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

2008 ◽  
Vol 52 (6) ◽  
pp. 2089-2096 ◽  
Author(s):  
Ritu Banerjee ◽  
Michael Gretes ◽  
Li Basuino ◽  
Natalie Strynadka ◽  
Henry F. Chambers
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Binding Protein ◽  
Plasmid Vector ◽  
High Volume ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Gene Encoding ◽  
Methicillin Resistant

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to β-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 μg/ml) for pAW8, 32-fold (from 4 to 128 μg/ml) for pYK20, and 8-fold (from 16 to 128 μg/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.

scholarly journals Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay.

1996 ◽  
Vol 40 (9) ◽  
pp. 2075-2079 ◽  
Author(s):  
S Roychoudhury ◽  
R E Kaiser ◽  
D N Brems ◽  
W K Yeh
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Methicillin Resistant ◽  
Chromogenic Assay ◽  
Enzymatic Acylation

We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.

scholarly journals LB11058, a New Cephalosporin with High Penicillin-Binding Protein 2a Affinity and Activity in Experimental Endocarditis Due to Homogeneously Methicillin-Resistant Staphylococcus aureus

2004 ◽  
Vol 48 (11) ◽  
pp. 4322-4327 ◽  
Author(s):  
Jacques Vouillamoz ◽  
José M. Entenza ◽  
Peter Hohl ◽  
Philippe Moreillon
Keyword(s):  
Staphylococcus Aureus ◽  
Serum Proteins ◽  
Binding Protein ◽  
Free Drug ◽  
Penicillin Binding Protein ◽  
Methicillin Resistant ◽  
Drug Concentrations ◽  
Mrsa Strains ◽  
Infusion Period

ABSTRACT LB11058 is a new synthetic cephalosporin with good affinity for staphylococcal penicillin-binding protein 2a (PBP2a). LB11058 was tested in vitro and in rats with experimental aortic endocarditis against three methicillin-resistant Staphylococcus aureus (MRSA) strains, one penicillinase-negative strain (strain COL), and two penicillinase-producing strains (COL-Bla+ and P8-Hom). The MICs of LB11058 for the organisms were 1 mg/liter. The MICs of vancomycin and ceftriaxone were 1 and ≥64 mg/liter, respectively. In population analysis profiles, none of the MRSA strains grew at ≥2 mg of LB11058/liter. Rats with endocarditis were treated for 5 days. LB11058 was highly bound to serum proteins in rats (≥98%). However, binding was saturable above a threshold of 250 mg/liter. Therefore, continuous concentrations of 250 mg/liter in serum were infused to ensure a free fraction (≥5 mg/liter) above the drug's MIC for the entire infusion period. Control treatments included simulation of human serum kinetics produced by intravenous vancomycin (1 g twice daily, free drug concentration above MIC, ≥90% of infusion period) or ceftriaxone (2 g/24 h, free drug concentrations above the MIC, 0% of infusion period). LB11058 successfully treated 10 of 10 (100%) and 13 of 14 (93%) of rats infected with COL-Bla+ and P8-Hom, respectively. This was comparable to vancomycin (sterilization of 8 of 12 [66%] and 6 of 8 [75%] rats, respectively). Ceftriaxone was inactive. Low concentrations of LB11058 (5 and 10 mg/liter, continuously infused) in serum were ineffective, as predicted by the pharmacodynamic parameters. At appropriate doses, LB11058 was highly effective both in vitro and in vivo. This finding supports the development of this beta-lactam with high PBP2a affinity for the treatment of MRSA infections.

scholarly journals β-Lactam Antibiotics Targeting PBP1 Selectively Enhance Daptomycin Activity against Methicillin-Resistant Staphylococcus aureus

2013 ◽  
Vol 57 (10) ◽  
pp. 5005-5012 ◽  
Author(s):  
Andrew D. Berti ◽  
George Sakoulas ◽  
Victor Nizet ◽  
Ryan Tewhey ◽  
Warren E. Rose
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Division ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Binding Protein ◽  
Membrane Insertion ◽  
Penicillin Binding Protein ◽  
Compensatory Response ◽  
Binding Profile ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThe activity of daptomycin (DAP) against methicillin-resistantStaphylococcus aureus(MRSA) is enhanced in the presence of subinhibitory concentrations of antistaphylococcal β-lactam antibiotics by an undefined mechanism. Given the variability in the penicillin-binding protein (PBP)-binding profiles of different β-lactam antibiotics, the purpose of this study was to examine the relative enhancement of DAP activity against MRSA by different β-lactam antibiotics to determine if a specific PBP-binding profile is associated with the ability to enhance the anti-MRSA activity of DAP. We determined that both broad- and narrow-spectrum β-lactam antibiotics known to exhibit PBP1 binding demonstrated potent enhancement of DAP anti-MRSA activity, whereas β-lactam antibiotics with minimal PBP1 binding (cefoxitin, ceftriaxone, cefaclor, and cefotaxime) were less effective. We suspect that PBP1 disruption by β-lactam antibiotics affects pathways of cell division inS. aureusthat may be a compensatory response to DAP membrane insertion, resulting in DAP hypersusceptibility.

scholarly journals Multiple Novel Inhibitors of the NorA Multidrug Transporter of Staphylococcus aureus

1999 ◽  
Vol 43 (10) ◽  
pp. 2404-2408 ◽  
Author(s):  
Penelope N. Markham ◽  
Eric Westhaus ◽  
Katya Klyachko ◽  
Michael E. Johnson ◽  
Alex A. Neyfakh
Keyword(s):  
Staphylococcus Aureus ◽  
Clinical Setting ◽  
In Vitro Selection ◽  
Antibacterial Activities ◽  
Multidrug Transporter ◽  
Wild Type ◽  
Chemical Library ◽  
Fluoroquinolone Antibiotics

ABSTRACT The multidrug transporter NorA contributes to the resistance ofStaphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

Degradation of Penicillin-Binding Protein 2′ in Methicillin-Resistant Staphylococcus aureus

Chemotherapy ◽  
1995 ◽  
Vol 41 (3) ◽  
pp. 172-177 ◽  
Author(s):  
Y. Sumita ◽  
M. Fukasawa ◽  
S. Mitsuhashi ◽  
M. Inoue
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Methicillin Resistant
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