Characterization and comparative genomic analysis of a novel bacteriophage against an extremely drug-resistant <i>Pseudomonas aeruginosa</i> ST1971

ABSTRACT Given the rising prevalence of multidrug-resistant strains, Pseudomonas aeruginosa necessitates alternative therapies, and phages are emerging as promising antibiotic alternatives. Our study introduces a novel lytic phage, CRPAP1904, exhibiting potent activity against an extremely drug-resistant P. aeruginosa ST1971 strain CRPA190. Phylogenetic and comparative genomic analyses validated the novelty of phage CRPAP1904, suggesting that it represents a new species of the Kochitakasuvirus genus. The phage CRPAP1904 has a double-stranded DNA genome of 62,430 bp base pairs, a GC content of 60.4% and predicted 83 open reading frames. Notably, no genes associated with virulence or antibiotic resistance were detected, suggesting its suitability for therapeutic applications. We further assessed the phage’s host range, optimal multiplicity of infection (MOI), one-step growth curves, physicochemical stability, and antibacterial activity, which are critical for its application in environmental and clinical settings. The phage exhibits a broad host range, lysing 83% (20/24) of tested strains. The optimal MOI was 0.01 with a large burst size of 109 PFU/cell. Additionally, the phage exhibited stability over a pH range of 6–8 and at temperatures ranging from −20°C to 60°C. In vitro , the phage significantly suppresses host bacterial growth at an MOI of 100 for the first 12 h, although resistant mutants gradually emerge during prolonged exposure. In conclusion, the newly discovered phage CRPAP1904 expands phage resources and supports medicinal phage library development, advancing phage therapy and biocontrol applications in MDR-PA infections. IMPORTANCE This study isolated a novel phage, CRPAP1904, from the hospital's sewage that specifically targets an extremely drug-resistant Pseudomonas aeruginosa ST1971 strain CRPA190. Genomic analysis identified CRPAP1904 as a new Kochitakasuvirus member lacking antibiotic resistance genes and virulence factors. CRPAP1904 shows excellent stability and lytic activity against MDR-PA clinical isolates. As the first phage against Pseudomonas aeruginosa ST1971, this work expands phage resources and supports medicinal phage library development, advancing phage therapy and biocontrol applications in MDR-PA infections.