The anti-biofilm mechanism of reuterin against Staphylococcus aureus and its potential application on milk-contact material surfaces

Reuterin, a potent antimicrobial compound derived from Limosilactobacillus reuteri , was investigated for its mechanism of action against Staphylococcus aureus biofilm and its efficacy in eliminating biofilms on four distinct milk-contact materials. Results showed that reuterin exhibited a minimum biofilm inhibitory concentration of 0.16 mg/mL against S. aureus ATCC 29213 after treatment for 48 h at 37 °C. Reuterin significantly reduced bacterial hydrophobicity, auto-aggregation and biofilm biomass by 39.36%, 25.49%, and 93.46%, respectively, effectively impeding biofilm formation and bacterial adhesion activity. For the mature biofilm, the minimum biofilm eradication concentration of reuterin incubated for 8 h at 37 °C was 0.31 mg/mL. At this concentration, reuterin significantly decreased the levels of extracellular proteins, polysaccharides and DNA of the biofilm as assayed by the BCA method, the phenol-sulfuric acid test and the spectrophotometric assay. Metabolic activity within the biofilm was reduced by 47.95% determined by the resazurin method, while the viable bacteria was not detected by plate counting. Laser confocal microscopy further confirmed the disruptive effects of reuterin on biofilm architecture. Moreover, RT-qPCR analysis showed that reuterin inhibited the relative expression of the accessory regulator ( sarA ) and bacterial adhesion factors ( clfA, clfB, fnbA, fnbB ). Additionally, reuterin was also found to interfere quorum-sensing genes within S. aureus , particularly targeting the LuxS/AI-2 system ( icaR, icaA, icaB, icaC, icaD ) and the agr system ( agrA, agrB, RNAIII ). Finally, reuterin eliminated biofilms formed on four milk-contact materials, including glass, stainless steel, polyvinyl chloride, and acrylonitrile-butadiene-styrene. Conclusively, reuterin emerges as a highly promising anti-biofilm agent in the food industry.