title: Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.
abstract: 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.
authors.affiliation: National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: Jennifer K
authors.identifier: http://orcid.org/0000-0001-5167-3836
authors.initials: JK
authors.lastname: Bender
authors.affiliation: National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Fleige
authors.affiliation: National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Klare
authors.affiliation: National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Fiedler
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany, and German Centre for Infection Research DZIF, Heidelberg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Mischnik
authors.affiliation: Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany, and German Centre for Infection Research DZIF, Heidelberg, Germany.
authors.forename: Nico T
authors.initials: NT
authors.lastname: Mutters
authors.affiliation: Nuffield Department of Clinical Medicine, Oxford University, John Radcliffe Hospital, Oxford, Oxfordshire, United Kingdom.|National Institute for Health Research, Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, Oxfordshire, United Kingdom.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Dingle
authors.affiliation: National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: Guido
authors.initials: G
authors.lastname: Werner
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D014644:Genetic Variation / Q000235:genetics*; D016680:Genome, Bacterial; D016908:Gram-Positive Bacterial Infections / Q000188:drug therapy* / Q000382:microbiology; D006801:Humans; D000069349:Linezolid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D000069349:Linezolid