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Influence of recombination on development of mutational resistance to linezolid in Enterococcus faecalis JH2-2.

ABSTRACT: We compared the propensities of Enterococcus faecalis JH2-2 and of the recombination-deficient JH2-2 recA strain to develop mutational resistance to linezolid. In both organisms, a mutation in a single rrl copy conferred resistance to linezolid. Delay in acquisition of the mutation by other rrl copies in JH2-2 recA showed that gene conversion contributed to the acquisition of resistance.

SUBMITTER: Boumghar-Bourtchai L

PROVIDER: S-EPMC2737844 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Influence of recombination on development of mutational resistance to linezolid in Enterococcus faecalis JH2-2.

Boumghar-Bourtchaï Leyla L Dhalluin Anne A Malbruny Brigitte B Galopin Sébastien S Leclercq Roland R

Antimicrobial agents and chemotherapy 20090622 9

We compared the propensities of Enterococcus faecalis JH2-2 and of the recombination-deficient JH2-2 recA strain to develop mutational resistance to linezolid. In both organisms, a mutation in a single rrl copy conferred resistance to linezolid. Delay in acquisition of the mutation by other rrl copies in JH2-2 recA showed that gene conversion contributed to the acquisition of resistance. ...[more]

PMID: 19546366

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Quantitative Proteomics Analysis of Membrane Proteins in Enterococcus faecalis With Low-Level Linezolid-Resistance.

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Dissemination of Linezolid Resistance Through Sex Pheromone Plasmid Transfer in <i>Enterococcus faecalis</i>.

Project description:Despite recent recognition of the ATP-binding cassette protein OptrA as an important mediator of linezolid resistance in Enterococcus faecalis worldwide, the mechanisms of optrA gene acquisition and transfer remain poorly understood. In this study, we performed comprehensive molecular and phenotypic profiling of 44 optrA-carrying E. faecalis clinical isolates with linezolid resistance. Pulse-field gel electrophoresis and DNA hybridization revealed the presence of optrA in the plasmid in 26 (59%) isolates and in the chromosome in 18 (41%) isolates. Conjugation experiments showed a successful transfer of optrA in 88.5% (23/26) of isolates carrying optrA in plasmids while no transfer occurred in any isolates carrying optrA in the chromosome (0/18). All 23 transconjugants exhibited in vitro resistance to linezolid and several other antibiotics and were confirmed to contain optrA and other resistance genes. Plasmid typing demonstrated a predominance (18/23,78%) of rep 9-type plasmids (pCF10 prototype) known to be the best studied sex pheromone responsive plasmids. Full plasmid genome sequencing of one isolate revealed the presence of drug resistance genes (optrA and fexA) and multiple sex pheromone response genes in the same plasmid, which represents the first sex pheromone responsive plasmid carrying optrA from a clinical isolate. PCR-based genotyping revealed the presence of three key sex pheromone response genes (prgA, prgB, and prgC) in 23 optrA-carrying isolates. Finally, functional studies of these isolates by clumping induction assay detected different degrees of clumping in 17 isolates. Our analysis suggests that optrA-mediated linezolid resistance can be widely disseminated through sex pheromone plasmid transfer.

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Enterococcus faecalis JH2-2

Project description:Enterococcus II

| PRJNA216995 | ENA

Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing.

Project description:Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.

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The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein.

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Characterization of monolaurin resistance in Enterococcus faecalis.

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Radezolid Is More Effective Than Linezolid Against Planktonic Cells and Inhibits Enterococcus faecalis Biofilm Formation.

Project description:The aim of this study was to compare the effects of radezolid and linezolid on planktonic and biofilm cells of Enterococcus faecalis. A total of 302 E. faecalis clinical isolates were collected, and the minimum inhibitory concentrations (MICs) of radezolid and linezolid were determined by the agar dilution method. Changes in the transcriptome of a high-level, in vitro-induced linezolid-resistant isolate were assessed by RNA sequencing and RT-qPCR, and the roles of efflux pump-related genes were confirmed by overexpression analysis. Biofilm biomass was evaluated by crystal violet staining and the adherent cells in the biofilms were quantified according to CFU numbers. The MIC50/MIC90 values of radezolid (0.25/0.50 mg/L) against the 302 E. faecalis clinical isolates were eightfold lower than those of linezolid (2/4 mg/L). The radezolid MICs against the high-level linezolid-resistant isolates (linezolid MICs ? 64 mg/L) increased to ? 4 mg/L with mutations in the four copies of the V domain of the 23S rRNA gene. The mRNA expression level of OG1RF_12220 (mdlB2, multidrug ABC superfamily ATP-binding cassette transporter) increased in the high-level linezolid-resistant isolates, and radezolid and linezolid MICs against the linezolid-sensitive isolate increased with overexpression of OG1RF_12220. Radezolid (at 1/4 or 1/8× the MIC) inhibited E. faecalis biofilm formation to a greater extent than linezolid, which was primarily achieved through the inhibition of ahrC, esp, relA, and relQ transcription in E. faecalis. In conclusion, radezolid is more effective than linezolid against planktonic E. faecalis cells and inhibits biofilm formation by this bacterium.

| S-EPMC7033516 | biostudies-literature

Linezolid resistance in Enterococcus faecium and Enterococcus faecalis from hospitalized patients in Ireland: high prevalence of the MDR genes optrA and poxtA in isolates with diverse genetic backgrounds.

Project description:ObjectivesTo investigate the prevalence of the optrA, poxtA and cfr linezolid resistance genes in linezolid-resistant enterococci from Irish hospitals and to characterize associated plasmids.MethodsOne hundred and fifty-four linezolid-resistant isolates recovered in 14 hospitals between June 2016 and August 2019 were screened for resistance genes by PCR. All isolates harbouring resistance genes, and 20 without, underwent Illumina MiSeq WGS. Isolate relatedness was assessed using enterococcal whole-genome MLST. MinION sequencing (Oxford Nanopore) and hybrid assembly were used to resolve genetic environments/plasmids surrounding resistance genes.ResultsoptrA and/or poxtA were identified in 35/154 (22.7%) isolates, the highest prevalence reported to date. Fifteen isolates with diverse STs harboured optrA only; one Enterococcus faecium isolate harboured optrA (chromosome) and poxtA (plasmid). Seven Enterococcus faecalis and one E. faecium harboured optrA on a 36 331 bp plasmid with 100% identity to the previously described optrA-encoding conjugative plasmid pE349. Variations around optrA were also observed, with optrA located on plasmids in five isolates and within the chromosome in three isolates. Nine E. faecium and 10 E. faecalis harboured poxtA, flanked by IS1216E, within an identical 4001 bp region on plasmids exhibiting 72.9%-100% sequence coverage to a 21 849 bp conjugative plasmid. E. faecalis isolates belonged to ST480, whereas E. faecium isolates belonged to diverse STs. Of the remaining 119 linezolid-resistant isolates without linezolid resistance genes, 20 investigated representatives all harboured the G2576T 23S RNA gene mutation associated with linezolid resistance.ConclusionsThis high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

| S-EPMC7303821 | biostudies-literature

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