Project Summary Progress in improving infection outcomes depends upon understanding factors that diminish antibiotic action. Recent work suggests antibiotic heteroresistance may be a key factor. Heteroresistant pathogens exhibit population-level resistance heterogeneity, meaning they contain highly resistant subpopulations, and often exhibit unstable resistance, meaning some cells become rapidly sensitive when grown without antibiotics. This proposal focuses on these two phenotypes as they are postulated to have major effects on susceptibility testing and antibiotic efficacy. Here we propose to study population-level resistance heterogeneity and unstable resistance in people with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa (Pa) infections who cycle on and off inhaled tobramycin. CF offers key advantages for these studies as it is a monogenic disease with uniform manifestations, the responses of a single bacterial species to the same antibiotic can be studied in sizable cohorts, and infrastructure exists to procure primary specimens from the infected site (sputum) during antibiotic "on" and "off" periods. These advantages, and methods developed in our preliminary work enable us to test the hypotheses that Pa from chronically-infected patients exhibit marked population-level resistance heterogeneity and unstable resistance; that parameters accounting for these factors predict clinical responses to treatment; and that unstable resistance in CF Pa is due to a core set of gene amplifications and fitness-compensating point mutations. This work will generate foundational knowledge about antibiotic heteroresistance that could improve understanding of antibiotic treatment failure in CF, and suggest new precision medicine approaches to select antibiotics and improve outcomes. The findings could also guide future work in infections where patient heterogeneity, pathogen and treatment diversity, and less developed infrastructure make studies more difficult.