Microbial composition of the rectal fecal samples collected from rats 10 days after MCAO/R treated with or without FMT using 16S rRNA gene sequencing analyses.

1. Method of profiling of the gut microbiota

  Using a previously described protocol, rectal feces were processed for the total DNA extraction using the cetyltrimethylammonium bromide/sodium dodecyl sulphonate method. DNA concentration and purity were monitored on 1% agarose gel, before being diluted to 1 ng/μl using sterile water. Subsequently, 16S ribosomal (r)RNA genes of distinct regions (16S V3-V4) were amplified using a specific primer pair (forward: 5’-CCTAYGGGRBGCASCAG-3’; reverse: 5’-GGACTACNNGGGTATCTAAT -3’). All PCR reactions were performed using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Inc.). Equal volumes of 1X loading buffer (SYB green) were then mixed with the PCR products and subjected to electrophoresis on 2% agarose gel for detection. Samples with bright main strips that are 400 to 450-bp long (16S) and 100 to 400-bp long (ITS) were chosen for further experiments. PCR products were first mixed in equidensity ratios. The mixture of PCR products was then purified using the Qiagen Gel Extraction Kit (Qiagen GmbH). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, Inc.) according to the manufacturer’s protocols, before the index codes were added. Library quality was assessed using the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc.). The library was then sequenced on an Illumina NovaSeq 6000 platform (Illumina, Inc.), where 250 bp paired-end reads were generated. The raw tags were double-ended reads, which could be accessed through fastq join (v1.3.1; https://code.google.com/p/ea-utils/) and pear (v0.9.11). Since the original sequence contained two primer sequences, Cutadapt (v1.18) was then used to isolate the sequences without primers and cut out the fully sequenced primers from the reads. Q30. Usearch (version 11.0.667) with no ambiguous bases was used to cluster according to 97% similarity. Alpha diversity was calculated using Mothur v1.42.1, beta diversity were calculated using the ‘vegan’ package in R and pathway enrichment was calculated using the PICRUSt2 software package (https:// github.com/picrust/picrust2).

2. FMT reverses the imbalance of gut microbiota induced by MCAO/R in rats

We next characterized the microbial composition of the rectal fecal samples collected from rats 10 days after treatment using 16S rRNA gene sequencing analyses. Microbial community barplots show that the microbiota composition of the genus and phylum was altered significantly (Figure 3A and 3B) after cerebral infarct. In terms of the microbial composition on a genus level, MCAO/R decreased the relative abundance of Ligilactobacillus from 56.21 to 14%, Romboutsia from 10.74 to 1.01% and HT002 from 22.75 to 5.68%, whilst increasing the relative abundance of Escherichia-Shigella from 0.43 to 16.49%, Staphylococcus from 0.01 to 2.15%, Lactobacillus from 6.29 to 26.38%, Streptococcus from 0.09 to 2.64%, Akkermansia from 0.17 to 18.74%, Corynebacterium from 0.26 to 2.00%, Bacteroides from 0.01 to 0.64%, Bilophila from 0.04 to 1.4% and Firmicutes_unclassified from 0.02 to 0.29% compared with the sham control rats. Compared with the MCAO/R model group, FMT appeared to have partially reversed the imbalance (Figure 3A).

On the phylum level, we analyzed the components and found that the relative abundance of Proteobacteria (0.59 to 17.14%), Verrucomicrobiota (0.17 to 18.74%), Actinobacteriota (0.34 to 2.06%), Bacteroidota (0.19 to 1.16%) and Desulfobacterota (0.14 to 1.47%) were significantly increased after MCAO/R compared with those in the sham control rats, whilst the relative abundance of Firmicutes (98.51 to 59.40%) was significantly decreased in the MCAO/R group (Figure 3B). FMT prevented these changes at the phylum level induced by MCAO/R injury.

1. 肠道菌群谱型分析（gut microbiota profiling）
采用已发表的实验方案，使用十六烷基三甲基溴化铵/十二烷基硫酸钠（cetyltrimethylammonium bromide/sodium dodecyl sulphonate）法对直肠粪便样本进行总DNA提取。通过1%琼脂糖凝胶电泳检测DNA浓度与纯度，随后使用无菌水将DNA稀释至1 ng/μl。接下来，利用特定引物对（正向引物：5’-CCTAYGGGRBGCASCAG-3’；反向引物：5’-GGACTACNNGGGTATCTAAT -3’）扩增16S核糖体RNA（16S rRNA）基因的高变区（16S V3-V4区）。所有PCR反应均采用Phusion®高保真PCR预混液（New England Biolabs, Inc.，新英格兰生物实验室公司）。随后将等体积的1×上样缓冲液（SYBR Green）与PCR产物混合，经2%琼脂糖凝胶电泳检测。选取目的条带明亮、片段长度为400~450 bp（16S）及100~400 bp（内部转录间隔区ITS）的样本用于后续实验。首先将PCR产物按等密度比例混合，随后使用Qiagen凝胶提取试剂盒（Qiagen GmbH，凯杰公司）纯化混合后的PCR产物。使用TruSeq® DNA PCR-Free样本制备试剂盒（Illumina, Inc.，因美纳公司）按照制造商操作规程构建测序文库，随后添加索引标签。文库质量通过Qubit® 2.0荧光定量仪（Thermo Fisher Scientific, Inc.，赛默飞世尔科技公司）与Agilent Bioanalyzer 2100系统（Agilent Technologies, Inc.，安捷伦科技公司）进行评估。随后在Illumina NovaSeq 6000测序平台（Illumina, Inc.）上完成测序，生成250 bp双端读长序列。原始标签为双端reads，可通过fastq join（v1.3.1; https://code.google.com/p/ea-utils/）与pear（v0.9.11）进行拼接。由于原始序列包含两段引物序列，使用Cutadapt（v1.18）去除引物序列，仅保留无引物的目标片段，并从测序reads中切除完整的引物区域。完成Q30质量控制后，使用无歧义碱基的Usearch（版本11.0.667）按照97%相似性进行聚类分析。使用Mothur v1.42.1计算α多样性，使用R语言的‘vegan’包计算β多样性，使用PICRUSt2软件包（https://github.com/picrust/picrust2）进行通路富集分析。

2. 粪便菌群移植（Fecal Microbiota Transplantation, FMT）可逆转大脑中动脉阻塞/再灌注（Middle Cerebral Artery Occlusion/Reperfusion, MCAO/R）诱导的大鼠肠道菌群失衡。
我们采用16S rRNA基因测序分析，对治疗10天后收集的大鼠直肠粪便样本的微生物组成进行表征。微生物群落条形图显示，脑梗死发生后，属水平与门水平的菌群组成均发生显著改变（图3A、3B）。在属水平上，与假手术对照组大鼠相比，MCAO/R模型组的Ligilactobacillus相对丰度从56.21%降至14%，Romboutsia从10.74%降至1.01%，HT002从22.75%降至5.68%；同时埃希氏菌-志贺氏菌属（Escherichia-Shigella）相对丰度从0.43%升至16.49%，葡萄球菌属（Staphylococcus）从0.01%升至2.15%，乳杆菌属（Lactobacillus）从6.29%升至26.38%，链球菌属（Streptococcus）从0.09%升至2.64%，Akkermansia菌属从0.17%升至18.74%，棒状杆菌属（Corynebacterium）从0.26%升至2.00%，拟杆菌属（Bacteroides）从0.01%升至0.64%，Bilophila菌属从0.04%升至1.4%，未分类厚壁菌门（Firmicutes_unclassified）从0.02%升至0.29%。与MCAO/R模型组相比，FMT可部分逆转这种菌群失衡状态（图3A）。

在门水平上，分析结果显示，与假手术对照组相比，MCAO/R模型组大鼠的变形菌门（Proteobacteria）相对丰度从0.59%升至17.14%，疣微菌门（Verrucomicrobiota）从0.17%升至18.74%，放线菌门（Actinobacteriota）从0.34%升至2.06%，拟杆菌门（Bacteroidota）从0.19%升至1.16%，脱硫杆菌门（Desulfobacterota）从0.14%升至1.47%；而厚壁菌门（Firmicutes）相对丰度从98.51%降至59.40%（图3B）。FMT可阻断MCAO/R损伤诱导的门水平菌群组成变化。