3-Hydroxyflavone Inhibits Biofilm Formation and Enhances Antibiotic Activity Against Enterococcus faecalis and Enterococcus faecium.

Background/Objectives: The increasing antibiotic resistance of pathogenic bacteria has created an urgent need for new therapies that target virulence factors. Biofilm formation is a major determinant of bacterial pathogenicity and antimicrobial resistance. Methods: Biofilm formation assays (using crystal violet staining) were performed in independent triplicates to evaluate the biofilm-inhibitory activity of 3-Hydroxyflavone. A bacterial viability assay (colony-forming unit counting) was used to assess the enhanced antibiotic activity, and qRT-PCR analysis was conducted to investigate the transcriptional modulation of biofilm-related genes. Results: 3-Hydroxyflavone inhibited biofilm formation in E. faecalis (IC50 = 1.025 μg/mL), E. faecium (IC50 = 0.19 μg/mL), S. aureus (IC50 = 1.21 μg/mL), C. acnes (IC50 = 0.132 μg/mL), S. sobrinus (IC50 = 5.49 μg/mL), P. aeruginosa (IC50 = 22.1 μg/mL), and E. coli (IC50 = 10.88 μg/mL). Notably, these effects were observed without significant inhibition of planktonic bacterial growth, indicating a biofilm-specific mechanism of action. 3-Hydroxyflavone also downregulated the expression of quorum-sensing genes (FsrB, FsrC, GelE, EbpA, EbpB, Acm, Scm, and Bps), the biofilm virulence gene Esp, and cytolysin genes (CylLs, CylR, and CylM), while exhibiting strong enhanced antibiotic activity in the viability assay. Conclusions: These in vitro findings suggest that 3-Hydroxyflavone may serve as a promising antibacterial adjuvant for controlling biofilm-associated bacterial infections. However, further studies, including in vivo validation, are required to determine its clinical applicability.