title: Whole-genome analysis of an oxacillin-susceptible CC80 mecA-positive Staphylococcus aureus clinical isolate: insights into the mechanisms of cryptic methicillin resistance.
abstract: OBJECTIVE
  The mec and bla systems, among other genetic factors, are critical in regulating the expression of methicillin resistance in Staphylococcus aureus. We examined by WGS a naturally occurring oxacillin-susceptible mecA-positive S. aureus isolate to identify the mechanism conferring oxacillin susceptibility.
  
  
  METHODS
  The mecA-positive oxacillin-susceptible S. aureus isolate GR2 (penicillin and oxacillin MICs 0.094 and 1 mg/L, respectively), belonging to clonal complex 80, was characterized. DNA fragment libraries were sequenced on Roche 454 and Illumina MiSeq sequencers and de novo assembly of the genome was generated using SeqMan NGen software. Plasmid curing was conducted by SDS treatment. Expression of mecA was quantified without/with β-lactam pressure.
  
  
  RESULTS
  The genome of GR2 consisted of a 2 792 802 bp chromosome and plasmids pGR2A (28 895 bp) and pGR2B (2473 bp). GR2 carried SCCmec type IV, with a truncated/non-functional mecR1 gene and no mecI. A single copy of the bla system, with an organization unique for S. aureus, was found, harboured by plasmid pGR2A. Particularly, the blaZ gene was orientated like its regulatory genes, blaI and blaR1, and a gene encoding transposase IS66 was integrated between blaZ and the regulatory genes deleting the 5'-end of blaR1; blaI, encoding blaZ/mecA repressor, was intact. After plasmid loss, GR2 became penicillin and oxacillin resistant (MICs 0.5 and 6 mg/L, respectively).
  
  
  CONCLUSIONS
  We can conclude that after exposure to β-lactams, the non-functional BlaR1 does not cleave the mecA repressor BlaI, derepression does not occur and mecA is not efficiently expressed. Removal of the bla system after curing of pGR2A allows constitutive expression of mecA, resulting in oxacillin and penicillin resistance.
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Artur J
authors.initials: AJ
authors.lastname: Sabat
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands Department of Microbiology, Medical School, University of Athens, Athens, Greece.
authors.forename: Spyros
authors.initials: S
authors.lastname: Pournaras
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Akkerboom
authors.affiliation: Department of Microbiology, Medical School, University of Athens, Athens, Greece.
authors.forename: Athanassios
authors.initials: A
authors.lastname: Tsakris
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Hajo
authors.initials: H
authors.lastname: Grundmann
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands alex.friedrich@umcg.nl.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Friedrich
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D005838:Genotype; D016106:Methicillin Resistance; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D058889:Molecular Typing; D010068:Oxacillin / Q000494:pharmacology*; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013203:Staphylococcal Infections / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; D010068:Oxacillin