For the microbiota composition analysis, DNA was isolated from the TIM-2 lumen samples using the QIAamp Fast DNA stool mini kit (Qiagen, Germany), with a bead-beating step. Sequencing of the V3-V4 region of the 16S rRNA gene was performed on the Illumina MiSeq system with the 341F and 805R primers (Illumina, Eindhoven, The Netherlands). Sequences were generated using the Illumina CASAVA pipeline (v1.8.3; Illumina), and subsequently converted into FASTQ files using the Binary Base Call text-based format for storing biological sequence and corresponding quality scores pipeline (BCL2FASTQ pipeline version 1.8.3, Illumina, USA).