DNA extraction and V3–V4 region of 16SrRNA gene sequencing
A commercial kit (Qiagen, Hilden, Germany) were used to extract faecal DNA. Faecal DNA was amplified by PCR using 16S amplicon PCR forward primer and 16S amplicon PCR reverse primer. After PCR amplification, the amplicons in each library were purified by Qiagen for library preparation. Subsequently, the qualified library was sequenced by Illumina Hiseq 2500 high-throughput sequencing platform. Sequences were clustered into operational taxonomic units (OTUs) based on Silva database v128, at a similarity level of 97%. Alpha and Beta diversity were generated in Quantitative Insights Into Microbial Ecology (QIIME). And the abundance of bacterial OTUs were divided into several levels (phyla, class, order, family and genus). The laboratory technicians were blinded to the clinical status (intervention or control group) of study participants.