Gam agar medium

DNA was extracted from fecal samples using a QIAamp DNA Mini kit (Qiagen, Tokyo, Japan). Each library was prepared according to “Illumina 16S Metagenomic Sequencing Library Preparation Guide” with a primer set (27Fmod:
5ʹ- AGR GTT TGA TCM TGG CTC AG-3ʹ and 338R: 5ʹ-TGC TGC CTC CCG TAG GAG T-3ʹ) targeting the V1–V2 region of the 16S rRNA gene. 250 bp paired end sequencing of the amplicon was performed on a MiSeq (Illumina) using MiSeq
v2 500 cycle kit. The resulting sequences were analyzed using the QIIME pipeline [10 (link)].
For determination of CFU in fecal samples, fecal samples were diluted with a buffer that consisted of 0.45 g KH₂PO₄ (Kishida Chemical, Osaka, Japan), 1.68 g
Na₂HPO₄•12H₂O (Kanto Chemical, Tokyo, Japan), 0.05 g ʟ-cysteine HCl•H₂O, 0.05 g Tween^® 80, and 0.1 g agar in 100 ml distilled water. Samples were
subjected to serial 10-fold (w/v) dilutions with dilution buffer and vortexed. In order to calculate the total number of bacteria, 50 µl of the diluted sample was applied to GAM agar medium (Nissui
Pharmaceutical, Tokyo, Japan) and cultured for 48 hr under anaerobic environment. Finally, the number of colonies was counted, and the log CFU/g was calculated.