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 byCandida albicansenablesPseudomonas aeruginosato become colistin-resistant. Here, we show that Mg²⁺ depletion drivesP. aeruginosato evolve greater colistin resistance through genetic changes in lipid A biosynthesis-modification pathways and a putative magnesium transporter. These mutations synergize with the Mg²⁺-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 inhtrB2, 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 ofhtrB2mutations without compromising membrane integrity. In both cases, reduced binding of colistin to the bacterial membrane underlies resistance. Our findings reveal that Mg²⁺scarcity unleashes two novel trajectories of colistin resistance evolution inP. aeruginosa. (160)