Efflux reprogramming promotes Staphylococcus aureus persistence with Pseudomonas aeruginosa in cystic fibrosis

Staphylococcus aureus persists in cystic fibrosis (CF) airways despite in vitro antibiotic susceptibility, suggesting that efflux reprogramming driven by environmental and polymicrobial interactions with Pseudomonas aeruginosa promotes survival in this setting. In this study, we investigated how CF-like conditions and co-infection with P. aeruginosa influence S. aureus physiology and antimicrobial response. Using artificial sputum medium (ASM) and co-culture models, we found that ASM induced a selective transcriptional response characterized by upregulation of the multidrug efflux pump tet38, while other pumps remained minimally expressed. In the presence of P. aeruginosa, S. aureus maintained tet38 expression but additionally induced norB, indicating a redistribution of efflux activity under polymicrobial stress. P. aeruginosa metabolites, including quinolines and phenazines, contributed to this response, and deletion of tet38 or norB significantly reduced S. aureus fitness in ASM and co-culture conditions. Analysis of S. aureus clinical isolates from people with CF revealed a similar hierarchy, with Tet38 and NorB as the predominant efflux pumps, particularly in S. aureus isolates co-recovered with P. aeruginosa. Despite exhibiting only low-level increases in MICs, these isolates demonstrated enhanced survival in ASM. Notably, S. aureus isolates paired with clinical P. aeruginosa strains showed improved survival compared with survival when paired with a P. aeruginosa reference strain, consistent with adaptation toward coexistence. Together, these findings demonstrate that CF-like environmental conditions and polymicrobial interactions drive selective reprogramming of efflux pumps that promote S. aureus persistence without conferring high-level resistance, highlighting a mechanism of antibiotic tolerance not captured by standard susceptibility testing.