Pseudomonas aeruginosa is a leading cause of chronic lung infections in cystic fibrosis (CF) patients. While bacteriophages hold potential as a treatment for antibiotic-resistant infections, the complex structure and heterogeneity of P. aeruginosa biofilms pose significant challenges to phage therapy. In this study, we investigate the adaptive evolution of the Pbunavirus phage PE1 to biofilms formed by a CF-derived P. aeruginosa isolate. Our findings reveal that biofilm-adapted PE1 mutants exhibit enhanced efficacy in controlling biofilms in vitro under conditions mimicking the CF lung environment. This improvement is attributed to the mutants' increased ability to recognize the diverse populations within the biofilm. Using a combination of cryo-EM, lipopolysaccharide (LPS) profiling, and adsorption assays, we demonstrate that mutations in tail fiber and baseplate genes of the phage improve adsorption and enable recognition of truncated LPS variants. This study highlights the critical role of biofilm heterogeneity in limiting phage effectiveness, identifies mechanisms to overcome this barrier, and pinpoints specific genomic targets for engineering phages tailored for therapeutic applications in CF patients.

CEB Centre of Biological Engineering University of Minho Braga Portugal

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Clinical Microbiology Rigshospitalet Copenhagen Denmark

Department of Molecular Bacteriology Helmholtz Centre for Infection Research Braunschweig Germany

Institute for Molecular Bacteriology TWINCORE Centre for Experimental and Clinical Infection Research Hannover Germany

LABBELS Associate Laboratory Braga Guimarães Portugal

Laboratory of Pharmaceutical Microbiology Ghent University Gent Belgium

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