Isolation and characterization of bacteriophages against antibiotic-resistance Pseudomonas aeruginosa

Abstract

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 re- newed 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 purifi- cation 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 peri- od, 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 num- ber 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 approxi- mately 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 Cau- doviricetes.

Conclusion: Hospital wastewater can serve as a practical source for the isolation of lytic bacteriophages active against anti- biotic-resistant P. aeruginosa. The in vitro properties of the isolated phages support their further evaluation as candidate an- tibacterial 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.