Objective: To explore the possibility that the intestinal flora profile in complex anal fistula patients is different to that of healthy controls. This was assessed by sequencing of 16S rDNA in fecal samples from cohorts representing these populations. Methods: Fecal samples were collected from 30 complex anal fistula patients and 30 matched healthy controls. Patients were included if they met the diagnostic criteria of cryptoglandular anal fistula and had exhibited symptoms for more than 3 months. Complex anal fistula is diagnosed under the following circumstances: if the fistula in question spans 2/3 or more of the diameter of the anal sphincter; if there are more than two external orifices or fistula tracks; or if recurrence is observed after previous anal fistula surgery. Patients were excluded if there were comorbities including inflammatory bowel disease (as assessed by colonoscopy), chronic diarrhea, chronic constipation, diabetes, gastrointestinal malignancies, liver/ kidney dysfunction, or cognitive impairment. Patients whose anal fistulas were caused by Crohn's disease, trauma, special infections (such as actinomycosis and tuberculosis) were also excluded, as were those who had used antibiotics, prebiotics, or probiotics that may affect intestinal microecology in the month prior to the study. Total bacterial genomic DNA was extracted by PCR amplification of the V4 hypervariable region of the 16S rRNA sequences. High-throughput sequencing and data analysis were performed on the Illumina Miseq platform. Finally, operational taxonomic unit (OTU) clustering, alpha diversity and LEfSE data analysis were carried out. The larger the Chao or ACE index is, the higher the species abundance of the microflora is expected to be. Similarly, a smaller value for the Simpson index or a larger value for the Shannon index indicates greater microflora diversity. There was no statistically significant difference in gender, age, body mass index (BMI), drinking history, or smoking history between the two groups (P>0.05), indicating that they were comparable. Results: The α-diversity analysis including ACE, Chao, Shannon and Simpson indexes indicated a richer diversity of intestinal microflora in complex anal fistula patients than in healthy controls. In both patients and controls, OUT cluster analysis demonstrated that 93.4%±32.0% and 87.4%±41.2% of sequences were from Firmicutes and Bacteroidetes spp., respectively. On a genus level, samples from anal fistula patients showed a greater abundance of Prevotella spp. (4.9%±7.4% vs. 0.1%±1.1%, P<0.001), Megamonas (3.9%±8.2% vs. 0.5%±4.2%, P<0.05) and Lachnospira (2.6%±5.7% vs. 0.1%±3.4%, P<0.05), while showing a lesser abundance of Proteobacteria spp. (0.02%±4.2% vs. 9.3%±14.4%, P<0.01), Enterococcus (0.02%±2.3% vs. 9.3%±19.6%, P<0.05), Bacteroides (24.7%±9.9% vs. 29.8%±9.1%, P<0.05) and Klebsiella (0.4%±4.2% vs. 3.9%±7.3%, P<0.05) compared with healthy controls. Intestinal flora diversity in the complex anal fistula group was richer than in controls, as indicated by a higher ACE index (293.30±44.00 vs. 218.75±33.83, t=102.069, P<0.001), a higher Chao index (318.40±41.99 vs. 250.00±46.38, t=77.818, P=0.028), a higher Shannon index (3.36±0.29 vs. 2.43±0.34, t=9.657, P=0.001), and a lower Simpson index (0.103±0.013 vs. 0.131±0.013, t=5.551, P=0.046). LDA effect size analysis suggests that the main strains of Veillonellaceae, Selenemondales and Negativicutes, which all belong to the phylum Firmicutes, have the greatest influence on the above difference (LDA>4). Conclusions: The diversity of intestinal flora in patients with complex anal fistula is greater than in healthy controls, suggesting that these bacteria or their metabolites may be involved in the occurrence and development of anal fistulas.
目的： 通过16S rDNA基因测序检测复杂性肛瘘患者的肠道菌群分布，探讨其与健康人的菌群分布是否存在差异。 方法： 采用病例对照研究方法，回顾性收集2020年6月至12月期间杭州市第三人民医院接受手术治疗的30例复杂性肛瘘患者临床资料（复杂性肛瘘组），并按1∶1配比选择本院健康体检人群30例作为对照组（健康对照组）。病例纳入标准：（1）符合腺源性肛瘘诊断的标准，且症状持续3个月以上；（2）术前已行电子结肠镜检查排除肠道炎性反应、炎性肠病等；（3）复杂性肛瘘的定义为符合下面情况之一者：瘘管跨越2/3及以上的肛门括约肌；包含两个以上的外口或瘘管；既往已行肛瘘手术后确认复发者。排除标准：（1）合并有炎性肠病、慢性腹泻或便秘、糖尿病、消化道恶性肿瘤、肝肾功能障碍等；（2）克罗恩病、外伤、特殊感染（如放线菌病和结核病）等引起的肛瘘；（3）近1个月内使用过抗生素、益生元、益生菌等可能影响肠道微生态制剂者；（4）认知缺陷不能配合者。提取研究对象的粪便样本总DNA，扩增细菌16S rDNA基因序列的V4高变区，利用Illumina Miseq平台进行高通量测序，最后进行操作分类单元（OTU）聚类、进行Alpha多样性和LEfSE数据分析，其中Chao指数或ACE指数越大，表明微生物区系的预期物种丰度越高，Simpson指数越小或Shannon指数越大，表明微生物区系多样性越高。两组间性别、年龄、体质指数（BMI）、饮酒吸烟史基线资料比较差异均无统计学意义（均P>0.05），说明两组具有可比性。本研究观察指标包括两组的测序和质量控制、Alpha多样性分析和物种及其丰度分析以及LEfSE分析。 结果： 复杂性肛瘘组与健康对照组的主要优势菌都是厚壁菌门和拟杆菌门，分别占93.4%±32.0%和87.4%±41.2%；在属水平上，复杂性肛瘘组的普雷沃菌属（4.9%±7.4%比0.1%±1.1%，P<0.001）、巨细胞菌属（3.9%±8.2%比0.5%±4.2%，P<0.05）和毛螺菌属（2.6%±5.7%比0.1%±3.4%，P<0.05）的丰度明显更高，而变形菌属（0.02%±4.2%比9.3%±14.4%，P<0.01）、肠球菌属（0.02%±2.3%比9.3%±19.6%，P<0.05）、拟杆菌属（24.7%±9.9%比29.8%±9.1%，P<0.05）和克雷伯菌属（0.4%±4.2%比3.9%±7.3%，P<0.05）相对更低。与健康对照组相比，复杂性肛瘘组肠道菌群多样性更丰富，表现为ACE指数更高［（293.30±44.00）比（218.75±33.83），t=102.069，P<0.001］，Chao指数也更高［（318.40±41.99）比（250.00±46.38），t=77.818，P=0.028］，Shannon指数更高［（3.36±0.29）比（2.43±0.34），t=9.657，P=0.001］；而Simpson指数更低［（0.103±0.013）比（0.131±0.013），t=5.551，P=0.046］，差异均有统计学意义（均P<0.05）。韦荣菌科、Selenemondales 及厌氧菌纲（均属于厚壁菌门）的菌株总体上对两组样本肠道菌群差异的影响最大（LDA 值均>4）。 结论： 与健康对照组相比，复杂性肛瘘患者肠道菌群多样性更丰富，提示某些肠道菌群及其代谢产物可能在肛瘘发病中发挥了一定的作用。.