title: PCR-based amplification of heterogeneous IS257-ileS2 junctions for molecular monitoring of high-level mupirocin resistance in staphylococci.
abstract: OBJECTIVE
  Plasmids encoding the ileS2 gene are responsible for the wide spread of high-level mupirocin resistance in staphylococci, and consequent clinical and epidemiological problems. We investigated the location of insertion sequence IS257 flanking ileS2 in different plasmids and developed a method for molecular typing of the IS257-ileS2 spacer regions.
  
  
  METHODS
  Nine ileS2-encoding plasmids (i.e. pPR1-pPR9) classified into distinct structural groups (i.e. S1-S4) were analysed. Complete DNA sequences between IS257s flanking ileS2 were determined. A PCR-based amplification scheme was designed for the simultaneous amplification of up- and down-stream IS257-ileS2 regions. The method was applied to a total of 90 high-level mupirocin-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).
  
  
  RESULTS
  pPR1-pPR9 possessed IS257s flanking ileS2. Plasmids of each structural group showed a unique configuration of IS257-ileS2 spacer regions. The PCR-based method permitted accurate typing of the heterogeneous IS257-ileS2 up- and down-stream junctions, and the differentiation of plasmids of each group. The results obtained corresponded with previous plasmid typing based on restriction enzyme analyses and ileS2 locus hybridization polymorphs. The application of the PCR-based method to a diverse collection of MRSA isolates carrying ileS2-encoding plasmids demonstrated its versatility and revealed extraordinary heterogeneity in the IS257-ileS2 spacers.
  
  
  CONCLUSIONS
  The devised PCR-based scheme offers a rapid, simple and effective approach for typing IS257-ileS2 configurations present on ileS2-encoding plasmids. Hopefully, it could serve as a useful molecular epidemiological tool to control high-level mupirocin resistance.
authors.affiliation: Unidad de Investigación, Hospital Universitario Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain. eperroth@ull.es
authors.forename: Eduardo
authors.initials: E
authors.lastname: Pérez-Roth
authors.forename: Estefanía
authors.initials: E
authors.lastname: Armas-González
authors.forename: Julia
authors.initials: J
authors.lastname: Alcoba-Flórez
authors.forename: Sebastián
authors.initials: S
authors.lastname: Méndez-Álvarez
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015373:Bacterial Typing Techniques / Q000379:methods; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics*; D008826:Microbial Sensitivity Tests / Q000379:methods; D058889:Molecular Typing / Q000379:methods; D016712:Mupirocin / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D016712:Mupirocin