Identification of Evolutionary Trade‐Offs Associated With High‐Level Colistin Resistance in <i>Acinetobacter baumannii</i>

Colistin (COL) belongs to the polymyxin group of drugs, which possesses a positive charge and interacts with lipopolysaccharide (LPS) of Gram-negative bacterial outer membranes. Acinetobacter baumannii, a bacterium in the "ESKAPE" group of "priority pathogens," has acquired resistance against the majority of available antibiotics, including the last resort antibiotic COL. Though plasmid-encoded acquisition of mcr-genes has been associated with clinical resistance, efflux, loss of LPS by inactivation of the biosynthetic pathway (lpxACD), and modifications of target LPS by products of chromosomal pmrCAB genes has been ascribed to resistance evolution. Systemic characterization of trade-offs and traits accompanying the evolution of COL resistance in the bacteria remains unaccomplished. Here we report adaptive evolution of extreme COL resistance of the reference strain A. baumannii ATCC19606. Systemic phenotypic characterization of the mutants revealed hyperbiofilm formation and a striking decrease in fitness as the major evolution-associated attributes. Comprehensive antibiotic susceptibility profiling indicated collateral sensitivity against vancomycin and fosfomycin. Whole genome sequencing of the resistant strains led to the identification of mutations associated with COL resistance. Phenotypic characterization of three COL-resistant clinical isolates of A. baumannii revealed similarity with experimentally evolved resistant mutants in one of the isolates, in which none of the mcr-genes could be detected.