In Vitro Selection and Characterization of Ceftobiprole-Resistant Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to ␤-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broadspectrum 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 g/ml) for pAW8, 32-fold (from 4 to 128 g/ml) for pYK20, and 8-fold (from 16 to 128 g/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.

Resistencia a antibióticos de última línea en cocos Gram positivos: la era posterior a la vancomicina

“…Se postularon tres mecanismos involucrados en la resistencia in vitro: la inhibición de la unión al substrato, la interferencia de la interacción proteína-proteína y la inhibición de la acilación. También se detectó resistencia en cepas que no presentaban el gen mecA y que, por lo tanto, no expresaban la proteína, por lo que se postuló que otros genes cromosómicos tendrían efecto en la resistencia a ceftobiprol (142).…”

A mec A -Negative Strain of Methicillin-Resistant S taphylococcus aureus with High-Level β-Lactam Resistance Contains Mutations in Three Genes

“…Our laboratory previously reported the emergence of ceftobiprole-resistant MRSA in vitro (1). We demonstrated that serial passage of MRSA strain COL in subinhibitory ceftobiprole concentrations selected for point mutations in mecA that conferred resistance to ceftobiprole and other ␤-lactams.…”

“…COLnex was transformed with a plasmid vector, pAW8, and serially passaged in increasing concentrations of ceftobiprole and 10 g/ml of tetracycline as previously described (1). A resistant mutant, CRB, was selected after 21 days of serial passage in ceftobiprole; MICs increased from 1 for the COL parent to 256 g/ml for mutant strain CRB.…”

Ceftobiprole- and Ceftaroline-Resistant Methicillin-Resistant Staphylococcus aureus

“…Ceftobiprole has been evaluated in clinical trials, and ceftaroline is FDA approved for treat-ment of skin and skin structure infections, including those caused by MRSA (5)(6)(7)(8)(9)(10). Previous studies from our group have shown that passage of the COL strain in ceftobiprole selects for resistant mutants with mutations in PBP2a (11). This study examines whether ceftaroline passage also selects for similar mutations.…”

“…It is present in ceftobiprole-passaged COL (among other mutations) (11), it is the only mecA mutation in the ceftaroline-passaged SF8300, and it has been reported in clinical isolates (17,18). Structurally, E447 resides in the penicillin-binding domain of PBP2a and interacts with the R2 group of ceftobiprole and other ␤-lactams (3, 19).…”