Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 4 2026 08 04 499 508 Parisa Abedi Elkhichi Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran Sara Bahonar Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Hanieh Shirazi Pediatric Pathology Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran Aghil Bahramian Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran Maryam Kazemi Aghdam Pediatric Pathology Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mona Ghazi Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2025 10 23 2026 07 03 Background and Objectives: As an important opportunistic pathogen, Pseudomonas aeruginosa is associated with severe infections and increasing antimicrobial resistance. The growing prevalence of multidrug-resistant (MDR) strains has renewed interest in bacteriophages as alternative antibacterial agents. The present study sought to isolate and characterize lytic bacteriophages from hospital wastewater active against antibiotic-resistant clinical P. aeruginosa isolates. Materials and Methods: Fifty clinical isolates of P. aeruginosa were obtained and tested for antimicrobial susceptibility. Bacteriophages were isolated from hospital wastewater by enrichment and spot assay screening, followed by plaque purification and titration using the double-layer agar method. The host range of purified phages was evaluated against the clinical isolates. Adsorption assays and one-step growth experiments were performed to determine adsorption efficiency, latent period, and burst size. Transmission electron microscopy (TEM) was used to examine phage morphology. Results: A considerable proportion of the clinical isolates showed multidrug-resistant phenotypes, and a considerable number were also classified as extensively drug-resistant. Four lytic bacteriophages, designated HSP1-HSP4, were successfully isolated and purified. All produced clear plaques and reached high titers. Host range analysis showed variable lytic spectra, with HSP1 exhibiting the broadest activity and showing lytic activity against 70% of the tested isolates, followed by HSP2 (58%), HSP3 (48%), and HSP4 (40%). Adsorption analysis indicated that over 80% of phage particles attached to their host cells within 10 minutes, while HSP1 exhibited the highest adsorption efficiency at 93.6%. A short latent period of approximately 20 minutes was observed for all four phages, and burst sizes ranged from 56 to 65 PFU/cell. Transmission electron microscopy revealed isometric heads and contractile tails, morphologically consistent with tailed phages of the class Caudoviricetes. Conclusion: Hospital wastewater can serve as a practical source for the isolation of lytic bacteriophages active against antibiotic-resistant P. aeruginosa. The in vitro properties of the isolated phages support their further evaluation as candidate antibacterial agents against resistant P. aeruginosa. However, genomic characterization, phage-antibiotic interaction analyses, and in vivo investigations are required before their broader applicability can be determined. https://ijm.tums.ac.ir/index.php/ijm/article/view/5878 https://ijm.tums.ac.ir/index.php/ijm/article/download/5878/1910