Sample processing and 16S rDNA sequencing
The collected EBC samples were stored in R-tubes at -20°C until sample processing for metagenomic sequencing. Due to the relatively low sample volume, 1x phosphate buffered saline solution (Welgene, Gyeongsan, Korea) was added to all EBC samples to attain a total volume of 1 mL for each sample. Subsequently, samples were centrifuged for 10 min (10,000 × g, 4 °C), the supernatant was collected, and passed through a 0.22-µm filter to sterilize the solution by removing any cells or unnecessary particles. The filtrate solution was subjected to a temperature of 100°C for 40 min and centrifuged (13,000 rpm, 4°C) for 30 min to extract DNA from the double membrane-enclosed EVs. The DNA within the subsequent supernatant was extracted according to the instructions provided in the DNeasy PowerSoil kit (QIAGEN, Germany) and quantified using the QIAxpert (QIAGEN, Germany) software. The V3-V4 hypervariable regions of the 16S rDNA contained in each sample were amplified and the libraries were prepared and quantified as described previously13. All resulting 16S rDNA amplicons were sequenced using the MiSeq (Illumina, USA).