title: Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M).
abstract: OBJECTIVE
  Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp.
  
  
  METHODS
  Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality.
  
  
  RESULTS
  Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus.
  
  
  CONCLUSIONS
  Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.
authors.affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, National Reference Centre for Staphylococci and Enterococci, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Fiedler
authors.affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, National Reference Centre for Staphylococci and Enterococci, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany benderj@rki.de.
authors.forename: J K
authors.initials: JK
authors.lastname: Bender
authors.affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, National Reference Centre for Staphylococci and Enterococci, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Klare
authors.affiliation: Division of Enteropathogenic Bacteria and Legionella, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Halbedel
authors.affiliation: Department of Life Sciences and Technology, Beuth University of Applied Sciences, Berlin, Germany Division of Infectious Diseases, University Medical Centre Freiburg, Freiburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Grohmann
authors.affiliation: Environmental Microbiology, Technical University Berlin, Berlin, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Szewzyk
authors.affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, National Reference Centre for Staphylococci and Enterococci, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Werner
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004352:Drug Resistance, Microbial / Q000235:genetics*; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics; D018628:Gene Dosage; D016905:Gram-Negative Bacterial Infections / Q000188:drug therapy* / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D008911:Minocycline / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013753:Tetracycline Resistance / Q000235:genetics; D000078304:Tigecycline; D015854:Up-Regulation / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C083138:Tet M resistance protein, Bacteria; C094988:tetL protein, Enterococcus faecalis; D000078304:Tigecycline; D008911:Minocycline