First Report of cfr - Encoding Plasmids in the Pandemic Sequence Type ( ST ) 22 Methicillin - 1

26 Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus 27 (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for 28 ribosomal proteins, cfr encoding phenicol, lincosamide, oxazolidinone, pleuromutilin and 29 streptogramin A (PhLOPS A) resistance, its homolgue cfr(B) or optrA conferring oxazolidinone 30 and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr even rarer. This study 31 investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from 32 patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPS A susceptibility 33 testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel 34 electrophoresis (PFGE), plasmid curing and conjugative transfer. Whole-genome sequencing was 35 used for single nucleotide variant (SNV) analysis, multilocus-sequence typing, L-protein 36 mutation identification, cfr-plasmid sequence analysis and optrA and cfr(B) detection. Isolates 37 M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and 38 harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 39 23S rRNA alleles compared to 1/5 in M13/0401. Both isolates were ST22-MRSA-IV/t032, 40 harbored cfr, exhibited the PhLOPS A phenotype and lacked optrA and cfr(B). They differed by 41 five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative 42 pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid.