MVs Extraction

The MVs extraction from L. reuteri was performed on biofilm and planktonic cultures as previously reported (Grande et al., 2015). Briefly, L. reuteri biofilms, scraped and suspended in PBS, were centrifuged (5000 × g, 20 min at 4°C) and the resultant supernatants were filtered through 0.22 μm cellulose membrane filters (Corning, United States). Two hundred microliters of both planktonic and biofilm filtrates were spread on MRS agar and incubated at 37°C on anaerobic conditions to confirm the total absence of L. reuteri colonies. The samples were further purified using a Beckman coulter Optima XL – 100K ultracentrifuge (Beckman coulter, United States) at 50000 rpm, for 2 h at 4°C, washed with PBS and ultra-centrifuged for the second time (50000 rpm, 2 h at 4°C). The pellets were then dissolved in 200 μl PBS and stored both at -80 and 4°C. To visualize pMVs and bMVs, samples were negative stained and analyzed through a Transmission Electron Microscopy (TEM). Briefly, a drop of vesicles suspension was placed onto a formvar–carbon–coated grid (Electron Microscopy Sciences, Hatfield, United Kingdom), and negatively stained with phosphotungstic acid solution (1% v/v). Samples were then analyzed with a Philips 208 TEM (2–120 kV, 480,000×) (FEI, Eindhoven, Netherlands).