Escape mutations circumvent a tradeoff between resistance to beta-lactams and a beta-lactamase inhibitor

Abstract

Beta-lactamase inhibitors are increasingly used to counteract microbial resistance to beta-lactam antibiotics mediated by beta-lactamase enzymes. These inhibitors compete with the beta-lactam drug for the same binding site of the beta-lactamase, thereby generating an inherent evolutionary tradeoff: enzyme mutations that increase its activity against the beta-lactam drug also increase its susceptibility towards the inhibitor. It is unclear how common and accessible are mutants that escape this adaptive tradeoff. Here, systematically constructing and phenotyping a deep mutant library of the ampC beta-lactamase gene of Escherichia coli, we identified escape mutations, which even in the presence of the enzyme inhibitor allow growth at beta-lactam concentrations far exceeding the native inhibitory levels of the wildtype strain. Importantly, while such escape mutations appear for combinations of avibactam with some beta-lactam drugs, for other drugs escape phenotypes are completely restricted. Amplicon sequencing of the selected mutant pool identified these escape mutations and showed that they are rare and drug specific. For the combination of avibactam with aztreonam, an escape phenotype was conferred via multiple substitutions in a single conserved amino acid (Tyr 150). In contrast, a different set of mutations showed an escape phenotype for cefepime, and no escape mutants appeared for piperacillin. The differential adaptive potential of ampC to combinations of avibactam and different beta-lactam drugs can help guide drug treatments that are more resilient to evolution of resistance.