Glycoside hydrolases enhance antibiotic activity against <i>Pseudomonas aeruginosa</i> biofilms from cystic fibrosis airways

<i>Pseudomonas aeruginosa</i> forms antibiotic-tolerant biofilms in cystic fibrosis (CF) lungs. Targeting exopolysaccharides such as Psl and Pel offers a potential strategy to disrupt biofilms and improve antibiotic efficacy. Fourteen clinical CF <i>P. aeruginosa</i> isolates were screened for Psl- and Pel-dependent biofilm formation using crystal violet assays. The adjunctive effects of glycoside hydrolases (GHs) PslG_h and PelA_h with colistin, levofloxacin, and tobramycin (at peak sputum concentrations following nebulization) were evaluated using a high-throughput assay. Confocal microscopy visualized effects in live biofilms at subinhibitory antibiotic levels for two representative strains. Enzyme stability in CF sputum supernatant at 37°C was assessed via Western blot. Nine of 14 isolates showed PslG_h-mediated disruption; only 3/14 were disrupted by PelA_h. PslG_h significantly enhanced antibiotic effects in 24 of 42 strain-antibiotic pairs (57%), compared with 5 of 42 (12%) for PelA_h. Adjunctive effects were most pronounced with tobramycin, yielding 18.1% biofilm reduction. Confocal imaging confirmed additive effects when the GH corresponded to the exopolysaccharide to which the isolate had demonstrated disruption in the screening assay, but showed no benefit when targeting the other exopolysaccharide, even at higher enzyme doses. PslG_h was more stable than PelA_h in sputum, with PelA_h degraded significantly by 4 and 20 h. GHs targeting exopolysaccharides associated with biofilm disruption in functional assays can enhance antibiotic efficacy against <i>P. aeruginosa</i> biofilms. PslG_h shows greater promise due to broader activity and better sputum stability. These findings support tailored GH-antibiotic strategies in CF lung infection treatment.IMPORTANCEBiofilms formed by <i>Pseudomonas aeruginosa</i> are a major obstacle to effective antibiotic treatment, particularly in chronic lung infections encountered in cystic fibrosis. This study explores the use of glycoside hydrolases (enzymes that degrade key biofilm components) as a way to enhance antibiotic activity against early-stage clinical isolates. Our findings show that targeting biofilm structure can potentiate the effects of multiple antibiotics, suggesting that biofilm-degrading compounds hold promise as future antibiotic adjuvants. However, variability among bacterial strains highlights the need for further research before these strategies can be reliably translated into clinical care.