Microenvironment-driven phenotypic shifts in Staphylococcus aureus from children with cystic fibrosis: a longitudinal cohort study

Staphylococcus aureus (SA) is a common pathogen in children with cystic fibrosis (CF), frequently preceding or coexisting with Pseudomonas aeruginosa (PA). However, the extent to which its phenotypic traits are shaped by transient changes in the airway microenvironment, including coinfection and antibiotic exposure, remains poorly understood. In this study, we characterized 546 SA isolates from a longitudinal cohort of 16 pediatric CF patients over 18 months. Based on clinical records, patients were classified as SA-monoinfected or SA-PA-coinfected. Isolates were analyzed for staphyloxanthin production, hemolytic activity, DNase activity, biofilm formation, resistance to PA. Using principal component and hierarchical cluster analyses, we identified three phenotypic clusters. Isolates from monoinfected patients were predominantly found in a cluster characterized by high pigment and biofilm production and absence of chronic PA infection. In contrast, transient PA detection was associated with a shift toward a cluster displaying reduced pigment and biofilm levels. Additionally, in two out of five monoinfected patients, this shift coincided with ciprofloxacin eradication treatment including inhaled tobramycin or colistin and was accompanied by a significant increase in methicillin-resistant SA (MRSA) isolates. No phenotypic transitions were observed in chronically coinfected patients. Molecular characterization of a set of isolates showed on the one hand heterogeneity in the SA lineages and on the other hand the phenotypic profile was not determined by the genetic background. In vitro experiments showed a higher survival of MRSA over MSSA isolates in presence of both PA and ciprofloxacin. These results suggest that the phenotypic adaptation of SA in pediatric CF airway is driven by microenvironment rather than clonal background, and that transient coinfection and antibiotic exposure can favor MRSA emergence even in early-stage disease.