Methicillin-resistant Staphylococcus aureus ST9 from a case of bovine mastitis carries the genes cfr and erm(A) on a small plasmid.

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide variety of infections in animals with bovine mastitis being the predominant staphylococcal infection in dairy cattle. MRSA isolates—mainly of multilocus sequence type (ST) 398—have recently been shown to be also involved in bovine mastitis. Although a variety of antimicrobial resistance genes have been identified in MRSA isolates from cases of bovine mastitis, the multidrug resistance gene cfr has not yet been identified in bovine CC398 isolates, although it has been found in a single porcine MRSA ST398 isolate and a single porcine methicillinsusceptible S. aureus ST9 isolate. Although pilot studies have indicated that MRSA ST9 isolates are commonly found among pigs in China, cfr-positive MRSA isolates of animal origin have not yet been reported in China. In 2010, during a routine surveillance study on antimicrobial resistance on dairy farms in China, an MRSA strain (designated SA16) was isolated from fresh raw milk of a cow suffering from mastitis. The MRSA strain was further characterized by multilocus sequence typing (MLST; http://saureus.mlst.net/), spa typing (http://spaserver.ridom.de) and SCCmec typing, as previously described. This strain belonged to ST9 (allelic profile 3-3-1-1-1-1-10), belonged to spa type t899 (allelic profile 07-16-23-02-34), and harboured an SCCmec element of type III. The strain displayed resistance to oxacillin (MIC1⁄4128 mg/L), erythromycin (MIC ≥64 mg/L) and clindamycin (MIC ≥64 mg/L), had a linezolid MIC of 4 mg/L and showed high florfenicol and tiamulin MICs of ≥128 mg/L and 256 mg/L, respectively. The genes mecA (oxacillin resistance), cfr and fexA (phenicol resistance) were detected by PCR and confirmed by sequencing of the respective amplicons. Plasmid preparation using the DNA midi kit (Qiagen, Hilden, Germany) identified an 7 kb plasmid, designated pMSA16. This plasmid was transformed into the recipient strain S. aureus RN4220 by electrotransformation using 0.2 cm cuvettes with a Gene Pulser apparatus at 2.5 kV (Bio-Rad, Munich, Germany). Transformants were selected on BHI agar containing erythromycin (15 mg/L) or florfenicol (10 mg/L). Southern blot analysis using the cfr and fexA amplicons as probes confirmed that the cfr gene was located on pMSA16 in the original strain and its transformants, while the fexA gene was most likely located on the chromosomal DNA. The pMSA16-harbouring transformants showed ≥4-fold increases in the MICs of chloramphenicol, florfenicol, clindamycin, linezolid and tiamulin, which are indicative for the cfr-associated resistance phenotype and demonstrated that the pMSA16-associated cfr gene is functionally active. In addition, the pMSA16-harbouring transformants showed an elevated MIC of erythromycin of ≥64 mg/L, which suggested that a macrolide resistance gene is also located on plasmid pMSA16. To gain insight into the structure and organization of plasmid pMSA16, the sequence of this 7054 bp plasmid was determined by primer walking. Using the open reading frame (ORF) finder software (http://www.ncbi.nlm.nih.gov/gorf/), five reading frames for proteins of ≥100 amino acids (aa) were identified in pMSA16 (Figure 1a). The rep gene codes for a 327 aa plasmid replication protein, which shares 100% and 98.5% aa identity with the same-sized RepU protein of a plasmid from the porcine S. aureus strain 7612628-4 (GenBank accession number JF968539) and the 326 aa RepU protein of the Staphylococcus saprophyticus plasmid pSES22, respectively. A macrolide-lincosamide–streptogramin B resistance gene coding for a 243 aa rRNA methylase was detected, the deduced aa sequence of which was indistinguishable from the same-sized Erm(A) protein of Tn554. Analysis of the erm(A)flanking regions identified a potential recombination site that included the translational stop codon of the erm(A) gene and might have contributed to the replacement of the pSES22-associated erm(C) gene by a Tn554-borne erm(A) gene (Figure 1b). At the other end, homology to Tn554 ended in the translational attenuator 135 bp upstream of the erm(A) translational start codon (Figure 1a and b). The erm(C) and erm(A) translational attenuators are composed of several pairs of inverted repeats, and areas characterized by inverted repeats are considered as preferential areas for illegitimate recombination events. Thus recombination in the erm(A)