Clinically Relevant Altered Antibiotic Responses and Mechanisms of β-Lactam Sensitization of MRSA in Cystic Fibrosis Artificial Sputum.

Staphylococcus aureus is the most common bacterial pathogen affecting pediatric patients with cystic fibrosis (CF), a genetic disorder that causes thick mucus buildup in the lungs, providing a scaffold for chronic infections. Antibiotic treatment is typically guided by standard in vitro antimicrobial susceptibility testing (AST) in Mueller-Hinton broth (MHB), which does not represent the infection site in CF lungs. Notably, discordances between AST predictions and antibiotic therapeutic outcomes have been reported in up to 50% of CF cases. To address this gap, we conducted ASTs against methicillin-resistant S. aureus (MRSA) in CF sputum-mimetic media compared with MHB, demonstrating ≥ 4-fold discordances across four of nine antibiotic classes. Most significantly, we observed unexpected β-lactam sensitization of MRSA strains (up to 128-fold) in CF sputum-like media, crossing the clinical breakpoint, suggesting this shift may alter therapeutic outcomes. Genome-wide screens and follow-up assays revealed underlying cell envelope remodeling and alterations to cell envelope stress responses. On the other hand, mucin binding to daptomycin may have led to an apparent 8-fold increase in resistance to this antibiotic in one of the CF sputum-like media. Overall, our AST results in CF sputum-mimetic conditions provide insights into bacterial responses during CF infections. Importantly, they suggest β-lactams may be effective in treating MRSA infections in CF patients, warranting further investigation in relevant in vivo systems.