A tigecycline-susceptible (TGC-S) Sequence Type (ST) 5 clinical methicillin-resistant Staphylococcus aureus (MRSA) strain was cultured in escalating levels of tigecycline, yielding mutants eightfold more resistant. Their genomes were sequenced to identify genetic alterations, resulting in resistance. Alterations in rpsJ, commonly related to tigecycline resistance, were also investigated. Tigecycline resistance was mediated by loss-of-function mutations in the transcriptional repressor mepR, resulting in derepression of the efflux pump mepA. Increased levels of resistance were obtained by successive mutations in mepA itself. No alterations in RpsJ were observed in selected strains, but we observed a K57M substitution, previously correlated with resistance, among TGC-S clinical strains. Thus, the pathway to tigecycline resistance in CC5 MRSA in vitro appears to be derepression of mep operon as the result of mepR loss-of-function mutation, followed by alterations in MepA efflux pump. This shows that other evolutionary pathways, besides mutation of rpsJ, are available for evolving tigecycline resistance in CC5 MRSA.

SUMMARYWe report the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) with resistance to tigecycline and to daptomycin isolated from intensive-care-unit patients in Brazil over a 6-month period. Thirty-six isolates (25 from infection sites, 11 from nasal sites) recovered from 23 patients who presented with MRSA infection during this period were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility profiling. Ten isolates from six patients and two isolates from different patients were resistant to tigecycline and daptomycin, respectively. Eight pulsotypes were identified and one, type A, accounted for 21 isolates from 12 patients; type A isolates were SCCmecII as were a further nine isolates of other pulsotypes. All but four of the total isolates were sequence type (ST) 5 or ST105 and classified as clonal complex (CC) 5; the historically prevalent lineage in Brazil, ST239-SCCmecIII, was identified in only three patients. Tigecycline-resistant strains were all ST105-SCCmecII and two patients were nasally colonized by strains of the same pulsotype found in infection sites. Two ST5-SCCmecII were daptomycin resistant after 48 h incubation. The origin and mechanism of these resistant strains remains unknown and further studies are warranted to determine whether such clones are becoming endemic in Brazilian hospitals and to assess their impact on infection control practice.

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