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

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.

Genetic and Structural Characterization of an L201P Global Suppressor Substitution in TEM-1 β-Lactamase

“…From the E28G:R244A library, a suppressor distant from the active site was isolated with a single nucleotide substitution resulting in a L201P mutation. Interestingly, the L201P substitution has been observed previously upon selection for inhibitor-resistant TEM-1 β-lactamases from an error-prone library of TEM-1 enzymes 37. In addition, the L201P substitution was recently identified as a suppressor among populations of highly mutagenized TEM-1 mutants propagated under conditions of neutral drift 38.…”

“…The L201P substitution was previously identified during an in vitro selection experiment for inhibitor resistant TEM-1 mutants37 and it was also recently identified as a suppressor from selection experiments with populations of highly mutagenized TEM-1 genes 38. Unlike the M182T substitution, however, the L201P change has not been identified in natural isolates.…”

PFunkel: Efficient, Expansive, User-Defined Mutagenesis

“…We predict that Y105N, Y105S, and S235T have the potential to emerge in the clinic. Their non-emergence to date, and the fact that they were not identified in previous selections for tazobactam resistance performed on error prone PCR libraries [31] may reflect the fact that the required base substitutions are not as common as the base substitutions for previously identified tazobactam resistance mutations. We speculate that we readily identified these mutations because PFunkel provides a less biased and much more comprehensive library of mutations than error prone PCR.…”

Network Models of TEM β-Lactamase Mutations Coevolving under Antibiotic Selection Show Modular Structure and Anticipate Evolutionary Trajectories

“…The extended-spectrum community contains mutations at eight positions that are known to extend the substrate spectrum of the enzyme: 39, 51, 104, 164, 173, 237, 238, 240 [9], [17], [19], [21], [24], [33]–[41], as well as four stabilizing mutations: 153, 182, 224, 268 [9], [25], [39], [41]–[43]. Likewise, the inhibitor community contains five positions known to confer inhibitor resistance: 69, 165, 244, 275, 276 [9], [13], [44]–[49] and three enhancer stabilizing mutations: 147, 201, 275 [9], [25], [43], [45], [50]–[52].…”