Gramicidin D as a Multi-Target Antimicrobial Against Staphylococcus aureus: Biofilm Disruption, Virulence...

Over the past decades, the threat of Staphylococcus aureus and multidrug-resistant S. aureus has escalated, creating an urgent need for novel therapeutic strategies that possess both bactericidal and antivirulence properties. We focused on gramicidin D, a conventional pore-forming antibiotic derived from Bacillus brevis, as a multi-target agent. The minimum inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC₅₀) against several Gram-positive pathogens were determined by broth microdilution assay. Furthermore, the combinatorial effect with other conventional antibiotics was also assayed via the checkerboard method. The effects on biofilm formation and mature biofilm were evaluated using the crystal violet staining method, supported by colony-forming unit (CFU) counts. Additionally, inhibition of staphyloxanthin biosynthesis was quantified by methanol extraction, followed by measurement of optical density via a microplate reader. Finally, in silico protein-peptide docking was performed to support and analyze the properties of the peptide. Gramicidin D demonstrated significant antibacterial efficacy against S. aureus and methicillin-resistant S. aureus (MRSA), with corresponding MIC values of 8 and 0.25 µg/mL, respectively. Moreover, gramicidin exhibited synergistic effects with oxacillin and gentamicin against S. aureus; however, it showed synergy with oxacillin only against MRSA. It inhibited S. aureus biofilm formation at sub-MICs, especially at 0.125 µg/mL, where 80.0% of biofilm was inhibited. Furthermore, it effectively reduced established biofilm at concentrations ranging from 128 to 4 µg/mL. Additional SEM images showed that membrane pore formation by gramicidin D was supported. It also inhibited staphyloxanthin synthesis at all concentrations ≥ 1 µg/mL. HDOCK server-based protein-peptide docking provided structural support for the in vitro experiments. These findings altogether illustrated the potential of gramicidin D as a multi-target agent against S. aureus.