Biofilm-Associated Agr and Sar Quorum Sensing Systems of <i>Staphylococcus aureus</i> Are Inhibited by 3-Hydroxybenzoic Acid Derived from <i>Illicium verum</i>

Abstract

Biofilm-producing <i>Staphylococcus aureus</i> (<i>S. aureus</i>) is less sensitive to conventional antibiotics than free-living planktonic cells. Here, we evaluated the antibiofilm activity of <i>Illicium verum</i> (<i>I. verum</i>) and one of its constituent compounds 3-hydroxybenzoic acid (3-HBA) against multi-drug-resistant <i>S. aureus</i>. We performed gas chromatography-mass spectroscopy (GC-MS) to identify the major constituents in the methanolic extract of <i>I. verum</i>. Ligand-receptor interactions were studied by molecular docking, and <i>in vitro</i> investigations were performed using crystal violet assay, spreading assay, hemolysis, proteolytic activity, and growth curve analysis. The methanolic extract of <i>I. verum</i> inhibited <i>S. aureus</i> at 4.8 mg/mL, and GC-MS analysis revealed anethole, m-methoxybenzaldehyde, and 3-HBA as the major constituents. Molecular docking attributed the antibiofilm activity to an active ligand present in 3-HBA, which strongly interacted with the active site residues of AgrA and SarA of <i>S. aureus</i>. At a subinhibitory concentration of 2.4 mg/mL, the extract showed biofilm inhibition. Similarly, 3-HBA inhibited biofilm activity at 25 μg/mL (90.34%), 12.5 μg/mL (77.21%), and 6.25 μg/mL (62.69%) concentrations. Marked attrition in bacterial spreading was observed at 2.4 mg/mL (crude extract) and 25 μg/mL (3-HBA) concentrations. The methanol extract of <i>I. verum</i> and 3-HBA markedly inhibited β-hemolytic and proteolytic activities of <i>S. aureus</i>. At the lowest concentration, the <i>I. verum</i> extract (2.4 mg/mL) and 3-HBA (25 μg/mL) did not inhibit bacterial growth. Optical microscopy and SEM analysis confirmed that <i>I. verum</i> and 3-HBA significantly reduced biofilm dispersion without disturbing bacterial growth. Together, we found that the antibiofilm activity of <i>I. verum</i> and 3-HBA strongly targeted the Agr and Sar systems of <i>S. aureus</i>.