Phage isolation and functional characterization reveal strong antibiofilm activity against Pseudomonas aeruginosa in a cystic fibrosis sputum model

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that forms persistent biofilms in the lungs of cystic fibrosis and other chronic pulmonary disease patients, contributing to antibiotic tolerance, recurrent infection, and clinical decline. The rise of multidrug-resistant <i>P. aeruginosa</i> underscores the urgent need for alternative therapies. Bacteriophages (phages) offer a powerful therapeutic approach by directly lysing bacteria, diminishing biofilm structures, and overcoming mechanisms that limit antibiotic efficacy. In this study, a library of 61 distinct <i>P. aeruginosa</i> phages was isolated and screened against 64 clinical isolates, identifying eight with broad host range and high lytic activity. These phages, including PA-319, PA-575, and PA-711, effectively prevented <i>P. aeruginosa</i> colonization on A549 human lung epithelial cells, inhibited bacterial biofilm formation as well as compromised established biofilms, surpassing the effects of high-concentration antibiotics. Genomic and transmission electron microscopy analyses revealed functional heterogeneity, including nucleus-forming and non-nucleus-forming jumbo phages and depolymerase-encoding genes. Our phage library provides a valuable resource for advancing research, developing combinatorial phage therapies, and optimizing treatment strategies against chronic, drug-resistant <i>P. aeruginosa</i> infections.