A two-phage cocktail targeting distinct receptors effectively controls Salmonella in milk and inhibits biofilm formation

Salmonella is an important foodborne pathogen. Bacteriophages (phages) have been considered as potential antimicrobials to control such pathogens. However, the use of phages as bactericidal alternatives to antibiotics can be problematic due to the emergence of resistant strains. To effectively monitor and prevent this resistance, a thorough investigation of phage resistance mechanisms is essential. A thymine deletion at position 77 of the Ug gene in Salmonella phage PH219-resistant mutant ZWSA409-C1 abolishes O-antigen synthesis, conferring phage resistance. This mutation reduces thermotolerance and antiphagocytosis while enhancing cell adhesion and antibiotic resistance. Unlike PH219, phage PH204 recognizes a non-LPS receptor and effectively lyses the Ug-deleted strain. A cocktail of PH219 and PH204 broadens the host range to cover Salmonella Typhimurium, I 4,[5],12:i:-, Gallinarum, Enteritidis, and Mbandaka, inhibits biofilm formation, and maintains bacteriostatic activity in milk and in vitro. This study highlights the trade-offs associated with phage resistance and presents a dual-phage strategy to overcome resistance while controlling Salmonella contamination in food systems.