The X Factor(s): New antibiotic resistance mechanisms in community-associated methicillin resistant Staphylococcus aureus
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Methicillin-resistant Staphylococcus aureus (MRSA) strains typically express low-level heterogeneous (HeR) resistance from which high-level homogeneously resistant (HoR) mutants can be isolated following oxacillin exposure. The HoR phenotype typically requires both increased expression of the methicillin resistance gene mecA, carried on the Staphylococcus cassette chromosome SCCmec, and additional mutations elsewhere on the chromosome that have previously been associated with increased c-di-AMP and ppGpp levels. An oxacillin hyper-resistant mutant of the community associated MRSA strain USA300 was isolated from a chemostat culture grown over 13 days in increasing concentrations of oxacillin. This mutant, designated HoR34, was found to contain multiple tandemly amplified copies of SCCmec on the chromosome, associated with increased mecA-encoded penicillin binding protein 2 a (PBP2A) expression. This is the first report of SCCmec amplification as a driver of high level methicillin resistance in MRSA. A screen of the Nebraska transposon mutant library revealed that mutation of pgl was accompanied by increased β-lactam resistance, independent of increased c-di-AMP levels. pgl encodes the second enzyme in the pentose phosphate pathway (PPP), 6-phosphogluconolactonase that converts 6-phosphogluconolactone to phosphogluconate. Disruption of the PPP may increase carbon flux through the glycolysis pathway, increasing the intracellular pool of fructose-6-phosphate enabling increased peptidoglycan biosynthesis following exposure to β-lactam antibiotics. Thus, these data suggest that increased β-lactam resistance is not solely reliant on increased c-di-AMP levels but can also be achieved by mutations in metabolic enzymes that enhance peptidoglycan biosynthetic capability. In contrast to the pgl mutation, disruption of the alanine/serine/glycine permease gene, cycA, was shown to increase susceptibility to β-lactam antibiotics and the alanine analogue d-cycloserine (DCS). Impaired alanine uptake in chemically defined media (CDM) containing glucose was accompanied by increased β-lactam susceptibility. In contrast, neither alanine uptake nor β-lactam susceptibility was affected in CDM media, indicating an essential role for alanine transport in cell wall integrity and consequently β-lactam resistance. DCS-mediated inhibition of alanine racemase and D-alanine ligase activity in the cycA mutant was also shown to aggravate the impact of impaired alanine uptake on β-lactam susceptibility under in vitro and in vivo conditions, indicating that DCS/oxacillin combinations offer an exciting new therapeutic option for the treatment of MRSA infections.