Localized hypermutation drives the evolution of colistin heteroresistance

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 some populations eventually recovered due to the growth of sub-populations of heteroresistant cells. Heteroresistance was unstable and was rapidly lost under culture in colistin-free medium. Genome sequencing revealed that heteroresistance was primarily driven by two hypermutable hotspot mutations in pmrB that occurred at a rate (2x10-5 per cell division) that was 103-104 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 highly mutable pmrB an adaptation to generate mutants that are resistant to host antimicrobial peptides that are secreted during infection.