated clinical isolates (46 Enterococcus faecium and 8 Enterococcus faecalis) including 42 vancomycin-resistant enterococci (VRE) (33 vanA and 9 vanB), 3 in vitro TGC-resistant mutants (E. faecium AusTig, HMtig1 and HMtig2) and 3 reference wild-type strains (E. faecium Aus0004 and HM1070, E. faecalis ATCC 29212). In vitro susceptibility was determined using Etest strips (for ERC) or by broth microdilution (for TGC, doxycycline, minocycline and tetracycline). Resistance genes [tet(M), tet(L), tet(O) and tet(S)] were screened by PCR for TGC- and/or ERC-resistant strains as well as sequencing of the rpsJ gene (encoding ribosomal protein S10). MIC 50/90 values were 0.016/0.08, ≤0.03/0.5, 4/32, 8/16 and 32/>32 mg/L for ERC, TGC, doxycycline, minocycline and tetracycline, respectively. According to EUCAST guidelines, nine strains were categorised as resistant to TGC (MIC, 0.5-8 mg/L), including four E. faecium vanA(+) strains also resistant to ERC (MIC, 0.19-1.5 mg/L). These four strains all possessed at least one mutation in rpsJ and two tet determinants: tet(M) + tet(L) (n = 2); and tet(M) + tet(S) (n = 2). Although ERC has excellent in vitro activity against enterococci (including VRE), emergence of resistance is possible due to combined mechanisms (rpsJ mutations + tet genes)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., β-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., (Copyright © 2020 American Society for Microbiology.)

Background: Linezolid-resistant enterococci (LRE) causing infections that are challenging to treat are rising, highlighting the need for reliable screening of LRE clinical isolates., Objectives: To evaluate the ability of the broth microdilution (BMD) method for LRE detection and to assess the performance of seven commercially available techniques for linezolid susceptibility testing., Methods: A collection of 100 clinical isolates (80 Enterococcus faecium and 20 Enterococcus faecalis), including 20 optrA-positive isolates, 17 poxtA-positive isolates and 1 optrA/poxtA-positive E. faecium isolate, were studied. MICs were determined after 18 h [Day 1 (D1)] and 42 h [Day 2 (D2)] of incubation and interpreted following EUCAST and CLSI guidelines, which currently provide different interpretative breakpoints. Performance of commercial techniques was compared with BMD results., Results: MIC50/D1 and MIC50/D2 were both 8 mg/L, while MIC90/D1 and MIC90/D2 were 16 and 32 mg/L, respectively. MICD1 values for poxtA-positive isolates were lower than those for optrA-positive isolates. Proportions of susceptible isolates at D1 and D2 were 48% and 41%, respectively, according to EUCAST breakpoints and 35% and 13%, respectively, according to CLSI criteria (the proportions of isolates categorized as intermediate following CLSI recommendations were 13% and 28% at D1 and D2, respectively). Percentage susceptibility assessed by the commercially available techniques was always higher. The four commercial methods allowing MIC determination provided an overall essential agreement of ≥90% at D1. Categorical agreement and error rates were generally improved at D2., Conclusions: Non-automated methods (Sensititre and UMIC) and, to a lesser extent, gradient strip Etest appear to show an acceptable correlation with the BMD reference method for the detection of isolates with low MICs of linezolid after prolonged incubation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France., Methods: The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A., Results: As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization., Conclusions: Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D)., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Vancomycin-resistant Enterococcus faecium is a leading cause of MDR hospital infection. Two genetically definable populations of E. faecium have been identified: hospital-adapted MDR isolates (clade A) and vancomycin-susceptible commensal strains (clade B). VanN-type vancomycin resistance was identified in two isolates of E. faecium recovered from blood and faeces of an immunocompromised patient. To understand the genomic context in which VanN occurred in the hospitalized patient, the risk it posed for transmission in the hospital and its origins, it was of interest to determine where these strains placed within the E. faecium population structure., Methods: We obtained the genome sequence of the VanN isolates and performed comparative and functional genomics of the chromosome and plasmid content., Results: We show that, in these strains, VanN occurs in a genetic background that clusters with clade B E. faecium, which is highly unusual. We characterized the chromosome and the conjugative plasmid that carries VanN resistance in these strains, pUV24. This plasmid exhibits signatures of in-host selection on the vanN operon regulatory system, which are associated with a constitutive expression of vancomycin resistance. VanN resistance in clade B strains may go undetected by current methods., Conclusions: We report a case of vancomycin resistance in a commensal lineage of E. faecium responsible for an atypical bacteraemia in an immunocompromised patient. A reservoir of transferable glycopeptide resistance in the community could pose a concern for public health.

Objectives: To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon., Methods: The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin., Results: E. faecium 16-346 was resistant to rifampicin (MIC >4 mg/L), erythromycin (MIC >4 mg/L), tetracycline (MIC >16 mg/L) and vancomycin (MIC 8 mg/L), but susceptible to teicoplanin (MIC 0.5 mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5 kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin., Conclusions: This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

Objectives: Daptomycin has become a first-line therapeutic option for vancomycin-resistant Enterococcus faecium infective endocarditis (IE). Although high doses (≥8 mg/kg) are often recommended, optimal doses, particularly for strains with MICs close to the susceptibility breakpoint (4 mg/L), are still debated., Methods: Daptomycin efficacy at doses equivalent to 8 mg/kg daptomycin (DAP8) and 12 mg/kg daptomycin (DAP12) in humans was evaluated in a rabbit model of aortic valve IE induced by 108 cfu of E. faecium reference strain Aus0004 (daptomycin MIC = 2 mg/L) or its in vitro mutant strain Mut4 (daptomycin MIC = 4 mg/L). Treatment began 48 h post-inoculation and lasted 5 days., Results: With Aus0004, the median log10 cfu/g of vegetations was significantly lower after DAP8 and DAP12 versus controls [6.05 (n = 12) and 2.15 (n = 10) versus 9.14 (n = 11), respectively; P < 0.001], with DAP12 being more effective than DAP8 concerning vegetation bacterial load (P < 0.001) and percentages of sterile vegetations (100% versus 0%, respectively; P < 0.001). Daptomycin-resistant Aus0004 mutants were detected in 8.3% of DAP8-treated vegetations. With Mut4, the median log10 cfu/g of vegetations was significantly lower after DAP8 and DAP12 versus controls [7.7 (n = 11) and 6.95 (n = 10) versus 9.59 (n = 11), respectively; P = 0.001 and P = 0.002], without any between-dose difference, but no vegetation was sterile. Moreover, 7 of 11 (63.6%) and 7 of 9 (77.8%) vegetations contained resistant mutants after DAP8 and DAP12, respectively., Conclusions: DAP12 was the most successful strategy against IE due to a WT E. faecium strain (daptomycin MIC = 2 mg/L). To treat IE strains with MIC = 4 mg/L, DAP8 or DAP12 monotherapy was poorly effective with the risk of resistant mutant emergence. Reassessment of the daptomycin susceptibility breakpoint for enterococci seems necessary.

Vancomycin-resistant Enterococcus faecium is a leading cause of hospital-acquired infections and outbreaks. Regulatory RNAs (sRNAs) are major players in adaptive responses, including antibiotic resistance. They were extensively studied in gram-negative bacteria, but less information is available for gram-positive pathogens. No sRNAs are described in E. faecium. We sought to identify a set of sRNAs expressed in vancomycin-resistant E. faecium Aus0004 strain to assess their roles in daptomycin response and resistance. Genomic and transcriptomic analyses revealed a set of 61 sRNA candidates, including 10 that were further tested and validated by Northern and qPCR. RNA-seq was performed with and without subinhibitory concentrations (SICs) of daptomycin, an antibiotic used to treat enterococcal infections. After daptomycin SIC exposure, the expression of 260 coding and srna genes was altered, with 80 upregulated and 180 downregulated, including 51% involved in carbohydrate and transport metabolisms. Daptomycin SIC exposure significantly affected the expression of seven sRNAs, including one experimentally confirmed, sRNA&#95;0160. We studied sRNA expression in isogenic mutants with increasing levels of daptomycin resistance and observed that expression of several sRNAs, including sRNA&#95;0160, was modified in the stepwise mutants. This first genome-wide sRNA identification in E. faecium suggests that some sRNAs are linked to antibiotic stress response and resistance.

Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo , with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB -positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004&#95;02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004&#95;02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy., (Copyright © 2017 American Society for Microbiology.)

Objectives: To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium., Methods: Daptomycin-resistant mutants (Mut4, Mut8, Mut16, Mut32, Mut64 and Mut128 with MICs from 4 to 128 mg/L) were obtained in vitro from E. faecium strain Aus0004 (MIC at 2 mg/L). The entire genome sequences of Mut64 and Mut128 were determined as well as those of liaFSR and cls genes for other mutants and corresponding revertants (named Rev4 to Rev128). The study of daptomycin resistance stability was performed without any selective pressure. The expression of liaF, liaS and liaR genes was quantified by quantitative RT-PCR., Results: By comparative genomic analysis, substitutions Asn13Ser in cls and Gly92Asp in liaS were identified in Mut64 and Mut128. Only the liaS mutation was found in Mut16 and Mut32 while Mut4 and Mut8 were devoid of any mutation. After 15 days, all mutants except Mut4 reverted to daptomycin hypersusceptibility (MICs from 0.12 to 0.25 mg/L). In all revertants (except Rev4 and Rev8), an IS was found in the liaFSR operon with a dramatic decrease of its expression: IS66 in the promoter region of liaF (Rev16 and Rev64), IS30 in liaR (Rev32) and IS982 in liaF (Rev128)., Conclusions: We demonstrated the stepwise and sequential acquisition of mutations in liaS and in cls leading to daptomycin resistance in E. faecium, and the instability of daptomycin resistance as well as the role of liaFSR inactivation in reversion to daptomycin hypersusceptibility., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67-92% of strains at recommended dosages (4-6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg)., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Tigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitro mutant AusTig and in vitro mutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strains E. faecium Aus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistant E. faecium clinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, the in vitro mutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJ gene (efau004&#95;00094, coding for the S10 protein of the 30S ribosomal subunit), efau004&#95;01228 (encoding a cation transporter), efau004&#95;01636 (coding for a hypothetical protein), and efau004&#95;02455 (encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The worldwide ratio of Enterococcus faecalis-Enterococcus faecium infections is currently changing in favor of E. faecium. Intrinsic and acquired antimicrobial resistance traits of this latter species can explain this evolution as well as the diffusion of hospital-adapted strains belonging to the clonal complex CC17. Like other enterococci, E. faecium is naturally resistant to cephalosporins and aminoglycosides (at low level). Because of its high genome plasticity, it can also acquire numerous other resistances. It is noteworthy that most modern isolates of E. faecium are highly resistant to ampicillin while a non-negligible proportion of them (depending on geographical locations) are resistant to glycopeptides (especially in the USA). Even if resistance to newer antimicrobial agents (linezolid, daptomycin, tigecycline) is still uncommon, some clinical isolates with reduced susceptibility or resistance have already been reported and better understanding of resistance mechanisms is needed for prediction and prevention of their dissemination.

As opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faecium is naturally susceptible. Since this phenotype may be selected for in vivo by quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitro mutants of E. faecium HM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitro mutant (E. faecium UCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitro and in vivo resistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for Enterococcus ABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)v from UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to β-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and adaptation for survival within the host, thereby contributes importantly to the opportunistic traits of E. faecium.

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleria mellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process., (Copyright © 2012 S. Karger AG, Basel.)

• Enterococci have become major human MDR nosocomial pathogens. • They are found ubiquitously in non-human habitats (animals and the environment). • The main threat for public health is the transfer of MDR enterococci and AMR genes. • Contamination of humans through the food chain seems to be minimal. • The environment (especially wastewaters) may play a critical role in AMR in humans. Enterococci are long-standing members of the gastrointestinal tract of humans and many animals and they are also ubiquitously distributed in natural environments. Classically as harmless bacteria, two main species (namely Enterococcus faecalis and Enterococcus faecium) have become a leading cause of human infections, especially in hospital settings, with the worldwide spread of multidrug-resistant isolates, especially vancomycin-resistant enterococci. In this review, it will be summarized what is known about genetic diversity and ecology of enterococci with a focus on E. faecalis and E. faecium from human and non-human habitats and related risks for public health. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the role of the regulatory small RNA (sRNA) Ern0160 in gastrointestinal tract (GIT) colonization by Enterococcus faecium. For this purpose, four strains of E. faecium were used, Aus0004 (WT), an ern0160 -deleted Aus0004 mutant (Δ0160), a trans -complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). Strains were studied both in vitro and in vivo , alone and in competitive assays. In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured single while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. Then, in vivo experiments were performed using a GIT colonization mouse model. Several existing models of GIT colonization were compared while a novel one, combining ceftriaxone and amoxicillin, was developed. A GIT colonization was performed with each strain alone, and no significant difference was noticed. By contrast, significant results were obtained with co-colonization experiments. With WT + Δ0160 suspension, a significant advantage for the WT strain was observed from day 5 to the end of the protocol, suggesting the involvement of ern 0160 in GIT colonization. With Δ0160_0160 + Δ0160_pAT29 suspension, the strain with the empty vector took the advantage from day 3 to the end of the protocol, suggesting a deleterious effect of ern0160 overexpression. Altogether, these findings demonstrate the potential implication of Ern0160 in GIT colonization of E. faecium. Further investigations are needed for the identification of sRNA target(s) in order to decipher underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output.Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins.Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance.Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered. [ABSTRACT FROM AUTHOR]

Objectives: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates.Methods: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments.Results: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases.Conclusions: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not). [ABSTRACT FROM AUTHOR]

Objectives: To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon.Methods: The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin.Results: E. faecium 16-346 was resistant to rifampicin (MIC >4 mg/L), erythromycin (MIC >4 mg/L), tetracycline (MIC >16 mg/L) and vancomycin (MIC 8 mg/L), but susceptible to teicoplanin (MIC 0.5 mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5 kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin.Conclusions: This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium. [ABSTRACT FROM AUTHOR]

Background: The ecological impact of ciprofloxacin on commensal enterococci is unknown.Methods: Forty-eight healthy volunteers received ciprofloxacin from day (D) 0 to D14; stools were collected on D7, D14 and D42. Fluoroquinolone-susceptible and -resistant enterococci (FQ-SE and FQ-RE) were detected and quantified by culture, and identified by MALDI-TOF MS. The relative abundance of FQ-RE over FQ-SE was determined. The genetic basis of fluoroquinolone resistance was deciphered by partial sequencing of gyrA and parC genes. Clonal relatedness was determined by random amplification of polymorphic DNA PCR. Clinical trial no.: NCT00190151.Results: Enterococci were carried by 47/48 (98%) subjects. Total counts were reduced during ciprofloxacin therapy (4.0 and 3.9 log cfu/g on D7 and D14 versus 5.9 log cfu/g before and 6.9 log cfu/g after treatment; P  < 0.05). Twenty-one out of 48 (44%) carried FQ-RE; among them, 21/21 carried Enterococcus faecium , 19 carried Enterococcus faecalis and 11 carried other species. Five out of 48 (10%) harboured FQ-RE (ciprofloxacin MIC >4 mg/L) before treatment (all E. faecium ), 6 on D7 (3 E. faecium and 3 E. faecalis ), 8 on D14 (4 E. faecium and 4 E. faecalis ) and 10 (21%) on D42 (9 E. faecium and 1 E. faecalis ). The relative abundance of FQ-RE increased from 44% on D0 to 73% and 75% on D7 and D14, respectively. No acquisition of fluoroquinolone resistance among endogenous D0 strains was evidenced. All (14/14) distinct Fluoroquinolone-resistant E. faecalis clones were gyrA / parC double mutants with high-level resistance (ciprofloxacin MIC >64 mg/L). In contrast, 34/35 E. faecium exhibited low-level resistance (ciprofloxacin MIC 4-32 mg/L) with no gyrA / parC mutation, but overexpressed the chromosomal Efm qnr gene. As compared with Fluoroquinolone-susceptible strains, Fluoroquinolone-resistant E. faecium were more frequently ampicillin resistant and Fluoroquinolone-resistant E. faecalis were more highly resistant to gentamicin.Conclusions: Although intrinsically poorly susceptible to fluoroquinolones, gut populations of enterococci are highly impacted both quantitatively and qualitatively by ciprofloxacin. [ABSTRACT FROM AUTHOR]

Twenty-five years ago, isolation of vancomycin-resistant Enterococcus faecium (VREm) was reported both in the UK and in France. Since then, VREm has spread worldwide in hospitals. Hospital outbreaks appeared to be related to the evolution since the end of 1980s of a subpopulation of E. faecium highly resistant to ampicillin and fluoroquinolones (the so-called clonal complex CC17) that later acquired resistance to vancomycin. CC17 isolates are presumably better adapted than other E. faecium isolates to the constraints of the hospital environment and most contain mobile genetic elements, phage genes, genes encoding membrane proteins, regulatory genes, a putative pathogenicity island and megaplasmids. Colonization and persistence are major features of VREm. Inherent characteristics of E. faecium including a remarkable genome plasticity, in part due to acquisition of IS elements, in particular IS16, have facilitated niche adaptation of this distinct E. faecium subpopulation that is multiply resistant to antibiotics. Quinupristin/dalfopristin and linezolid are licensed for the treatment of VREm infections, with linezolid often used as a first-line treatment. However, the emergence of plasmid-mediated resistance to linezolid by production of a Cfr methyltransferase in Enterococcus faecalis is worrying. Daptomycin has not been extensively evaluated for the treatment of VREm infections and resistant mutants have been selected under daptomycin therapy. Although control of VRE is challenging, a laissez-faire policy would result in an increased number of infections and would create an irreversible situation. Although so far unsuccessful, dissemination of glycopeptide-resistant Staphylococcus aureus with van genes acquired from resistant enterococci cannot be ruled out. [ABSTRACT FROM AUTHOR]

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleriamellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To determine if hospital effluent input has an ecological impact on downstream aquatic environment, antibiotic resistance in Enterococcus spp. along a medical center-retirement home-wastewater treatment plant-river continuum in France was determined using a culture-based method. Data on antibiotic consumption among hospitalized and general populations and levels of water contamination by antibiotics were collected. All isolated enterococci were genotypically identified to the species level, tested for in vitro antibiotic susceptibility, and typed by multilocus sequence typing. The erm(B) and meflA) (macrolide resis- tance) and tet(M) (tetracycline resistance) genes were detected by PCR. Along the continuum, from 89 to 98% of enterococci, according to the sampled site, were identified as Enterococcusfaecium. All E. faecium isolates from hospital and retirement home effluents were multiply resistant to antibiotics, contained erm(B) and meflA) genes, and belonged to hospital-adapted clonal complex 17 (CC17). Even though this species remained dominant in the downstream continuum, the relative proportion of CC17 isolates progressively decreased in favor of other subpopulations of E. faecium that were more diverse, less resistant to antibiotics, and devoid of the classical macrolide resistance genes and that belonged to various sequence types. Antibiotic concentrations in waters were far below the MICs for susceptible isolates. CC17 E. foecium was probably selected in the gastrointestinal tract of patients under the pressure of administered antibiotics and then excreted together with the resistance genes in waters to progressively decrease along the continuum. [ABSTRACT FROM AUTHOR]