We previously generated a ceftobiprole-resistant Staphylococcus aureus strain after high inoculum serial passage of a mecA-negative variant of strain COL (R. Banerjee, M. Gretes, L. Basuino, N. Strynadka, and H. F. Chambers, Antimicrob. Agents Chemother. 52: [2089][2090][2091][2092][2093][2094][2095][2096] 2008). Genome resequencing of this strain, CRB, revealed that it differs from its parent by five single-nucleotide polymorphisms in three genes, specifically, those encoding PBP4, a low-molecular-weight penicillin-binding protein, GdpP, a predicted signaling protein, and AcrB, a cation multidrug efflux transporter. CRB displayed resistance to a variety of -lactams but was hypersusceptible to cefoxitin.The efficacy of antimicrobials currently used to treat methicillin-resistant Staphylococcus aureus (MRSA) is decreasing (2,7,12,20,26). Novel, investigational -lactams with activity against MRSA, including ceftobiprole and ceftaroline, are in clinical development and bind with high affinity to staphylococcal PBP2a, the penicillin-binding-protein encoded by the gene mecA (5,6,18,22). Widespread clinical use of these newer cephalosporin antibiotics will likely generate organisms resistant to them.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. This is not surprising, given that ceftobiprole's anti-MRSA activity is due to its ability to bind to the active site of all PBPs, including that of PBP2a. However, an unexpected result was that passage of a mecA-negative COL variant (COLnex) also selected for high level -lactam resistance in a derivative, named CRB (1). We undertook genome resequencing of strain CRB to identify the molecular basis for mecA-independent resistance to -lactams.Strain COLnex (a tetracycline-and methicillin-susceptible, -lactamase negative variant of COL) lacks chromosomal mecA, which had been eliminated by complete excision of the SCCmec cassette element and selection for loss of methicillin resistance (14). 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.CRB displayed reduced growth rate and colonies that are smaller, less hemolytic, and less pigmented than those of the parent strain. These multiple phenotypic abnormalities suggest that CRB has alterations of global gene expression and/or cell signaling pathways. Scanning electron microscopy of CRB and COLnex in the presence and absence of -lactam did not reveal any gross ultrastructural differences between the strains. CRB also displayed high-level resistance to all -lactams tested but demonstrated hypersensiti...
The new nomenclature should stimulate the crossfertilization of ideas among parasitologists and with the broader redox research community. The diverse parasite developmental stages and host environments present complex systems in which to explore the variety of roles played by Prxs, with a view toward parlaying what is learned into novel therapies and vaccines that are urgently needed.
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.
The most important mode of bacterial resistance to beta-lactam antibiotics is the expression of beta-lactamases. New cyclobutanone analogues of penams and penems have been prepared and evaluated for inhibition of class A, B, C, and D beta-lactamases. Inhibitors which favor conformations in which the C4 carboxylate is equatorial were found to be more potent than those in which the carboxylate is axial, and molecular modeling studies with enzyme-inhibitor complexes indicate that an equatorial orientation of the carboxylate is required for binding to beta-lactamases. An X-ray structure of OXA-10 complexed with a cyclobutanone confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. An unsaturated penem analogue was also found to enhance the potency of meropenem against carbapenem-resistant MBL-producing strains of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia. These cyclobutanones represent the first type of reversible inhibitors to show moderate (low micromolar) inhibition of both serine- and metallo-beta-lactamases and should be considered for further development into practical inhibitors.
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