SELECTION AND CHARACTERIZATION OF BETA-LACTAM-BETA-3-LACTAMASE INACTIVATOR-RESISTANT MUTANTS FOLLOWING PCR MUTAGENESIS OF THE TEM-1 BETA-LACTAMASE GENE

Mechanism-based inactivators of beta-lactamases are used to overcome t he resistance of clinical pathogens to beta-lactam antibiotics. This s trategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutag enesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven di fferent mutant genes from these strains contained from 1 to 10 mutatio ns, 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 mul tiple amino acid substitutions generated in this study conferred highe r levels of resistance to ampicillin alone or ampicillin with beta-lac tamase inactivators (clavulanate, sulbactam, or tazobactam) than the l evels of resistance conferred by the corresponding single-mutant enzym es. Of the four enzymes with just a single mutation (Ser130Gly, Arg244 Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of r esistance to ampicillin in the presence of inhibitors. Site-directed r andom mutagenesis of the Ser130 codon yielded no other mutant with rep lacement of Ser130 besides Ser130Gly that produced ampicillin-clavulan ate resistance. Thus, despite PCR mutagenesis we found no new mutant T EM beta-lactamase that conferred a level of resistance to ampicillin p lus inactivators greater than that produced by the single-mutation enz ymes that have already been reported in clinical isolates. Although th is is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.