Milk-Derived Extracellular Vesicles Inhibit Staphylococcus aureus Growth and Biofilm Formation

Staphylococcus aureus is a key pathogen in bovine mastitis, and antibiotic therapy is challenged by resistance and residue concerns. Milk-derived extracellular vesicles emerge as promising natural antimicrobials. This study aimed to evaluate the antimicrobial activity and explore potential associated mechanisms of milk-derived extracellular vesicles against S. aureus. Milk-derived EV-enriched fractions (mEVs) from healthy (HmEVs) and mastitic (MmEVs) bovine milk suppressed S. aureus growth in vitro and were associated with oxidative imbalance, with MmEVs showing stronger inhibition. In addition, MmEVs significantly reduced biofilm biomass, extracellular matrix production, and the expression of key biofilm-associated genes (sarA, icaB, fnbA, clfB, cidA). Small RNA sequencing revealed distinct miRNA profiles between HmEVs and MmEVs; in particular, MmEVs were enriched in miRNAs predicted to target the S. aureus biofilm-associated gene clfB. Although we did not directly demonstrate uptake of mEV-derived miRNAs by bacteria or their regulation of bacterial gene expression in this study, our small RNA sequencing data together with subsequent bioinformatic predictions suggest that vesicular miRNAs should be regarded as candidate contributors, rather than demonstrated mediators, of the observed antibacterial and antibiofilm effects. Taken together, these findings indicate the potential of mEVs as residue-free adjuncts for controlling bovine mastitis, while recognizing that the present conclusions are mainly derived from in vitro experiments with S. aureus and bioinformatic analyses. Therefore, functional validation of candidate miRNAs, in vivo studies, and evaluation of activity against other mastitis-associated pathogens are still required to clarify the underlying mechanisms, therapeutic potential, and spectrum of activity of mEVs.