Kurarinone and sophoraflavanone G from Sophora flavescens suppress Streptococcus agalactiae biofilm via dual antibiofilm mechanisms involving adhesion protein binding and ABC transporter pathway perturbation.

INTRODUCTION: Biofilm formation by Streptococcus agalactiae (S. agalactiae) is a major driver of chronic bovine mastitis that resists conventional antibiotic treatment, yet targeted strategies that disarm its biofilm machinery remain limited. Sophora flavescens (S. flavescens) is a medicinal plant rich in prenylated flavonoids with antimicrobial properties, but the antibiofilm mechanisms of these compounds against S. agalactiae remain unexplored. METHODS: The antibacterial activity of 15 prenylated flavonoids from S. flavescens was evaluated against major mastitis-associated bacteria, and minimum inhibitory concentrations (MIC₉₀) were determined. Biofilm inhibition was assessed by crystal violet and acridine orange staining. Molecular docking, quantitative real-time PCR (qPCR), untargeted metabolomics, and phenotypic validation assays were integrated to elucidate the antibiofilm mechanisms. A mouse mastitis model was employed to evaluate in vivo efficacy, and cytotoxicity and acute toxicity assays were performed to assess safety. RESULTS: Kurarinone and sophoraflavanone G exhibited potent antibacterial activity against S. agalactiae, Streptococcus dysgalactiae, and Staphylococcus aureus, with MIC₉₀ values of 3.12 μg/mL against S. agalactiae, superior to norfloxacin (6.25 μg/mL). Mechanistically, these compounds acted through two complementary pathways: (1) strong binding affinity to the adhesion-related proteins FbsA, FbsB, FbsC, and Lmb in molecular docking analysis, with transcriptional downregulation of their encoding genes confirmed by qPCR, and (2) perturbation of the ATP-binding cassette (ABC) transporter pathway, as suggested by untargeted metabolomics, leading to markedly reduced secretion of extracellular polysaccharides (by up to 60%), capsular polysaccharides, and extracellular DNA. Notably, this dual antibiofilm activity occurred at sub-MIC concentrations, indicating an antibiofilm mechanism that operates independently of direct bacterial killing and is distinct from the previously reported membrane-disruptive effects of these compounds. In a mouse mastitis model, intragastric administration of both compounds significantly ameliorated mammary tissue histopathology, reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8), and decreased myeloperoxidase activity, with favorable safety profiles (LD₅₀ > 2000 mg/kg). CONCLUSION: These findings suggest that adhesion proteins and the ABC transporter pathway represent potential dual targets against S. agalactiae biofilms, and position kurarinone and sophoraflavanone G as promising leads for antibiofilm drug development targeting biofilm-associated bovine mastitis.