Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance

Abstract Antimicrobial resistance poses a significant global health threat. Experimental evolution studies are crucial in understanding resistance mechanisms and thereby informing strategies to preserve antibiotic efficacy. We developed a novel continuous experimental evolution system enabling uninterrupted medium exchange with a rising antibiotic gradient, using standard laboratory equipment. We applied this system to three Enterobacter cloacae complex strains isolated from urinary tract infections in Germany between 1990 and 1992, which therefore had no prior exposure to cefepime, a fourth-generation cephalosporin approved in Germany in 2004. After four days of exposure to a cefepime gradient, resistant mutants emerged in all three strains. Notably, one mutant exhibited cross-resistance to the novel antibiotics cefiderocol and ceftazidime-avibactam, due to a single missense mutation in the β-lactamase gene bla MIR-11 . Our study demonstrates the effectiveness of this novel approach for investigating antimicrobial resistance development and cross-resistance mechanisms, as well as identifying and characterizing a mutation attributed to major cross-resistance.