Rationale: Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation have been described for Pseudomonas aeruginosa, resulting in improved diagnosis and treatment, these features remain more poorly defined for S. aureus.

Objectives: To characterize the molecular epidemiology and genetic adaptation of S. aureus during chronic CF airway infection and in response to antibiotic therapy.

Methods: We performed whole-genome sequencing of 1,382 S. aureus isolates collected longitudinally over a mean 2.2 years from 246 children with CF at five U.S. centers between 2008 and 2017. Results were integrated with clinical and demographic data to characterize bacterial population dynamics and identify common genetic targets of in vivo adaptation.

Measurements and Main Results: Results showed that 45.5% of patients carried multiple, coexisting S. aureus lineages, often having different antibiotic susceptibility profiles. Adaptation during the course of infection commonly occurred in a set of genes related to persistence and antimicrobial resistance. Individual sequence types demonstrated wide geographic distribution, and we identified limited strain-sharing among children linked by common household or clinical exposures. Unlike P. aeruginosa, S. aureus genetic diversity was unconstrained, with an ongoing flow of new genetic elements into the population of isolates from children with CF.

Conclusions: CF airways are frequently coinfected by multiple, genetically distinct S. aureus lineages, indicating that current clinical procedures for sampling isolates and selecting antibiotics are likely inadequate. Strains can be shared by patients in close domestic or clinical contact and can undergo convergent evolution in key persistence and antimicrobial-resistance genes, suggesting novel diagnostic and therapeutic approaches for future study.