Beyond the matrix: rethinking antibiotic tolerance in CF biofilms using 3D models

Area of Science:

• Microbiology
• Biomaterials Science
• Drug Discovery

Background:

• Chronic lung infections in cystic fibrosis (CF) patients often involve Pseudomonas aeruginosa biofilms.
• These biofilms display significant antibiotic tolerance, a major challenge in CF treatment.
• The precise mechanisms underlying this tolerance, particularly the role of the biofilm matrix, remain unclear.

Purpose of the Study:

• To investigate the role of biofilm matrix components, specifically acetylated alginate and DNA, in Pseudomonas aeruginosa antibiotic tolerance.
• To assess the impact of these matrix components on antibiotic diffusion and efficacy using 3D biofilm models.
• To challenge existing hypotheses regarding diffusion limitation and exopolysaccharide binding as primary drivers of biofilm resilience.

Main Methods:

• Development of 3D biofilm models using bioinks composed of acetylated alginate and DNA, mimicking mucoid biofilms.
• Seeding these models with P. aeruginosa (PAO1) to support in vivo-like microcolony formation.
• High-throughput assessment of antibiotic diffusion (tobramycin, ciprofloxacin, colistin) and efficacy within the 3D models.
• Comparison with mucoid biofilms to evaluate antibiotic penetration dynamics.

Main Results:

• Antibiotic diffusion was not significantly impeded by alginate acetylation or DNA incorporation in the 3D models.
• Bacterial tolerance to antibiotics increased substantially upon encapsulation in alginate, with acetylation further enhancing tolerance.
• DNA incorporation mitigated antibiotic tolerance in a drug-specific manner.
• While mucoid biofilms showed slower antibiotic penetration, they became saturated within 20 hours, indicating diffusion limitation is not the sole factor.

Conclusions:

• Direct interaction with alginate or DNA does not fully explain slow antibiotic diffusion in mucoid P. aeruginosa biofilms.
• Biofilm matrix components contribute to bacterial adaptation and enhanced antibiotic tolerance, independent of diffusion limitations.
• Targeting matrix-induced bacterial adaptation, rather than solely diffusion barriers, is crucial for developing effective antibiofilm therapies for cystic fibrosis.