Cryptic beta-Lactamase Evolution Is Driven by Low beta-Lactam Concentrations

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary beta-lactamases is limited. Using the widespread 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 developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs 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-MICs of ceftazidime, they demonstrated strong selectable fitness bene-fits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazi-dime. These changes are likely caused by enhanced flexibility of the X-and beta 5-beta 6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of beta-lactams can drive the evolution of beta-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance. IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolu-tion of antimicrobial resistance. While substantial progress has been made to under -stand the driving role of low concentrations during resistance development for dif-ferent antimicrobial classes, the importance of beta-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low beta-lactam concentrations on the widespread -lactamase OXA-48. Our data indi-cate that the exposure to beta-lactams at very low concentrations enhances beta-lacta-mase diversity and drives the evolution of beta-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolu-tion of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.