Isolation and Characterization of River-Derived Bacteriophages Targeting Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a critical priority pathogen responsible for chronic infections throughout the body, and is commonly found in the wounds of diabetic patients. Bacteriophages represent a promising alternative antimicrobial strategy against such pathogens. This study isolated and characterized a lytic bacteriophage from river water samples capable of infecting P. aeruginosa InaCC B3, and evaluated its antibacterial and anti-biofilm potential. Plaque morphology analysis revealed a distinctive three-zone bullseye pattern consisting of a central clear lysis zone, a lytic plaque zone, and an outer halo zone, suggesting strict lytic activity and putative extracellular polysaccharide depolymerase activity. In vitro killing curve assays demonstrated significant bacterial growth suppression at multiplicity of infection (MOI) 10 at 8 hours (p < 0.05), while lower MOI levels (1, 0.1, 0.01, 0.001) did not achieve statistical significance. Stability testing against 70% ethanol, 10% povidone-iodine, and 3% hydrogen peroxide resulted in complete phage inactivation across all agents tested, with full viability retained in the saline control. Biofilm inhibition assays demonstrated a consistent MOI-dependent reduction trend, though without statistical significance. Biofilm eradication assays produced a statistically significant reduction in pre-formed biofilm biomass at MOI 10 (p < 0.05). Host range analysis revealed lytic activity against 10 of 15 tested P. aeruginosa strains, including multiple CRPA clinical isolates. These findings establish the isolated phage as a biologically active, moderately broad-spectrum lytic candidate warranting further genomic, structural, and in vivo characterization for therapeutic development against P. aeruginosa infections.