Gut microbiota composition

Cecal DNA was extracted from frozen samples using the FastDNA SPIN kit for feces (MP Biomedicals, Santa Ana, CA, USA) according to manufacturer instructions. Bacteria (L. salivarius JCM1044, L. johnsonii JCM2012, L. acidophilus JCM1028, and L. gasseri JCM1131) was obtained from the Japan Collection of Microorganisms of RIKEN BRC (Tokyo, Japan) and used as a standard specifically for the DNA-based determination of intestinal lactic acid bacterial count. Bacterial DNA was isolated using the MonoFas bacterial genomic kit IV (GLC science, Tokyo, Japan) according to manufacturer instructions. Quantitative real-time PCR analysis was performed using SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan) and the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA). Standard curves for quantification comprised a series of 10-fold serial dilutions in the range of 10⁸ to 10⁰ copies of target 16 S rRNA genes. Bacterial primer sequences were as follows: Lactobacillus genus, 5′-AGCAGTAGGGAATCTTCCA-3′ (forward) and 5′- CACCGCTACACATGGAG-3′ (reverse)³²; L. salivarius, 5′-CGAAACTTTCTTACACCGAATGC-3′ (forward) and 5′-GTCCATTGTGGAAGATTCCC-3′ (reverse); L. johnsonii, 5′- CACTAGACGCATGTCTAGAG-3′ (forward) and 5′-AGTCTCTCAACTCGGCTATG-3′ (reverse); L. acidophilus, 5′-GTCTGATGGAGCAACGCCGC-3′ (forward) and 5′-ACCTTGCGGTCGTACTCCCC-3′ (reverse); and L. gasseri, 5′-GCCACATTGGGACTGAGACA-3′ (forward) and 5′-TTGCTCCATCAGACTTGCGT-3′ (reverse). Partial 16 S rRNA gene sequences were amplified targeting the hypervariable regions v4 using primers 515 F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3′) and 806 R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′). Amplicons generated from each sample were subsequently purified using AMPure XP (Beckman Coulter, Brea, CA, USA). The 16 S rRNA sequence data generated by the MiSeq sequencer (Illumina, San Diego, CA, USA) using the MiSeq Reagent kit (version 3.0; 600 cycles) were processed by the quantitative insights into microbial ecology (QIIME 1.8.0; http://qiime.org/) pipeline. Data analysis was performed using MiSeq Reporter software with the SILVA database (Illumina). Diversity analyses were performed using the QIIME script core_diversity_analyses.py. The statistical significance of sample groupings was assessed using permutational multivariate analysis of variance (QIIME script compare_categories.py).