Small plasmids carrying vga(A) or vga(C) genes mediate resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-resistant Staphylococcus aureus ST398 from swine

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) isolates of the sequence type ST398 have been identified recently from cases of exudative epidermitis in swine, but also as colonizers of apparently healthy swine in Portugal. A considerable number of these isolates displayed an unusual resistance phenotype, namely resistance to the lincosamide clindamycin, but susceptibility to the macrolide erythromycin. Since the genetic basis of this resistance phenotype is unknown, we selected five representative isolates to identify the respective resistance genes, to determine whether they are transferable and to investigate their genetic environment. Of the five MRSA ST398 isolates, three (E32, E33 and E49) were obtained from dust samples in three different holdings of breeding pigs during the European Community baseline study, one (E8) was from the nasal swab of an apparently healthy sow and the remaining one (E18) was from a skin lesion of a piglet suffering from exudative epidermitis. PCR screening for the presence of the lincosamide resistance gene lnu(A) yielded negative results in repeated experiments using plasmid DNA or chromosomal DNA as targets. A full antibiogram of these isolates, as conducted by broth microdilution according to CLSI document M31-A3, revealed that besides the previously reported resistance to oxacillin, clindamycin and tetracycline, all five isolates displayed high MIC values of ≥128 mg/L for the pleuromutilin tiamulin and ≥32 mg/L for the streptogramin A antibiotic virginiamycin M1. Combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics has recently been described to be associated with the resistance genes vga(A) and vga(C), both of which are located on plasmids. Plasmid profiling revealed 1–3 plasmids in each of the five MRSA ST398 isolates. The plasmids were subjected to transformation into the plasmid-free recipient strain S. aureus RN4220, with subsequent selection of the transformants on medium containing clindamycin (2 mg/L). All five isolates yielded transformants that harboured a single plasmid of either 5.3 (E49) or 5.7 kb (E8, E18, E32 and E33). Susceptibility testing confirmed that all transformants displayed resistance or high MICs (in cases where no applicable breakpoints are available to classify an isolate as resistant) to lincosamides, pleuromutilins and streptogramin A antibiotics. Restriction analysis with the enzymes PvuII, DraI, PstI and BglII showed that the 5.3 kb plasmid, designated pCPS49, differed distinctly from the 5.7 kb plasmids, which were indistinguishable based on restriction analysis with the aforementioned endonucleases, but also with HindIII, PstI, ClaI, EcoRI, XhoI and EcoRV. The plasmid obtained from the E32 transformant, designated pCPS32, was chosen as representative of this group of plasmids for further analysis. HindIII fragments of plasmids pCPS32 and pCPS49 were cloned into pBluescript II SK+, and sequenced completely on both strands. Plasmid pCPS32 was 5718 bp in size (accession no. FN806791). It showed 99.9% sequence identity to the 5713 bp plasmid pVGA obtained from a human clinical S. aureus in Portugal. Sequence analysis revealed the presence of three open reading frames (Figure 1). The first reading frame coded for a Rep protein of 312 amino acids involved in replication of plasmid pCPS32. The second reading frame encoded a Vga(A) ABC transporter protein of 522 amino acids that is responsible for combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The third reading frame coded for a Mob protein of 325 amino acids involved in plasmid mobilization. While the Rep and Vga(A) proteins of pCPS32 were indistinguishable from those of pVGA, the Mob protein showed a single amino acid exchange, Thr50 (pCPS32) versus Ala50 (pVGA). Plasmid pCPS49 was 5292 bp in size (accession no. FN806792). It also comprised three reading frames for proteins involved in plasmid replication, antimicrobial resistance and mobilization. Plasmid pCPS49 harboured a vga(C) gene encoding a 522 amino acid ABC transporter identical to that described in the revised sequence of plasmid pKKS825 (accession no. FN377602). The Vga(C) protein also conferred combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The pre/mob gene for plasmid recombination and