Synergistic effects of two bacteriophages with distinct infection patterns and broad host specificity against multidrug-resistant Salmonella Typhimurium and their potential applications in the poultry industry

Controlling Salmonella Typhimurium in poultry requires effective alternatives to conventional antimicrobials. This study evaluated the efficacy of a two-phage cocktail composed of S. Typhimurium-infecting phages SLAM_phiST45 and SLAM_phiST56 and investigated its mechanistic basis. Host-range profiling of 61 phages against 10 S. Typhimurium strains revealed two major host-associated clusters (swine-human vs. poultry). Notably, the cocktail exhibited broad host ranges that crossed these clusters. The cocktail showed clear synergy, supported by complementary receptor usage and reduced emergence of phage-resistant mutants. In egg-based food application assays, the cocktail achieved rapid bacterial suppression even at an MOI of 1 and fully eliminated S. Typhimurium under refrigerated conditions. In a chick infection model, the cocktail improved growth performance, reduced intestinal and systemic bacterial loads, alleviated intestinal lesions, and restored inflammatory and barrier-related gene expression. While the overall gut microbiota structure remained stable, subtle alterations in low-abundance taxa indicated that phage treatment may cause minor off-target shifts. Overall, this study demonstrates that the phage cocktail is a potent and practical biocontrol tool capable of reducing Salmonella in both food matrices and live birds. These findings support its application in poultry production and highlight the importance of monitoring microbiome-level impacts in future phage-based interventions.