title: Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus.
abstract: BACKGROUND
  We used 2 in vitro experimental systems to compare phenotypic and genotypic changes that accompany selection of mutants of methicillin-resistant Staphylococcus aureus (MRSA) strain JH1 with low-level vancomycin resistance similar to the type found in vancomycin-intermediate S. aureus (VISA).
  
  
  METHODS
  The previously described MRSA strain JH1 and its vancomycin-intermediate mutant derivative JH2, both of which were recovered from a patient undergoing vancomycin chemotherapy, were used in this study. Mutants of JH1 were selected in vitro by means of a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs) and by exposure to vancomycin in laboratory growth medium. Phenotypic abnormalities of JH1 mutants generated by each in vitro experimental system were compared to those of JH2, and whole genomes of 2 in vitro JH1 mutants were sequenced to identify mutations that may be associated with an increased vancomycin minimum inhibitory concentration.
  
  
  RESULTS
  JH1R1 was selected from the PK/PD model, and JH1R2 was selected in laboratory growth medium. Both mutants displayed reduced vancomycin and daptomycin susceptibility and phenotypic alterations (eg, thicker cell walls and abnormal autolysis) that are typical of in vivo VISA mutants. Genome sequencing of JH1R1 identified point mutations in 4 genes, all of which were different from the mutations described in JH2, including 1 mutation in yycG, a component of the WalKR sensory regulatory system. Sequencing of the JH1R2 genome identified mutations in 7 genes, including 2 in rpoB.
  
  
  CONCLUSIONS
  Our findings indicate that JH1 is able to develop VISA-type resistance through several alternative genetic pathways.
authors.affiliation: Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA.
authors.forename: Celine
authors.initials: C
authors.lastname: Vidaillac
authors.forename: Susana
authors.initials: S
authors.lastname: Gardete
authors.forename: Ryan
authors.initials: R
authors.lastname: Tewhey
authors.forename: George
authors.initials: G
authors.lastname: Sakoulas
authors.forename: Glenn W
authors.initials: GW
authors.lastname: Kaatz
authors.forename: Warren E
authors.initials: WE
authors.lastname: Rose
authors.forename: Alexander
authors.initials: A
authors.lastname: Tomasz
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rybak
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D005838:Genotype; D006801:Humans; D066298:In Vitro Techniques; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D013203:Staphylococcal Infections / Q000188:drug therapy; D014640:Vancomycin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D014640:Vancomycin
keywords: JH1; PK/PD model; VISA; cell wall thickness; genetic alterations; genome sequencing; pharmacokinetics/pharmacodynamics; phenotypic alteration; vancomycin resistance
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