Fong, S.A.Bouras, G.Houtak, G.Nepal, R.Feizi, S.Morales, S.Psaltis, A.J.Wormald, P.-J.Vreugde, S.Van Tyne, D.2025-07-242025-07-242025Microbiology Spectrum, 2025; 13(5):e02149-24-1-e02149-24-222165-04972165-0497https://hdl.handle.net/2440/146308Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.en© 2025 Fong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.bacteriophagecystic fibrosismultidrug resistancePseudomonas aeruginosaBiofilmsPseudomonas PhagesPseudomonas InfectionsAnti-Bacterial AgentsMutationGenome, BacterialGenetic VariationPhage TherapyWhole Genome SequencingJournal article10.1128/spectrum.02149-24734306Bouras, G. [0000-0002-5885-4186]Feizi, S. [0000-0001-5489-4650]Psaltis, A.J. [0000-0003-2197-0797] [0000-0003-2967-1855]Wormald, P.-J. [0000-0001-7753-7277]Vreugde, S. [0000-0003-4719-9785]