Takuma Yoshida The key markers of Staphylococcus aureus membrane vesicles (MVs) are:

• Immunostimulatory DNA, RNA, and peptidoglycan that activate innate immune receptors like Toll-like receptors (TLR2, TLR7, TLR8, TLR9) and NOD2
• Small RNAs (sRNAs) detected in the RNA content of S. aureus MVs.
• Variable protein cargo composition, with 131 proteins identified in MVs from S. aureus grown in Luria-Bertani broth and 617 proteins in MVs from brain-heart infusion broth.
• The number, morphology, and cargo composition of S. aureus MVs can vary depending on the bacterial strain and growth conditions.

To ensure your culture medium contains S. aureus MVs, you should:

1. Use either Luria-Bertani broth or brain-heart infusion broth as the growth medium.

2. Analyze the culture supernatant for the presence of spherical nanovesicles by electron microscopy.

3. Detect the immunostimulatory nucleic acids (DNA, RNA) and peptidoglycan in the vesicle fraction using PCR and immunoassays.

4. Identify the variable protein cargo composition by mass spectrometry proteomics.

The presence of these markers will confirm that your S. aureus culture is producing membrane vesicles that can modulate host immune responses and bacterial pathogenesis.