Background: Within the mammary gland, S. aureus establishes biofilm communities that contribute to treatment failure, recurrence and increased antimicrobial use in bovine mastitis. Strategies that disrupt biofilm-associated survival mechanisms may therefore improve therapeutic outcomes while supporting antimicrobial stewardship. 
 
Methods: In this study, we integrated robust computational screening of seventeen biofilm-associated S. aureus proteins implicated in adhesion, extracellular matrix production and transcriptional regulation against selected plant-derived polyphenolic compounds, followed by in vitro and in vivo validation to identify adjunctive therapeutic strategies for mastitis management. 
 
Result: Reserpine emerged as a multi-target candidate, with predicted interactions across key biofilm determinants, including adhesins and regulatory proteins. In vitro, reserpine significantly reduced biofilm biomass (68.6-71.5% inhibition) and decreased viable bacterial counts by &gt;95%, indicating disruption of both biofilm matrix integrity and bacterial persistence. In a murine mastitis model, reserpine monotherapy modestly reduced intramammary bacterial load. However, when combined with ceftiofur, bacterial burden declined by approximately three log units relative to untreated controls, accompanied by reduced inflammatory infiltration and improved preservation of mammary architecture. Targeting intrinsic persistence pathways alongside conventional antibiotic therapy may present a clinically relevant approach for improving therapeutic outcomes in biofilm driven bovine mastitis while reducing reliance on escalating antimicrobial use in dairy systems.

Therapeutic Evaluation of Reserpine against Biofilm-forming Staphylococcus aureus of Bovine Mastitis in a Murine Model