Butenolide synergises with vancomycin to eradicate pre-formed biofilm of <i>Staphylococcus aureus</i> by interfering with energy-associated metabolism

Bacterial biofilms significantly contribute to persistent infections and the emergence of drug resistance of <i>Staphylococcus aureus</i>. Integrating conventional antibiotics with antibiofilm agents represents a promising strategy for combating biofilm-associated infections. This study systematically investigated the antibiofilm activity and underlying mechanisms of butenolide (BU) against methicillin-resistant <i>S. aureus</i> (MRSA), with a focus on the synergistic effects between BU and vancomycin (VAN). BU exhibited dual antibiofilm activities by efficiently preventing biofilm formation and eradicating established biofilms. Phenotypic characterisation revealed that 200 μg/mL of BU suppressed extracellular DNA production and autoaggregation of MRSA, leading to a significant reduction in biofilm thickness, biovolume, and coverage by up to 30%, 98%, and 96%, respectively. Transcriptome and quantitative-PCR analyses showed that BU treatment downregulated the expression of genes involved in energy metabolism. Notably, BU exhibited promising synergistic and additive effects with VAN in eradicating pre-formed biofilms, achieving synergy or additivity in five out of six <i>S. aureus</i> clinical strains tested, with a minimal fractional inhibitory concentration index as low as 0.375. These results highlight the potential of BU as an effective antibiofilm agent for preventing <i>S. aureus</i>-related infections.