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

Laura A. Gallagher, Simone Coughlan, Nikki S. Black, Pierce Lalor, Elaine M. Waters, Bryan Wee, Mick Watson, Tim Downing, J. Ross Fitzgerald, Gerard T. A. Fleming, James P. O'Gara

Hospital-associated methicillin-resistant Staphylococcus aureus strains typically express high level, homogenous (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 mecA gene, carried on the Staphylococcus cassette chromosome SCCmec element, 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 gdpP, clpX, guaA and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ∼10 times over-represented compared to other parts of the chromosome. qPCR confirmed >10-fold higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that sub-culture of HoR34 in varying concentrations of oxacillin (0--100 μg/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than USA300, HoR34 out-competed the parent strain in the presence of sub-inhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.