This study aimed to investigate the effects of dietary bacteriophage administration on diarrhea and intestinal impairment induced by enterotoxigenic Escherichia coli (ETEC) in a newly weaned mouse model. Forty-four newly weaned C57BL/6 mice were divided into four treatment groups, where they were provided either the control diet or the bacteriophage-supplemented diet, with or without ETEC infection. The results show that the bacteriophage administration resulted in increased body weight, decreased diarrhea score, and improved jejunal histopathology in ETEC-infected mice. The bacteriophage administration enhanced the intestinal barrier function of the ETEC-infected mice, as indicated by the reduced serum DAO level and the increased expression of Claudin-1, Occludin, and ZO-1 at both the mRNA and protein levels in the jejunum. Also, the bacteriophage administration resulted in a decrease in serum TNF-α and IL-1β levels, a down-regulation of TNF-α and IL-6 mRNA levels in the jejunum, and the inhibition of jejunal TLR-4/NF-κB pathway activation induced by ETEC infection. Moreover, the bacteriophage administration increased the levels of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in the caecum content. The bacteriophage administration increased the Shannon index, increased the abundance of Bacteroidota and Muribaculaceae, and decreased the abundance of Verrucomicrobiota and Akkermansiaceae in the colon contents of the ETEC-infected mice. Spearman’s correlation analysis indicates that the protective effects of bacteriophage on ETEC-induced intestinal impairment, inflammation, and intestinal barrier function are associated with regulating the abundance of Bacteroidota and Muribaculaceae in the colon contents of mice. Collectively, bacteriophage administration alleviates ETEC-induced diarrhea and intestinal impairment through regulating intestinal inflammation and gut microbiota in newly weaned mice.

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.