Cryptic β-lactamase evolution is driven by low β-lactam concentrations

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary {beta}-lactamases is limited. Using the wide-spread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to develop clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-minimum inhibitory concentrations (sub-MIC) of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MIC of ceftazidime, they showed strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies towards ceftazidime. These changes are likely caused by enhanced flexibility of the {Omega}- and {beta}5-{beta}6 loops. In conclusion, low-level concentrations of {beta}-lactams can drive the evolution of {beta}-lactamases through cryptic phenotypes which may act as stepping-stones towards clinical resistance.