Localized hypermutation drives the evolution of unstable colistin resistance inPseudomonas aeruginosa

Colistin has emerged as an important last line of defence for the treatment of infections caused by antibiotic resistant Gram-negative pathogens. Here we investigate the responses of ≈1,000 populations of an MDR strain of P. aeruginosa to a high dose of colistin. Colistin exposure resulted in rapid cell death, but a sub-set of populations eventually recovered due to the outgrowth of heteroresistant cells. Genome sequencing revealed that heteroresistance was primarily driven by mutations in the PmrAB two-component system that occurred at a rate (≈2×10 -5 per cell division) that was 10 3 -10 4 fold higher than typical resistance mutation rates. Crucially, this elevated mutation rate was only found in pmrB , demonstrating that hypermutability is localized to this gene. PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a high mutation rate as an adaption to generate mutants that are resistant to host antimicrobial peptides that are secreted during infection. Interestingly, we found no mutations in 1/3 of populations that recovered from colistin treatment, suggesting that phenotypic plasticity and/or persister cells contribute to the ability of Pseudomonas to adapt to colistin.