Phenotypic and genomic characterization of two novel lytic bacteriophages targeting multidrug-resistant Pseudomonas aeruginosa

BACKGROUND: The increasing prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa highlights the urgent need for innovative therapeutics beyond conventional antibiotics. METHOD: In this study, two novel lytic bacteriophages, Pseudomonas phage_SW_PA2862_14_24 (N2) and Pseudomonas phage_SW_PA2862_11_24 (N3) of the myovirus morphology, were isolated and comprehensively characterized for activity against P. aeruginosa isolates. Transmission electron microscopy was used to determine morphotypes, while adsorption assays, one-step growth curves, and time-kill assays assessed infection dynamics. Genomic characterization included whole-genome sequencing and bioinformatic analysis for safety profiling and comparative genomics. RESULTS: Electron microscopy revealed distinct morphologies: N2 exhibited a contractile tail, and N3 displayed a flexible, non-contractile tail. Adsorption assays showed that N3 achieved rapid host binding (∼95% within 5 min), while N2 demonstrated a higher burst size of 231 PFU/cell. Genomic analysis identified genome sizes of ∼92.8 kb with 202-203 coding sequences, 15 tRNA genes, and a high proportion (∼73%) of hypothetical proteins. Both phages contained complete lytic modules and conserved genes related to replication and DNA metabolism. Comparative analysis demonstrated high genomic homology with established therapeutic phages (SPA01, EPA1, OMKO1). Time-kill assays confirmed dose-dependent bactericidal efficacy, with N2 showing superior and sustained lytic activity at moderate to high MOIs. Importantly, both genomes lacked lysogeny, antimicrobial resistance, and virulence genes. CONCLUSION: These novel phages constitute biosafe, genetically defined lytic agents with distinct infection kinetics and potent activity against MDR P. aeruginosa. Their complementary features and favorable genomic profiles support their potential use in precision phage therapy to combat antibiotic-resistant infections.