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Mechanisms of Resistance

Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene

Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Sergei B. Vakulenko
Departments of Medicine and
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Bruce Geryk
Departments of Medicine and
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Lakshmi P. Kotra
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
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Shahriar Mobashery
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
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Stephen A. Lerner
Departments of Medicine and
Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, and
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DOI: 10.1128/AAC.42.7.1542
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ABSTRACT

Mechanism-based inactivators of β-lactamases are used to overcome the resistance of clinical pathogens to β-lactam antibiotics. This strategy can itself be overcome by mutations of the β-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 β-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with β-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp β-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM β-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.

  • Copyright © 1998 American Society for Microbiology
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Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene
Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Antimicrobial Agents and Chemotherapy Jul 1998, 42 (7) 1542-1548; DOI: 10.1128/AAC.42.7.1542

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Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene
Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Antimicrobial Agents and Chemotherapy Jul 1998, 42 (7) 1542-1548; DOI: 10.1128/AAC.42.7.1542
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KEYWORDS

Drug Resistance, Multiple
Escherichia coli
beta-lactam resistance
beta-lactamases

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