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Colistin is a last-resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous-culture device known as a morbidostat. As a result, colistin resistance reproducibly increased 10-fold within 10 days and 100-fold within 20 days, along with highly stereotypic yet strain-specific mutation patterns. The majority of mutations hit the pmrAB two-component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC , pmrE , and migA We tracked the frequencies of all arising mutations by whole-genome deep sequencing every 3 to 4 days to obtain a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant subpopulations. In 7 out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture