Dissemination of the linezolid-resistant Staphylococcus epidermidis clone ST2 exhibiting the G2576T mutation in the 23S rRNA gene in a tertiary-care hospital, Brazil.

Sir, Linezolid is the first agent of the oxazolidinone class to be introduced clinically. It has been approved for the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus spp. and vancomycin-resistant enterococci (VRE). This oxazolidinone inhibits protein biosynthesis by preventing the formation of the initiation complex that consists of the mRNA, the f-Met tRNA and the 50S subunit of the ribosome. Although linezolid resistance has been mediated by the cfr-encoded product or by ribosomal proteins (L3, L4 and L22), the most common mechanism of resistance involves mutations in the central loop of domain V of the 23S rRNA gene. In this regard, the G2576T mutation is the most frequently reported in staphylococci, – 4 although other resistance mutations in 23S rRNA have been identified in clinical strains of this genus. Linezolid resistance in Staphylococcus epidermidis has been especially mediated by G2576T, G2447T, T2504A, C2534T and G2631T. In this study, we report the first dissemination of linezolid-resistant S. epidermidis ST2 clone exhibiting the G2576T mutation in Brazil. From March 2008 to November 2009, 9 S. epidermidis strains exhibiting resistance to linezolid were isolated from skull, blood and catheter cultures from patients in a tertiarycare hospital in Brazil, and were included in this study for the ascertainment of the resistance mechanisms to this antimicrobial agent. Additionally, a linezolid-susceptible S. epidermidis strain (23/1116), isolated in January 2010, was included as the comparative control. Initially, identification and antimicrobial susceptibility testing were performed by the Vitek 2 system (bioMérieux, St Louis, MO, USA). The susceptibility of the bacterial strains was tested using the disc diffusion method. The agar dilution method and Etests (AB Biodisk, Solna, Sweden) were used for linezolid, oxacillin and vancomycin. The interpretive criteria used were those specified by the CLSI guidelines. The presence of the cfr gene and mutations in the rplC, rplD and rplV genes were investigated as described previously. The domain V region of 23S rRNA was amplified by PCR using the primers F (5′ –GCTTTACTGTAGCCTGATATTGA–3′) and R (5′ –GACAACTGGTACACCAGAGGTA–3′), corresponding, respectively, to bases 2097–2119 and 2703–2724 of the S. aureus 23S rRNA gene (GenBank accession no. X68425). The PCR products were sequenced and aligned with the corresponding nucleotide sequence from a linezolid-susceptible S. epidermidis strain (GenBank accession no. CP000029). The clonal relatedness of the strains was determined by PFGE using the restriction enzyme FastDigest SmaI (Fermentas Life Sciences, Canada) and by multilocus sequence typing (MLST), which was carried out as previously described (http:// sepidermidis.mlst.net). The strains exhibited high-level resistance to linezolid (MICs 32–128 mg/L) and all were multidrug resistant, remaining susceptible only to vancomycin and teicoplanin. Some isolates were susceptible to tetracycline (Table 1). The G2576T mutation in domain V region of 23S rRNA was identified in all isolates, except in the linezolid-susceptible S. epidermidis control strain, which was obtained from the same hospital. On the other hand, all isolates, including the linezolidsusceptible S. epidermidis control strain, showed the L3 Leu101Val mutation, suggesting that this alteration is probably not involved in linezolid resistance. Mutations in ribosomal proteins L4 and L22 were not detected. Furthermore, the cfr gene was not identified in any isolate. PFGE analysis revealed the presence of three clones (.85% similarity cut-off value). Regarding MLST analysis, all S. epidermidis strains belonged to the sequence type ST2. Linezolid was approved in Brazil in June 2000. Just 2 years after the drug was introduced, linezolid resistance was reported in a clinical isolate of S. aureus. This strain belonged to the Brazilian endemic MRSA clone and had the G2576T mutation in domain V of the 23S rRNA gene. In the Beneficência Portuguesa Hospital, linezolid was introduced in November 2000 and is currently the drug of choice for the treatment of VRE infections, which are highly prevalent. Linezolid-resistant S. epidermidis strains have been disseminated in this institution from 2008 and represented 0.04% of all S. epidermidis isolated in this