Biochanin A, a Plant Isoflavone, Disrupts Peptidoglycan Biosynthesis by Downregulating femA and femB, and Impairs Cell Wall Integrity in Multidrug-Resistant Staphylococcus aureus

Background/Objectives: The global rise in multidrug-resistant Staphylococcus aureus (MDR-SA) threatens the efficacy of existing antibiotics and necessitates alternative antibacterial strategies. Plant-derived isoflavones represent a promising but underexplored source of novel antimicrobials. Biochanin A, isolated from Cajanus cajan seeds, exhibits antibacterial activity and may act via noncanonical mechanisms. This study elucidates the mechanism of action and safety profile of Biochanin A against MDR-SA using integrated experimental and computational approaches. Methods: Antibacterial activity was assessed by minimum inhibitory concentration (MIC) testing. Membrane integrity and morphological alterations were evaluated using flow cytometry and scanning electron microscopy (SEM), respectively. Target gene modulation was analyzed by qRT-PCR, while molecular interactions were examined through in silico docking. Cytotoxicity was evaluated in normal mammalian kidney, liver, and cardiac cells. Results: Biochanin A inhibited MDR-SA with an MIC80 of 64 µg/mL. Flow cytometry showed membrane disruption in 74.46 ± 13.19% of treated cells, and SEM revealed a 20% reduction in cell size (561.95 ± 21.99 nm). Biochanin A markedly downregulated femA (94%) and femB (67%), with minimal effect on femX (10%). Docking analyses supported preferential binding to FemA (−7.7 kcal/mol) and FemB (−7.5 kcal/mol) proteins. No cytotoxic effects were observed in normal mammalian cells. Conclusions: Biochanin A is a promising plant-derived antibacterial candidate against MDR-SA, targeting key cell wall biosynthesis genes while maintaining mammalian safety. These findings position Biochanin A as a viable lead for further biochemical, structural, and in vivo pharmacological validation, highlighting the translational potential of plant-derived isoflavones in combating antibiotic resistance.