The Inhibitory Effect of Mannosylerythritol Lipids on <i>Staphylococcus aureus</i> Biofilm via the Agr Quorum-Sensing System

Abstract Mannosylerythritol lipids (MELs) are glycolipid biosurfactants with pronounced anti-biofilm activity, yet their molecular mechanism of action remains incompletely understood. To identify regulatory pathways involved in MEL-mediated biofilm inhibition, we first conducted comparative transcriptomic analysis of Staphylococcus aureus biofilms exposed to MELs, which identified the quorum-sensing (QS) response regulator agrA as a key transcriptionally repressed component. Functional validation using an isogenic ΔagrA mutant demonstrated that loss of agrA markedly attenuated the anti-biofilm efficacy of MELs. In wild-type (WT) biofilms, MELs caused a significant reduction in biomass (approximately 59.1% on average) and embedded cell viability (up to 1.59 log CFU/mL at 512 µg/mL), accompanied by pronounced disruption of extracellular polymeric substances (EPS). In contrast, these effects were substantially weakened in the ΔagrA mutant. The agrA-dependent inhibitory effect of MELs was consistently maintained across food-relevant conditions, including varied temperatures, stainless steel surfaces, and milk matrices. This regulatory dependency further persisted in mixed-species biofilms formed with Bacillus cereus, indicating that agrA represents a stable vulnerability under both simplified and ecologically complex conditions. Subsequent transcriptomic and pathway enrichment analysis revealed that agrA mediates structured yet context-dependent transcriptional responses to MELs exposure. Core virulence-associated pathways, including QS and S. aureus infection, were consistently downregulated in both mono- and mixed-species biofilms. In contrast, ribosome-related pathways were selectively upregulated in mono-species biofilms, whereas mixed-species biofilms exhibited a distinct metabolic reprogramming characterized by enhanced energy-supply pathways. Collectively, these findings demonstrate that MELs suppress S. aureus biofilm formation in an agrA-dependent manner, with agrA serving as a central regulatory node, resulting in conserved virulence repression alongside ecologically driven metabolic remodeling.