title: Sequential steps of daptomycin resistance in Enterococcus faecium and reversion to hypersusceptibility through IS-mediated inactivation of the liaFSR operon.
abstract: To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), F-14032 Caen, France.
authors.forename: Clara
authors.initials: C
authors.lastname: Sinel
authors.affiliation: Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), F-14032 Caen, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Cosquer
authors.affiliation: CHU de Caen, Service de Microbiologie, F-14033 Caen, France Centre National de Référence sur la Résistance aux Antibiotiques (laboratoire associé 'Entérocoques'), F-14033 Caen, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Auzou
authors.affiliation: Université de Caen Basse-Normandie, CMAbio, F-14032 Caen, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Goux
authors.affiliation: Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), F-14032 Caen, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Giard
authors.affiliation: Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), F-14032 Caen, France CHU de Caen, Service de Microbiologie, F-14033 Caen, France Centre National de Référence sur la Résistance aux Antibiotiques (laboratoire associé 'Entérocoques'), F-14033 Caen, France cattoir-v@chu-caen.fr.
authors.forename: Vincent
authors.initials: V
authors.lastname: Cattoir
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D004251:DNA Transposable Elements; D017576:Daptomycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016984:Enterococcus faecium / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009876:Operon; D020713:Vancomycin Resistance
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D017576:Daptomycin