In vitro selection and characterization of ceftobiprole-resistant methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to beta-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broad-spectrum cephalosporin, ceftobiprole (BPR), binds PBP2a with high affinity and is active against MRSA. We hypothesized that BPR resistance could be mediated by mutations in mecA, the gene encoding PBP2a. We selected BPR-resistant mutants by passage in high-volume broth cultures containing subinhibitory concentrations of BPR. We used strain COLnex (which lacks chromosomal mecA) transformed with pAW8 (a plasmid vector only), pYK20 (a plasmid carrying wild-type mecA), or pYK21 (a plasmid carrying a mutant mecA gene corresponding to five PBP2a mutations). All strains became resistant to BPR by day 9 of passaging, but MICs continued to increase until day 21. MICs increased 256-fold (from 1 to 256 microg/ml) for pAW8, 32-fold (from 4 to 128 microg/ml) for pYK20, and 8-fold (from 16 to 128 mug/ml) for pYK21. Strains carrying wild-type or mutant mecA developed six (pYK20 transformants) or four (pYK21 transformants) new mutations in mecA. The transformation of COLnex with a mecA mutant plasmid conferred BPR resistance, and the loss of mecA converted resistant strains into susceptible ones. Modeling studies predicted that several of the mecA mutations altered BPR binding; other mutations may have mediated resistance by influencing interactions with other proteins. Multiple mecA mutations were associated with BPR resistance in MRSA. BPR resistance also developed in the strain lacking mecA, suggesting a role for chromosomal genes.