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.

The class C ␤-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum ␤-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 ␤-lactamase conferring a higher MIC of cefepime (MIC, 8 g/ml, compared with 0.5 g/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other ␤-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 ␤-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat /K m ) of the Leu-293-Pro mutant ␤-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 ␤-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant ␤-lactamase.