Abstract Aim Bacteriophages are effective natural antimicrobial agents against drug‐resistant pathogens. Therefore, identification and detailed characterization of bacteriophages become essential to explore their therapeutic potential. This study aims to isolate and characterize a lytic bacteriophage against drug‐resistant Pseudomonas aeruginosa. Methods and Results The Pseudomonas phage AIIMS‐Pa‐A1, isolated from the river Ganga water against drug‐resistant P. aeruginosa, showed a clear lytic zone on spot assay. The phage revealed an icosahedral head (58.20 nm diameter) and a small tail (6.83 nm) under a transmission electron microscope. The growth kinetics showed an adsorption constant of 1.5 × 10−9 phage particles cell−1 ml−1 min−1 and a latent period of approximately 15 min with the burst size of 27 phages per infected cell. The whole‐genome sequencing depicted a GC‐rich genome of 40.97 kb having a lysis cassette of holin, endolysin and Rz protein, with features of the family Autographiviridae. The comparative genome analysis, Ortho‐average nucleotide identity value, and phylogenetic analysis indicated the novelty of the phage AIIMS‐Pa‐A1. Conclusions The study concludes that the Pseudomonas phage AIIMS‐Pa‐A1 is a novel member of the Autographiviridae family, truly lytic in nature for drug‐resistant P. aeruginosa. Significance and Impact of the Study The Pseudomonas phage AIIMS‐Pa‐A1 is having promising potential for future therapeutic intervention to treat drug‐resistant P. aeruginosa infections.
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