title: Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries.
abstract: BACKGROUND
  spa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003).
  
  
  METHODS
  Phylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping techniques.
  
  
  RESULTS
  Fourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities.
  
  
  CONCLUSIONS
  canSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods.
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, USA. dengelthaler@tgen.org
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
authors.forename: Erin
authors.initials: E
authors.lastname: Kelley
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Driebe
authors.forename: Jolene
authors.initials: J
authors.lastname: Bowers
authors.forename: Carl F
authors.initials: CF
authors.lastname: Eberhard
authors.forename: Jesse
authors.initials: J
authors.lastname: Trujillo
authors.forename: Frederic
authors.initials: F
authors.lastname: Decruyenaere
authors.forename: James M
authors.initials: JM
authors.lastname: Schupp
authors.forename: Joel
authors.initials: J
authors.lastname: Mossong
authors.forename: Paul
authors.initials: P
authors.lastname: Keim
authors.forename: Jos
authors.initials: J
authors.lastname: Even
mesh_terms: D015373:Bacterial Typing Techniques; D005060:Europe / Q000453:epidemiology; D016680:Genome, Bacterial; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D008189:Luxembourg / Q000453:epidemiology; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013203:Staphylococcal Infections / Q000453:epidemiology