Fig. 1. Identification of escape mutations by site-directed mutagenesis followed by selection and sequencing.

a The bla_ampC gene of E. coli is mutated using MAGE technology to yield a mutant library of 790 different single amino-acid substitutions in the Bla_ampC pocket. b The mutant library is then selected on a gradient of beta-lactam antibiotics with or without avibactam (AVI) and cell density is measured (Gray shade). At the antibiotic concentration that inhibits the growth of the WT bacteria ($MI{C}_{AB}^{\mathrm{W}T}$, white cells), only resistant mutants grow and a decline in cell density is observed (light gray). When growth medium is supplemented with avibactam (+AVI) the growth of all mutants may be inhibited at drug concentrations lower than ${\mathrm{MIC}}_{\mathrm{AB}}^{\mathrm{WT}}$ (middle row, left-pointing arrow) unless escape mutants are present, which grow on concentrations higher than ${\mathrm{MIC}}_{\mathrm{AB}}^{\mathrm{WT}}$ even in the presence of avibactam (bottom row, right-pointing arrow). c Such escape mutations, as well as resistance-conferring mutations, are identified by high-throughput sequencing of the resulting culture.