Experimental evolution ofPseudomonas aeruginosato colistin in spatially confined microdroplets identifies evolutionary trajectories consistent with adaptation in microaerobic lung environments
Authors: Disney-McKeethen Saoirse , Seo Seokju , Mehta Heer , Ghosh Karukriti , Shamoo Yousif 
Published: June 2023

Antibiotic resistance is a continuing global health crisis. Identifying the evolutionary trajectories leading to increased antimicrobial resistance can be critical to the discovery of biomarkers for clinical diagnostics and new targets for drug discovery. While the combination of patient data andin vitroexperimental evolution has been remarkably successful in extending our understanding of antimicrobial resistance, it can be difficult forin vitromethods to recapitulate the spatial structure and consequent microenvironments that characterizein vivoinfection. Notably, in cystic fibrosis (CF) patients, changes to either the PmrA/PmrB or PhoP/PhoQ two-component systems have been identified as critical drivers for high levels of colistin and polymyxin resistance. When using microfluidic emulsions to provide spatially structured, low-competition environments, we found that adaptive mutations tophoQwere more successful thanpmrBin increasing colistin resistance. Conversely, mutations topmrBwere readily identified using well-mixed unstructured cultures. We found that oxygen concentration gradients within the microdroplet emulsions favored adaptive changes to the PhoP/PhoQ pathway consistent with microaerobic conditions that can be found in the lungs of CF patients. We also observed mutations linked to hallmark adaptations to the CF lung environment, such as loss of motility (fleQ, fliC, fleS, flg, flh, andfleQ) and loss of O antigen biosynthesis (wbpL). Mutation towbpL, in addition to causing loss of O antigen, was additionally shown to confer moderately increased colistin resistance. Taken together, our data suggest that distinct evolutionary trajectories to colistin resistance may be shaped by the microaerobic partitioning and spatial separation imposed within the CF lung.

Importance

Antibiotic resistance remains one of the great challenges confronting public health in the world today. Individuals with compromised immune systems or underlying health conditions are often at an increased for bacterial infections. Patients with Cystic Fibrosis (CF) produce thick mucus that clogs airways and provides a very favorable environment for infection by bacteria that further decrease lung function and, ultimately, mortality. CF patients are often infected by bacteria such asPseudomonas aeruginosaearly in life and experience a series of chronic infections that, over time, become increasingly difficult to treat due to increased antibiotic resistance. Colistin is a major antibiotic used to treat CF patients. Clinical and laboratory studies have identified PmrA/PmrB and PhoP/PhoQ as responsible for increased resistance to colistin. Both have been identified in CF patient lungs, but why, in some cases, is it one and not the other? In this study, we show that distinct evolutionary trajectories to colistin resistance may be favored by the microaerobic partitioning found within the damaged CF lung.