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Antimicrobial Agents and Chemotherapy, July 1998, p. 1542-1548, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Sergei B. Vakulenko,1 Bruce Geryk,1 Lakshmi P. Kotra,2 Shahriar Mobashery,2 and Stephen A. Lerner1,3,*

Departments of Medicine1 and Biochemistry and Molecular Biology,3 Wayne State University School of Medicine, Detroit, Michigan 48201, and Department of Chemistry, Wayne State University, Detroit, Michigan 482022

Received 27 October 1997/Returned for modification 9 February 1998/Accepted 16 April 1998

Mechanism-based inactivators of beta -lactamases are used to overcome the resistance of clinical pathogens to beta -lactam antibiotics. This strategy can itself be overcome by mutations of the beta -lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta -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 beta -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 beta -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 beta -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.

* Corresponding author. Mailing address: Division of Infectious Diseases, Harper Hospital, 3990 John R, Detroit, MI 48201. Phone: (313) 745-9131. Fax: (313) 993-0302. E-mail: slerner{at}oncgate.roc.wayne.edu.

Antimicrobial Agents and Chemotherapy, July 1998, p. 1542-1548, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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