Magnesium depletion unleashes two unusual modes of colistin resistance with different fitness costs.

Increasing bacterial resistance to colistin, a vital last-resort antibiotic, is an urgent challenge. We previously reported that magnesium sequestration by Candida albicans enables Pseudomonas aeruginosa to become colistin-resistant. Here, we show that Mg²⁺ depletion drives P. aeruginosa to evolve greater colistin resistance through genetic changes in lipid A biosynthesis-modification pathways and a putative magnesium transporter. These mutations synergize with the Mg ²⁺ -sensing PhoPQ two-component signaling system to remodel lipid A structures of the bacterial outer membrane in previously uncharacterized ways. One predominant mutational pathway relies on early mutations in htrB2 , a non-essential gene involved in lipid A biosynthesis, which enhances resistance but compromises outer membrane integrity, resulting in fitness costs and increased susceptibility to other antibiotics. A second pathway achieves increased colistin resistance independently of htrB2 mutations without compromising membrane integrity. In both cases, reduced binding of colistin to the bacterial membrane underlies resistance. Our findings reveal that Mg ²⁺ scarcity unleashes two novel trajectories of colistin resistance evolution in P. aeruginosa . (160).