Abstract: Background: α-pinene, a naturally occurring monoterpene found in many essential oils, has demonstrated notable antimicrobial potential. This study aimed to evaluate both the in silico binding interactions and the in vitro antibacterial efficacy of α-pinene against clinical mastitis isolates of Escherichia coli and Staphylococcus aureus. Methods: In silico molecular docking was performed using AutoDock Vina to predict the interaction of α-pinene with key bacterial proteins, including DNA gyrase (PDB ID: 1KZN) and topoisomerase IV (PDB ID: 4PU3) in E. coli, as well as penicillin-binding protein 2 (PBP2, PDB ID: 3UDI) and sortase A (PDB ID: 2W1J) in S. aureus. For in vitro evaluation, α-pinene (≥98% purity, Sigma-Aldrich) was evaluated against three clinical E. coli and three S. aureus isolates obtained from bovine mastitis, with reference strains (E. coli ATCC 25922 and S. aureus ATCC 25923). The minimum inhibitory concentrations (MICs) were determined by the broth microdilution as CLSI guidelines, and time-kill assays were performed at MIC/2, MIC, and 2MIC concentrations. Results: Docking results showed strong affinity of α-pinene for bacterial targets, with binding energies ranging from -5.8 to -6.9 kcal/mol for E. coli proteins and -6.1 to-7.3 kcal/mol for S. aureus targets, indicating stable interactions through hydrophobic and van der Waals forces. In vitro, α-pinene exhibited MIC values of 256-512 µg/ml for E. coli isolates and 128-256 µg/ml for S. aureus isolates. Time-kill study revealed concentration-dependent bactericidal effects: at 2× MIC, bacterial counts decreased by 3.2-3.8 log10 CFU/ml after 24 h for S. aureus, and by 2.5-3.0 log₁₀ CFU/ml for E. coli. Conclusion: α-pinene displayed favorable binding to essential bacterial enzymes and notable antibacterial activity against both Gram-positive and Gram-negative bovine mastitis isolates. These findings suggest that α-pinene may serve as a promising natural antimicrobial candidate or adjuvant for controlling mastitis-associated pathogens.
