Beyond the Matrix: Rethinking Antibiotic Tolerance in CF Biofilms Using 3D Models

Abstract Chronic lung infections in cystic fibrosis (CF) patients are associated with Pseudomonas aeruginosa biofilms exhibiting high antibiotic tolerance, with no clear explanation for this phenomenon. We investigate the role of the biofilm matrix in antibiotic tolerance using 3D biofilm models based on acetylated alginate and DNA, which mimic mucoid biofilms. Printed from these bioinks seeded with P. aeruginosa (PAO1), these models support robust microcolony formation, as observed in vivo , and enable high-throughput assessment of antibiotic diffusion and efficacy. Surprisingly, antibiotic diffusion is not significantly impeded by acetylation or extracellular DNA incorporation. Despite this, bacterial tolerance increases tremendously upon encapsulation in alginate. Acetylation further enhances tolerance, particularly to tobramycin, ciprofloxacin, and colistin. Addition of DNA mitigates this effect in a drug-specific manner. While mucoid biofilms, in contrast to the biofilm models, exhibit significant retardation of antibiotic penetration, they also become saturated with all tested antibiotics within 20 hours. This demonstrates that direct interaction with alginate or DNA does not explain the slow diffusion of antibiotics in mucoid P. aeruginosa biofilms. Our findings challenge the view that diffusion limitation or antibiotic binding by biofilm exopolysaccharides dominates biofilm resilience, highlighting the need to target matrix-induced bacterial adaptation in the development of antibiofilm therapies.