Characterization, genomic insights and anti-biofilm potential of phage vB_PaeM_PKMS3, a lytic Pbunavirus capable of infecting clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa, a leading opportunistic nosocomial pathogen, is a major contributor to antimicrobial resistance (AMR)-associated morbidity and mortality. The global surge of multidrug-resistant (MDR) P. aeruginosa strains necessitates the use of reliable alternative therapeutic strategies like phage therapy. Here, we report the isolation and comprehensive characterisation of a myoviral bacteriophage, vB_PaeM_PKMS3, effective against multiple clinical P. aeruginosa isolates. The phage exhibited potent lytic activity, a relatively broad intra-species host range, and stability across diverse temperature and pH conditions. One-step growth curve analysis revealed a short 20-min latent period, a rapid 10-min rise period, and a moderately high burst size of ~ 82 PFU/infected cell. vB_PaeM_PKMS3 showed excellent in vitro cytocompatibility and was well tolerated in the Danio rerio model during safety assessment. The exceptional in vitro biofilm-degradative ability of the phage, exemplified by a three-fold reduction in established biofilms, was corroborated by fluorescent and electron microscopic analyses. Genome sequencing revealed that phage vB_PaeM_PKMS3 possesses a linear dsDNA genome of ≈66 kbp, belongs to the Pbunavirus genus, and encodes several antibacterial proteins, including holin, depolymerase, and endolysin, while lacking virulence, lysogenic, and antibiotic resistance genes. Collectively, these results identify vB_PaeM_PKMS3 as a safe and favourable candidate for potential inclusion in phage cocktail formulations targeting MDR P. aeruginosa. This work also highlights the importance of systematic characterisation of locally isolated bacteriophages, particularly in regions burdened by drug-resistant infections.