Fecal Collection and 16S rRNA Sequencing

At the end of the study, fresh fecal samples were collected from all 75 mice and stored at −80°C. A total of 30 individual feces (10 individual feces in each group) were used in this study. In accordance with the manufacturer’s instructions, microbial genomic DNA was extracted from fecal samples using the E.Z.N.A.^® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). The V3–V4 region of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) and an ABI Gene Amp^® 9700 PCR thermocycler (ABI, Vernon, CA, USA). The PCR products were separated on agarose gels then sequenced via paired-end sequencing and an Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) using PE300 (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) in accordance with the manufacturer’s protocol.

The resulting sequences were quality-filtered using fastp (0.19.6) (Chen S. et al., 2018) and merged via FLASH (v. 1.2.11) (Magoc and Salzberg, 2011). The high-quality sequences were then denoised using the DADA2 plugin (Callahan et al., 2016) in Qiime2 (v. 2020.2) (Bolyen et al., 2019) to obtain amplicon sequence variants (ASVs). Taxonomic assignment of ASVs was performed using the naive Bayes consensus taxonomy classifier implemented in Qiime2 and the SILVA 16S rRNA database (v. 138). Alpha diversity was calculated based on richness (Chao and ACE index) and diversity (Shannon and Simpson’s diversity index) to evaluate microbial community diversity, and principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA) were calculated to assess beta diversity. Linear discriminant analysis effect size (LEfSe) analysis of the 16S rRNA microbiome sequencing data was performed to identify discriminative bacterial taxa and KEGG categories among the groups, only when linear discriminant analysis (LDA) score >2.0 and p-value <0.05 were considered significant (http://huttenhower.sph.harvard.edu/galaxy) (Segata et al., 2011).