Comprehensive Evaluation of Capparis Spinosa-Derived γ-Sitosterol Against Methicillin-Resistant Staphylococcus aureus...

Methicillin-resistant Staphylococcus aureus (MRSA) represents a major bacterial pathogen due to its resistance to β-lactam antibiotics and its strong biofilm-forming ability. This study evaluated the antibacterial, antibiofilm, and antivirulence activity of γ-Sitosterol, identified as the major bioactive constituent isolated from Capparis spinosa, specifically against MRSA. Gas chromatography-mass spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FT-IR) analyses confirmed γ-Sitosterol as the major constituent in C. spinosa extracts. Antimicrobial assays revealed 150 µg/mL minimum inhibitory concentration (MIC) and 320 µg/mL minimum bactericidal concentration (MBC) of γ-Sitosterol. Time-kill kinetics demonstrated complete bacterial eradication within 12 h. γ-Sitosterol significantly inhibited MRSA biofilm formation (86%), exopolysaccharide synthesis (EPS) (73%), and virulence factors such as hemolysin and catalase. Scanning electron microscopy (SEM) and fluorescence imaging revealed substantial membrane disruption in biofilm cells. Quantitative polymerase chain reaction (qPCR) analysis showed marked downregulation of Saes, glyB, and aur genes, corroborating its antibiofilm and antivirulence effects. Molecular docking confirmed high binding affinity of γ-Sitosterol to key virulence proteins, suggesting a direct inhibitory mechanism. Furthermore, γ-Sitosterol exhibited strong antioxidant activity across 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), ferric reducing antioxidant power (FRAP), and hydrogen peroxide (H₂O₂) scavenging assays. These findings support further in vivo validation and molecular dynamics simulations to explore membrane-associated protein interactions and broaden its potential clinical application in combatting multidrug-resistant pathogens.