Evolution of Antimicrobial Resistance in Pseudomonas aeruginosa in Patients with Bronchiectasis Infection

Abstract

Non-cystic fibrosis bronchiectasis (NCFB) is a chronic airway disease in which Pseudomonas aeruginosa often establishes long-term, multidrug-resistant infection. Compared with cystic fibrosis (CF), little is known about how resistance emerges, persists, and evolves within NCFB patients, particularly over clinically relevant timescales. This thesis investigates the phenotypic diversity, genetic basis, and longitudinal dynamics of antimicrobial resistance (AMR) in P. aeruginosa using isolates collected from 20 participants in the ORBIT-3 trial of inhaled liposomal ciprofloxacin. Across 1,800 baseline isolates, high-resolution phenotyping revealed striking within-patient diversity, with individual patients harbouring multiple coexisting resistance profiles. Co-resistance was structured, dominated by strong within-class associations and occasional cross-class links. Resistance to fluoroquinolones and aminoglycosides was common, while β-lactam resistance remained low. Growth assays showed that fluoroquinolone resistance carried measurable fitness costs, whereas most other traits had minimal impact on growth. Whole-genome sequencing of 345 baseline isolates demonstrated that infections were largely clonal within individual patients, with limited within-patient genomic variation and only occasional hypermutator lineages. Genomic diversity correlated poorly with resistance, indicating that AMR heterogeneity often arises within single lineages. In the longitudinal component (3,840 isolates across nine timepoints), cyclic ciprofloxacin exposure repeatedly selected for predictable fluoroquinolone resistance through canonical gyrA/parC mutations, narrowed resistotype diversity, and did not drive consistent cross-resistance to other drug classes. Strain replacement was rare; adaptation occurred mainly within existing lineages. Together, these findings show that AMR evolution in NCFB is strongly shaped by patient-specific conditions and constrained genetic pathways. This underscores the need for multi-isolate diagnostics, up-to-date susceptibility testing, and treatment strategies that account for predictable within-host evolutionary trajectories.