Colistin resistance dynamics in Pseudomonas aeruginosa under biofilm and planktonic growth.

Pseudomonas aeruginosa is a major pathogen in chronic biofilm-associated lung infections, particularly in patients with cystic fibrosis. Colistin is commonly used to treat these infections, although there is little understanding of how resistance evolves when cells are grown within biofilms. The current study compared the phenotypic dynamics and genetic adaptations of colistin resistance between planktonic and biofilm-grown P. aeruginosa. Using an in vitro experimental evolution approach, we passaged planktonic and biofilm cultures over 10 days under static or progressively increasing colistin concentrations. Population analysis profiling was performed daily to track resistance dynamics and heterogeneity. Whole-genome sequencing was conducted on evolved lineages. Biofilm-grown populations exhibited significantly slower resistance rates than planktonic cultures, particularly under treatments above 0.5 mg/L (1×MIC). Despite this initial delay, both biofilm- and planktonic cultures ultimately evolved similar frequencies of resistant subpopulations. Genetically, we observed shared mutations in canonical colistin resistance determinants such as phoQ and qseC. We also identified growth-mode-specific patterns: oprH mutations were primarily found in biofilm-evolved populations, while nfeD mutations were pervasive in planktonic cultures but rare in biofilms. Taken together, our results provide key insights into the role of biofilm in shaping the evolutionary trajectories of colistin resistance evolution in P. aeruginosa.