Antibacterial Activity of Rosmarinic Acid Against Streptococcus agalactiae and Its Anti-Inflammatory Effects in Mouse Mastitis

Mastitis severely hinders dairy farming, with Streptococcus agalactiae (S. agalactiae) serving as a major causative pathogen. Prolonged antibiotic administration promotes the emergence of multidrug-resistant strains, thereby compromising mastitis control. Consequently, novel natural antibacterial agents are urgently needed. In this study, bacteriostatic assays identified the MIC of rosmarinic acid (RA) against S. agalactiae as 8 mg/mL. At the MIC, RA reduced the adhesion capacity of S. agalactiae by 16.6% and inhibited biofilm formation by 25.09%. RA also disrupted bacterial membrane architecture and markedly downregulated the expression of virulence factors. A mouse model of mastitis treated with varying doses of RA (25, 50, and 100 mg/kg) indicated that RA attenuated pathological damage to the mammary duct architecture and ameliorated inflammatory responses in S. agalactiae-infected mice. ELISA revealed that RA significantly reduced pro-inflammatory mediators, including IL-1α, IL-1β, IL-6, and TNF-α. RT-qPCR results indicated that mRNA expression levels of TLR2, NF-κB, AKT, and PI3K were substantially reduced following RA treatment, whereas apoptosis-related genes p53, RUNX1, and Bim exhibited distinct expression patterns. Western blotting analysis further indicated that RA modulated the phosphorylation of Bim, RUNX1, and p53 in mastitis tissues, suppressed the TLR2/NF-κB inflammatory signaling pathway, and regulated the PI3K/AKT cascade. By targeting multiple pathways, RA suppressed S. agalactiae proliferation and attenuated mammary gland inflammation, highlighting its potential as a green therapeutic agent for sustainable animal husbandry.