Isolation and Characterization of Phage Cocktails (Phps1 + Phps4) with Broad Lytic Activity Against Multidrug-Resistant <i>Pseudomonas aeruginosa</i> Strain PSM01, Isolated from Dairy Products

Abstract

Background: Bacteriophages are viruses that specifically target bacteria and offer a promising strategy to control resistant and spoilage-causing strains in dairy products, such as Pseudomonas spp. Their application can improve food safety, extend shelf life, and provide a natural alternative to chemical preservatives and antibiotics. Objectives: In this study, 100 milk and cream samples were aseptically collected and screened for Pseudomonas spp. using biochemical tests and 16S rRNA analysis. Methods: Five strains were isolated, showing high resistance to multiple antibiotics and variable susceptibility to meropenem and amikacin. Three isolates were further analyzed by 16S rRNA sequencing to confirm their phylogenetic relationships with reference Pseudomonas spp. Six lytic bacteriophages were isolated from wastewater and characterized by transmission electron microscopy (TEM). Phages were evaluated for host range, one-step growth, multiplicity of infection (MOI)-dependent efficacy, thermal and pH stability, and lytic activity. Results: Six distinct lytic bacteriophages targeting Pseudomonas spp. were successfully isolated from wastewater and designated Phps1, Phps2, Phps3, Phps4, Phps01, and Phps04. Phages Phps1, Phps3, and Phps4 exhibited broad host ranges and strong lytic activity, while others were more strain-specific. Individual phages and phage cocktails were tested in pasteurized milk at 4°C and 25°C. Phps01 significantly reduced bacterial counts at 4°C, though efficacy declined at 25°C. Phage cocktails, particularly combinations such as Phps1 + Phps4, displayed very strong lytic activity against all tested strains. Conclusions: These results demonstrate that bacteriophages, especially in cocktail formulations, are effective biocontrol agents against Pseudomonas spp. in dairy products. Their stability under various conditions and strong antibacterial activity highlight their potential as natural, safe, and efficient tools to enhance dairy quality, extend shelf life, and mitigate antibiotic-resistant spoilage bacteria.