design: Total DNA from stool samples was extracted with a QIAamp DNA Stool Mini Kit (QIAGEN, Germantown, MD, USA) following the manufacturers instructions. PCR amplification was performed spanning the V3 and V4 regions using the forward primers 5-CCTACGGRRBGCASCAGKVRVGAAT-3 and reverse primers 5-GGACTACNVGGGTWTCTAATCC-3 of the 16S rDNA gene and subsequently sequenced using 250 bp paired-end sequencing (Illumina MiSeq, Illumina, San Diego, USA).