基于16S rDNA序列分析天疱疮患者皮肤菌群的变化  

Analysis of skin microbiota diversity in patients with pemphigus based on 16S rDNA sequences

作　　者：李丽莉[1] 孙秀坤[1] 沈宏[1] Li Lili;Sun Xiukun;Shen Hong(Department of Dermatology,The Third People′s Hospital of Hangzhou,Hangzhou 310009,China)

机构地区：[1]杭州市第三人民医院皮肤科,310009

出　　处：《中华皮肤科杂志》2021年第3期212-219,共8页Chinese Journal of Dermatology

基　　金：杭州市科技发展计划项目(20191203B112)。

摘　　要：目的采用16S rDNA测序技术研究寻常型天疱疮(PV)患者皮肤菌群多样性。方法收集杭州市第三人民医院皮肤科10例PV患者及10例健康对照,取皮损旁(PV组)及对侧无皮损处皮肤菌群样本(PVn组),正常对照组取相应部位菌群样本。采用16S rDNA扩增子测序技术,对所有样本的菌群基因进行测序与分类,通过Usearch软件对数据聚类分析,得到可操作分类单元(OTU),获得门、纲、目、科、属水平的物种丰度。以Observed species指数、Shannon指数和Simpson指数等反映α多样性,使用主坐标分析法(PCoA)分析β多样性。采用线性判别分析效应量(LEfSe)分析比较各组的差异物种。利用PICRUSt软件进行基因功能预测。2组非参数检验采用Wilcoxon秩和检验,3组间非参数检验采用Kruskal Waills检验。结果注释到门、纲、目、科、属的OTU分别有2002、1869、1751、1611、1120个,3组均以厚壁菌门、放线菌门、拟杆菌门和变形菌门为主,在属水平上,PV组及PVn组葡萄球菌属丰度最高,正常对照组为棒状杆菌属。α多样性分析显示,3组间Observed species指数、Shannon指数、Simpson指数差异均有统计学意义(均P<0.05),PV组Shannon指数(3.24±1.30)和Simpson指数(0.70±0.19)均低于正常对照组(P<0.05)。PCoA分析显示,3组间β多样性差异无统计学意义(P=0.054)。秩和检验显示,3组间丰度差异有统计学意义的物种共32个,其中相对丰度较高的有富集于PV组的芽孢杆菌纲及富集于正常对照组的微球菌属、短波单胞菌属等。按病程<3个月、≥3个月分组,PV长病程组中富集的物种有梭状芽孢杆菌目、颤杆菌属及鞘氨醇单胞菌属等;短病程组γ变形菌纲富集。功能预测显示,与感染性疾病相关的基因功能富集于天疱疮组。结论16S rDNA测序技术应用于天疱疮微生物组学研究,证实PV皮肤菌群多样性及组成结构与正常人存在一定差异。Objective To analyze the skin microbiota diversity in patients with pemphigus vulgaris(PV)using 16S rDNA sequencing.Methods Ten patients with PV and 10 healthy controls were collected from the Department of Dermatology,the Third People′s Hospital of Hangzhou.Skin swabs were collected from perilesional skin(PV group)and contralateral non-lesional skin(PVn group)of the patients with PV,as well as from the corresponding body sites of the healthy controls(normal control group).The 16S rDNA amplicon sequencing technology was used for gene sequencing and classification in all microbiota samples,and Usearch software for data cluster analysis to obtain operational taxonomic units(OTUs)and assess species abundance at the phylum,class,order,family and genus levels.Observed species index,Shannon index and Simpson index were used to estimateαdiversity,and principal coordinate analysis(PCoA)was performed to analyzeβdiversity.Linear discriminant analysis effect size(LEfSe)analysis was conducted to identify differentially abundant species in each group.PICRUSt software was used for gene function prediction.Wilcoxon rank sum test was used as nonparametric test for comparisons between 2 groups,and Kruskal Waills test as nonparametric test for comparisons among 3 groups.Results There were 2002,1869,1751,1611 and 1120 OTUs at phylum,class,order,family and genus levels respectively.Cluster analysis showed that skin microbiome in the 3 groups mainly consisted of Firmicutes,Actinobacteria,Bacteroidetes and Proteobacteria at the phylum level.At the genus level,Staphylococcus was the most abundant in the PV group and PVn group,and Corynebacterium was the most abundant in the normal control group.The observed species index,Shannon index and Simpson index all significantly differed among the 3 groups(all P<0.05),and the Shannon index and Simpson index were significantly lower in the PV group(3.24±1.30,0.70±0.19,respectively)than in the normal control group(P<0.05).PCoA analysis showed no significant difference inβdiversity among th

关 键 词：天疱疮 葡萄球菌属 皮肤菌群 微生物组 16S rDNA测序 功能预测 

分 类 号：R758.66[医药卫生—皮肤病学与性病学]