Stainless steel bead

Total DNA was isolated from EF samples by performing a pre-digestion step at 37 °C for 30 min with 25 ug/μL lysozyme, 0.12 U/μL lysostaphin, 0.4 U/μL mutanolysin and 1.8% Triton X-100 to degrade the bacterial cell walls. DNA was then extracted with the QIAamp DNA Blood Mini kit (Qiagen) following the manufacturer’s instructions. Finally, the DNA was eluted with 35 μL of nuclease free water and quantified using a photometric technology (Nanodrop, Waltham, MA, USA).
Total DNA was isolated from the EB samples by performing a pre-digestion step for difficult-to-lyse bacteria. For this digestion, 25 mg of tissue was cut into small pieces and treated with proteinase K at 56 °C for 3 h under agitation. Samples were then mixed with ATL buffer and disrupted mechanically in a TissueLyser LT for 5 min at 50 Hz using stainless-steel beads (all acquired from Qiagen). After these pretreatments, bacterial nucleic acids were purified using the DNA tissue programme V7-200-LC of the QIAsymphony (Qiagen) following the manufacturer’s instructions. Finally, the DNA was eluted with 50 μL of nuclease free water and quantified using MultiskanGO (Thermo Scientific, Waltham, MA, USA).

Plasma exosomes were extracted using the magnetic bead sorting method. Briefly, 1 ml plasma was mixed with 5 ml magnetic bead (Stainless Steel Beads, Qiagen, Germany). After being washed for three times, exosomal proteins were lysed using Lysis Buffer 3 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM ethylenediaminetetraacetic acid (EDTA). After vortexing (vortex-genie 2, Shanghai, China) and standing for 5 min, dithiothreitol (DTT) was added into the mixture to final concentration of 10 mM. The mixture was then centrifuged at 25,000g for 20 min at 4°C, and the supernatant was collected. After being incubated at 56°C for 1 h, the proteins were precipitated by adding four times volume of cold acetone and incubating at room temperature for 45 min in the dark. After centrifugation at 25,000g for 20 min, the pellets were collected and resuspended in X buffer. The concentration of plasma exosomal proteins was measured using the Bradford assay (Bio-Rad, Hercules, CA). The integrity quality control of the collected proteins was verified by protein electrophoresis on 12% uniform SDS-polyacrylamide gels using the Ettan DALT II system (Amersham Bioscience, Uppsala, Sweden) at 120 V for 120 min. The separated proteins were visualized by Coomassie blue staining.