Phage Administration Protects Against Staphylococcus aureus-Induced Mastitis Through Direct Pathogen Lysis and Subsequent Alleviation of Host Inflammatory Responses in a Murine Model

Staphylococcus aureus (S. aureus)-induced mastitis poses a severe threat to the global dairy industry and raw milk safety. This study aimed to evaluate the protective effects of lytic phage JXAU-SA-P1, recently isolated in our laboratory, against S. aureus-induced mastitis in mice and to elucidate its underlying mechanisms. A total of 42 lactating BALB/c mice (7–10 days postpartum) were allocated into seven groups (n = 6) to assess bacterial reduction, tissue pathology, and host immune responses following intramammary S. aureus challenge. The results showed that JXAU-SA-P1 significantly reduced S. aureus burdens and alleviated mammary histopathological damage, with better efficacy than antibiotic therapy. Safety assessments showed that JXAU-SA-P1 was steadily cleared in the absence of host bacteria without triggering inflammation response. Transcriptomic profiling identified 448 overlapping differentially expressed genes (DEGs) modulated by high-dose phage administration. Gene set enrichment analysis (GSEA) revealed that phage administration suppressed major pro-inflammatory pathways, including the TNF, NF-kappa B, and IL-17 signaling pathways. Core regulatory hub genes, including IL-1β, Ptprc, Itgam, Fcgr3, and Itgb2, were identified through protein–protein interaction network analysis. Subsequent ELISA confirmed significant reductions in pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), the neutrophil infiltration marker myeloperoxidase (MPO), and serum immunoglobulins (IgA, IgM, and IgG). In conclusion, phage JXAU-SA-P1 effectively alleviated mastitis through direct pathogen lysis and subsequent alleviation of the host inflammatory response, providing a strong experimental foundation for the development of phage-based products as safe, antibiotic-free biocontrol agents for dairy production.