Tandem Amplification of the Staphylococcal Cassette Chromosome <i>mec</i> Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus

Abstract

Hospital-associated methicillin-resistant <i>Staphylococcus aureus</i> (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 <i>mecA</i> gene, carried on the staphylococcal cassette chromosome <i>mec</i> element (SCC<i>mec</i>), 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 <i>gdpP</i>, <i>clpX</i>, <i>guaA</i>, and <i>camS</i> Closer inspection of the genome sequence data further revealed that reads covering SCC<i>mec</i> were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of <i>mecA</i> DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCC<i>mec</i> 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<i>mec</i> 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<i>mec</i> element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.