Tandem Amplification of the Staphylococcal Cassette Chromosome Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus.

Hospital-associated methicillin-resistant (MRSA) strains typically express high-level, homogeneous (HoR) β-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the gene, carried on the staphylococcal cassette chromosome element (SCC), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 μg/ml to 130 μg/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in , , , and Closer inspection of the genome sequence data further revealed that reads covering SCC were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCC element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 μg/ml) was accompanied by accordion-like contraction and amplification of the SCC element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCC element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.