Synergistic therapeutic effect of isochlorogenic acid (ICGA) combined with the bacteriophage lyase Lys40 against Salmonella-induced mastitis: In vitro and in vivo evaluations

Background Bovine mastitis caused by Salmonella spp. presents a significant challenge to the dairy industry because of increasing antimicrobial resistance (AMR) and the risk of antibiotic residues in milk. Bacteriophage-derived endolysins are promising alternatives, but their efficacy against gram-negative bacteria is often limited by the outer membrane (OM) barrier. Isochlorogenic acid (ICGA), a plant-derived polyphenol, has membrane-permeabilizing properties. This study investigated the synergistic antibacterial activity of ICGA combined with the recombinant phage lyase Lys40 against Salmonella and its therapeutic potential in mastitis models. Methods The synergistic effect was evaluated via the checkerboard method to determine the fractional inhibitory concentration index (FICI). The antibacterial spectrum was assessed against 24 mastitis-associated isolates. In vitro efficacy was determined via the use of a Salmonella -infected bovine mammary epithelial cell (bMEC) model, in which cell viability (CCK-8), lactate dehydrogenase (LDH) release, bacterial adhesion/invasion, and cellular morphology were analyzed. In vivo therapeutic effects were evaluated in a Salmonella -induced murine mastitis model via histopathology, bacterial load quantification, and immunohistochemical analysis of apoptotic markers (Caspase-3, Caspase-9, and Cyt-c). Results Lys40 alone showed weak activity against Salmonella CVCC-529 (MIC > 1 mg/mL). However, combination with ICGA significantly reduced the MIC of Lys40 to 0.200 mg/mL (FICI = 0.45), indicating strong synergy, particularly at pH 8–9. The combination demonstrated broad-spectrum activity against both gram-negative (e.g., E. coli and Salmonella ) and gram-positive pathogens (e.g., S. aureus ). In bMECs, the combination significantly inhibited bacterial adhesion and invasion (p < 0.05), reduced LDH release, and preserved cellular morphology. Compared with the untreated infection, the combined treatment significantly decreased the mammary tissue bacterial load and histopathological damage in the murine model (p < 0.01). Furthermore, the treatment downregulated the expression of proapoptotic proteins (caspase-3, caspase-9, and Cyt-c), protecting mammary tissue from apoptosis. Conclusions The combination of ICGA and Lys40 exerts a potent synergistic effect by overcoming the outer membrane barrier, suggesting a promising antibiotic-free therapeutic strategy for controlling Salmonella -induced mastitis.