基于QIIME 2平台对先天性巨结肠患儿与正常儿童肠道菌群的比较分析

Comparative analysis of intestinal flora between children with Hirschsprung disease and normal children based upon QIIMIE 2technology

目的 比较先天性巨结肠患儿与正常儿童肠道菌群差异,阐明先天性巨结肠对肠道菌群组成的影响。方法 收集2019年4月至2020年12月来河北省儿童医院就诊的先天性巨结肠患儿与体检的正常儿童粪便样本。10例先天性巨结肠患儿纳入先天性巨结肠患儿组(HDB_Nor组),其中男7例,女3例;年龄为(1.22±0.65)岁;14例正常儿童纳入对照组,即正常儿童组(Normal组),其中男8例,女6例;年龄为(1.67±0.84)岁。利用细菌16S rRNA基因序列测序,通过QIIME 2平台对测序数据进行统计分析并绘图,对比分析两组肠道菌群组成、多样性、菌群差异、菌群物种间相关性以及菌群与疾病之间联系。结果 在肠道菌群组成、Beta多样性方面先天性巨结肠患儿与正常儿童肠道菌群差异有统计学意义;LEfSe差异分析结果显示假小链双歧杆菌、两歧双歧杆菌、瘤胃菌属等在先天性巨结肠患儿组中物种显著增加;放线菌属、粘滑罗斯菌属及弗氏柠檬酸杆菌等三个细菌种属在先天性巨结肠患儿组中显著减少。通过肠道菌群与先天性巨结肠相关性分析发现AKK菌属、大肠埃希-志贺氏菌属、肠球菌属和伯克霍尔德菌属等四个种属与疾病关联性影响较大。结论 先天性巨结肠患儿与正常儿童肠道菌群组成及多样性方面存在显著差异,先天性巨结肠影响肠道菌群的组成和多样性,尤其是对AKK菌属和大肠埃希-志贺氏菌属两个菌属的影响更显著。

Objective To compare the differences of intestinal flora between children with Hirschsprung disease(HD)and normal children,to explore the relationship between HD and changes in intestinal flora with species and to clarify the influence of HD on the composition of intestinal flora.Methods Samples were collected from HD children and normal children from 2019 to 2020.The diagnostic criteria of HD,healthy controls and flora-related factors were listed as inclusion or exclusion criteria.High-throughput sequencing was employed for analyzing the bacterial 16S rRNA gene and QIIME 2 platform for processing the sequencing data and examining the composition and diversity of intestinal flora of two groups.Gender characteristics,flora differences,correlation between flora species and relationship between flora and disease were examined for exploring the composition characteristics of intestinal flora in HD children.Results Intestinal flora of HD children and normal children varied significantly in composition of intestinal flora and Beta diversity.The genus of bacteria and other species increased significantly including Bifidobacterium pseudosmallerans,Bifidobacterium bifidum and Ruminococcus by LEfSe differential analysis.And Actinomyces,Rossella glutinosa and Citrobacter freundii decreased markedly in HD children.Regarding the correlation between intestinal flora and HD,four species of AKK,Escherichia coli-Shigella,Enterococcus and Burkholderia spp.exerted a greater impact on disease.Conclusions Significant differences exist in composition and diversity of intestinal flora between HD and normal children.The major differential bacterial species have been explored and the influence of HD on the change of intestinal flora has been discovered,especially Akkermansia and Escherichia-Shigella.