Supplementary Information

<i>Pseudomonas aeruginosa</i> is an opportunistic bacterium frequently infecting the lower airways of individuals with cystic fibrosis (iwCF). This study examines colistin (CST) resistance mechanisms in <i>Psa</i>-ST3351 and their effects on bacterial virulence. <i>Psa</i>-ST3351 represents a novel sequence type, CST-resistant isolate from a child with CF. We show that <i>Psa</i>-ST3351 CST-resistance requires the addition of 4-amino-4-deoxy-L-arabinose to lipid A. Surprisingly, CST-exposure induced a non-mucoid-to-mucoid transition characterized by enhanced swarming motility, decreased aminoglycoside susceptibility, impaired ExoS secretion via the type-3 secretion system, and increased bacterial competition, all hallmarks of a chronic infection phenotype. CST-treated <i>Psa</i>-ST3351 exhibited reduced phagocytosis by macrophages, although the induction of pro-inflammatory cytokines remained intact. Moreover, CST exposure enhanced the pathogenicity of <i>Psa</i>-ST3351 in a murine pneumonia model. We conclude that CST-mediated transition from a non-mucoid-to-mucoid phenotype in <i>Psa</i>-ST3351 may have implications, particularly when CST-resistance and mucoid conversion remain undetected in iwCF treated with nebulized CST.