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

## TL;DR

Milk extracellular vesicles can inhibit Staphylococcus aureus growth and biofilm formation, offering a natural treatment for bovine mastitis.

## Contribution

The study identifies milk-derived extracellular vesicles as potential residue-free antimicrobial agents for bovine mastitis.

## Key findings

- Milk-derived extracellular vesicles inhibit Staphylococcus aureus growth and disrupt oxidative balance.
- Mastitic milk-derived vesicles reduce biofilm formation and expression of biofilm-related genes.
- Mastitic milk vesicles are enriched in miRNAs predicted to target biofilm-associated genes.

## Abstract

Bovine mastitis is a common and costly disease in dairy cows, caused by Staphylococcus aureus, which is hard to treat due to its ability to form biofilms and resist antibiotics. In this study, we explored the potential of milk-derived extracellular vesicles as natural antimicrobial agents. We compared exosomes from healthy and mastitic milk and found that both inhibited bacterial growth and were associated with disrupted oxidative balance, with stronger effects from mastitic milk. These vesicles also reduced biofilm formation and were associated with lower expression of biofilm-related genes. Small RNA sequencing suggested that vesicular miRNAs might be involved in these effects, and mastitic milk-derived vesicles were enriched in miRNAs predicted to target biofilm-associated genes; however, bacterial uptake and direct functional regulation of these miRNAs were not assessed in this study. Our findings indicate that milk-derived extracellular vesicles have potential as residue-free adjuncts for controlling bovine mastitis, but these conclusions are based on in vitro data and require further validation in animal models before clinical application.

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.