Whole-genome Sequencing of MRSA Isolates

We cultured MRSA from screening swabs and clinical specimens by plating on to MRSA selective medium (Brilliance MRSA chromogenic medium, Oxoid, Basingstoke, UK). We identified bacteria with a commercial latex agglutination kit (Pastorex Staph Plus, Bio Rad Laboratories, Hemel Hempstead, UK). We did antimicrobial susceptibility testing with disk diffusion¹² for the following drugs: cefoxitin, ciprofloxacin, erythromycin, fusidic acid, gentamicin, mupirocin, neomycin, rifampicin, tetracycline, and vancomycin. All isolates were assigned unique strain identification numbers. We prepared sequencing libraries from 500 ng of DNA extracted from each MRSA isolate as previously described,¹³ with amplification using Kapa Hifi polymerase (Kapa Biosystems, Woburn, MA, USA). Whole-genome sequencing was done with an Illumina MiSeq (Illumina, San Diego, CA, USA) to generate 150 bp paired end reads, and interpreted by an investigator (SRH) who was masked to all clinical, epidemiological, and microbiological information. The genome data has been deposited in the European Nucleotide Archive (appendix). We aligned sequence reads to the chromosome (accession number HE681097) and plasmid (CP002148) of a reference isolate (HO 5096 0412) to identify single-nucleotide polymorphisms (SNPs) and insertions or deletions. This reference isolate was defined by multilocus sequence typing as sequence type (ST) 22.