Abstract
Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.
Highlights
From the ‡Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and ¶Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
The S130G and S130T -lactamases expressed in E. coli were ampicillin resistant (MICs Ն 32 g/ml)
None of the variant -lactamases expressed in E. coli conferred more resistance than SHV-1 against ampicillin/tazobactam or piperacillin/tazobactam
Summary
Bacterial Strains and Plasmids—The chromosomal SHV-1 -lactamase with its native promoter and ribosomal binding site was cloned into the recombinant phagemid vector, pBC SK(Ϫ) (Stratagene, La Jolla, CA) as described previously [14]. Antibiotic Susceptibility—E. coli DH10B expressing the mutated blaSHV genes were phenotypically characterized by LB agar dilution minimum inhibitory concentrations (MICs) using a “Steers replicator” that delivered 104 colony forming units/spot. Antibiotics used and their suppliers were described previously [8, 9, 15]. A direct competition assay was performed to determine the dissociation constant for the preacylation complex, KI, of the inhibitors (clavulanic acid and tazobactam). We incubated increasing amounts of inhibitor (clavulanic acid or tazobactam) with a fixed concentration of SHV-1 or S130G -lactamase in a total volume of 100 l of 20 mM phosphate-buffered saline, pH 7.4, at room temperature. The proportion of clavulanic acid or tazobactam relative to SHV-1 or S130G that resulted in 90% inactivation after 24 h was used to determine the ratio of inhibitor to enzyme in
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