title: Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS.
abstract: OBJECTIVE
  Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms.
  
  
  METHODS
  Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains.
  
  
  RESULTS
  Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant.
  
  
  CONCLUSIONS
  We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureus.
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Bonn, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Berscheid
authors.affiliation: Genomic Research Laboratory, Infectious Diseases Service, Geneva University Hospitals and the University of Geneva, Geneva, Switzerland.
authors.forename: Patrice
authors.initials: P
authors.lastname: François
authors.affiliation: Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Strittmatter
authors.affiliation: Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gottschalk
authors.affiliation: Genomic Research Laboratory, Infectious Diseases Service, Geneva University Hospitals and the University of Geneva, Geneva, Switzerland.
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
authors.affiliation: Institute for Pharmaceutical Biology, University of Düsseldorf, Düsseldorf, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Sass
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Bonn, Germany g.bierbaum@uni-bonn.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Bierbaum
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics*; D056426:Genetic Loci; D016680:Genome, Bacterial; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000235:genetics; D020125:Mutation, Missense; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics*; D014640:Vancomycin / Q000494:pharmacology*; D020713:Vancomycin Resistance
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; C478169:VraS protein, Staphylococcus aureus; D014640:Vancomycin
keywords: antibiotic resistance; daptomycin; glycopeptides; teicoplanin; two-component regulatory systems