Dual-stage assessment of Salmonella-specific bacteriophage formulation: Antibiofilm activity on food matrices and in vivo efficacy against the murine salmonellosis model.

Foodborne infections caused by Salmonella infection remain a major global concern due to increasing multidrug resistance and biofilm formation, resulting in significant morbidity and mortality. Thus, the development of potential alternatives, including bacteriophage cocktail formulations, is emerging as a promising strategy. In this direction, we developed a Salmonella-specific phage formulation (BPF-Sal) and evaluated its stability, biocontrol efficacy, in vitro safety, antibiofilm activity and protective potential in an in vivo model. Interestingly, BPF-Sal remained stable across a wide range of pH values and temperatures while maintaining significant lytic activity. Further, it effectively reduced Salmonella contamination on chicken breast and mixed fruit matrices to below detection limits (<1 CFU/100 μL) within 6 h and 10 h, respectively, compared to conventional preservatives. In HT-29 cells, BPF-Sal (102-101⁰ PFU/mL) exhibited no cytotoxicity, preserved cellular morphology, and showed efficient phage internalization. It also displayed antibiofilm activity, reducing preformed Salmonella biofilms by 90-92% at MOI 100 and up to 98% at MOI 1000, as confirmed by crystal violet assay, scanning electron and fluorescence microscopy. In a murine salmonellosis model, oral administration of BPF-Sal conferred significant protection, preventing weight loss and reducing bacterial loads along with improved health status and histopathological outcomes. Metagenomic analysis revealed infection-induced gut dysbiosis, characterized by enrichment of Proteobacteria and depletion of beneficial taxa. BPF-Sal partially restored microbial balance, while combination therapy further improved microbiota normalization. Thus, our findings establish BPF-Sal as a safe, effective, multifunctional phage-based strategy for Salmonella biocontrol and other phage-based applications.