Sequencing mutant library in the E. coli beta-lactamase gene ampC selected on different drugs

Beta-lactamase inhibitors are increasingly used to counteract microbial resistance 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 discover 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 drug combinations completely prevent such escape phenotypes. Amplicon sequencing of the selected mutant pool identified these escape mutations and showed that they are rare and drug specific. 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.