EVs in human feces were isolated via centrifugation, as reported previously34,35. Human stool samples were filtered via a cell strainer after incubation in 10 mL of phosphate-buffered saline for 24 h. To separate EVs from stool samples, EVs in the samples were isolated via centrifugation at 10,000×g for 10 min at 4 °C to obtain the pellet of stool samples containing bacterial cells, while the supernatant of stool samples contained EVs. Bacteria and foreign particles were eliminated from the sample supernatants via sterilization using a 0.22 μm filter. To extract DNAs from bacterial cells and bacterial EVs, bacteria and EVs were boiled for 40 min at 100 °C. To eliminate the residual floating particles and debris, the supernatant was collected after centrifugation at 13,000 rpm for 30 min at 4 °C. DNA was extracted using a PowerSoil DNA Isolation Kit (MO BIO, Carlsbad, CA, USA), following the standard manufacturer’s protocol. The DNA extracted from the bacterial cells and EVs in each sample was quantified using a QIAxpert system (QIAGEN, Hilden, Germany). In this study, we analyzed the DNAs from bacterial cells (microbiome originating from bacterial cells) and DNAs from bacterial EVs (microbiome originating from bacteria-derived EVs) separately.

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