Localized pmrB hypermutation drives the evolution of colistin heteroresistance

Colistin has emerged as an important last line of defense for the treatment of infections caused by antibiotic-resistant gram-negative pathogens, but colistin resistance remains poorly understood. Here, we investigate the responses of ≈1,000 populations of a multi-drug-resistant (MDR) strain of P. aeruginosa to a high dose of colistin. Colistin exposure causes rapid cell death, but some populations eventually recover due to the growth of sub-populations of heteroresistant cells. Heteroresistance is unstable, and resistance is rapidly lost under culture in colistin-free medium. The evolution of heteroresistance is primarily driven by selection for heteroresistance at two hotspot sites in the PmrAB regulatory system. Localized hypermutation of pmrB generates colistin resistance at 10³-10⁴ times the background resistance mutation rate (≈2 × 10⁻⁵ per cell division). PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a highly mutable pmrB as an adaptation to host immunity.