基于16SrDNA及ITS高通量测序分析甲真菌病病甲微生物群落的变化

Analysis of the nail microbiome in patients with onychomycosis by high-throughput sequencing of 16S rDNA and internal transcribed spacer regions

目的比较甲真菌病患者病甲与健康甲细菌和真菌微生物菌群差异。方法收集2020年8月至2021年6月于大连市皮肤病医院门诊就诊的31例甲真菌病患者的病甲及健康甲的甲屑样本,提取甲屑菌群总DNA,以细菌16S rDNA的V3-V4区序列及真菌内转录间隔区(ITS)作为目标序列进行基因扩增及Illumina测序,对所测序列采用USEARCH和mothur软件进行OTU聚类分析,采用Wilcoxon rank sum test方法进行α多样性分析,采用相似性分析(Anosim)进行β多样性分析,采用LEFSe方法进行物种差异分析。结果入组甲真菌病患者31例,男16例,女15例,按年龄分青年组(18~35岁)10例,中年组(36~60岁)11例,老年组(大于60岁)10例。α多样性分析显示,病甲组标本Shannon指数明显低于健康甲组(W=290,P=0.007),而Simpson指数明显高于健康甲组(W=663,P=0.010),病甲组细菌菌群的多样性及均匀度低于健康甲组,病甲在真菌菌群的多样性与健康甲无明显区别。β多样性分析显示,基于unweighted-unifrac距离矩阵分析结果,病甲组与健康甲组细菌和真菌微生物的组成差异无统计学意义(细菌菌群:R=0.0052,P=0.331;真菌菌群:R=0.0036,P=0.337);基于weighted-unifrac距离矩阵分析结果,病甲组与健康甲组在细菌菌群及真菌菌群的丰度方面差异均有统计学意义(均P=0.001)。在物种组成上,病甲组较健康甲组丰度显著降低的细菌菌群有拟杆菌门及变形菌门、β-变形菌纲、伯克氏菌目、罗尔斯顿菌属、鞘氨醇单胞菌属、链球菌属,而芽孢杆菌纲、芽孢杆菌目、葡萄球菌属明显高于健康甲组。病甲组显著升高的真菌菌群有散囊菌纲、爪甲团囊菌目、未分类锤舌菌目、关节皮肤真菌科、黑团孢属、白粉菌属、铁艾酵母属、毛癣菌属、十字花科白粉菌、红色毛癣菌、合轴马拉色菌,而酵母菌纲-酵母菌目-酵母菌科、隐囊菌科、念珠菌、链格孢菌较健康甲组显著降低。结论甲真菌病患者病甲与健康甲细菌菌群的多样性及丰度均有明显的差异,真菌菌群仅丰度存在一定的差异,其中某些真菌和细菌菌属可能具有相关性。

Objective To investigate differences in bacterial and fungal microbiome between infected nails and healthy nails among patients with onychomycosis.Methods Nail scraping samples were collected from infected nails and healthy nails of 31 patients with onychomycosis,who visited Dalian Dermatosis Hospital from August 2020 to July 2021.The total DNA of nail microbiota was extracted,and the V3-V4 regions of the bacterial 16S rDNA gene and the fungal internal transcribed spacer(ITS)region were amplified and sequenced using Illumina technology.The USEARCH and mothur softwares were used for data cluster analysis to obtain the operational taxonomic units(OTUs),Wilcoxon rank sum test was used to analyzeαdiversity,analysis of similarities(ANOSIM)was performed to analyzeβdiversity,linear discriminant analysis of effect size(LEfSe)was performed to evaluate the species difference.Results Among the 31 patients with onychomycosis,16 were males and 15 were females.According to the age,they were divided into young group(18-35 years old,10 cases),middle-aged group(36-60 years old,11 cases),and elderly group(over 60 years old,10 cases).As theα-diversity analysis revealed,the infected nail group showed significantly decreased Shannon index(W=290,P=0.007),but significantly increased Simpson index(W=663,P=0.010)compared with the healthy nail group,suggesting that the diversity and evenness of bacterial communities were lower in the infected nail group than in the healthy nail group;however,there was no significant difference in the diversity of fungal communities between the infected nail group and healthy nail group.Theβ-diversity analysis based on the unweighted-UniFrac distance matrix showed no significant difference in the fungal or bacterial community composition between the infected nail group and healthy nail group(bacterial communities:R=0.0052,P=0.331;fungal communities:R=0.0036,P=0.337);theβ-diversity analysis based on the weighted-UniFrac distance matrix showed significant differences in the abundance of bacterial and fungal communities between the two groups(both P=0.001).In terms of the species composition,the bacterial flora with significantly decreased abundance in the infected nail group compared with the healthy nail group included Bacteroidetes,Proteobacteria,Betaproteobacteria,Burkholderiales,Ralstonia,Sphingomonas and Streptococcus,while the abundance of Bacilli,Bacillales and Staphylococcus was significantly higher in the infected nail group than in the healthy nail group.Compared with the healthy nail group,the fungal flora with significantly increased abundance in the infected nail group included Eurotiomycetes,Onygenales,Leotiomycetes-ord-incertae-sedis,Arthrodermataceae,Periconia,Erysiphe,Tilletiopsis,Trichophyton,Erysiphe cruciferarum,Trichophyton rubrum,Malassezia sympodialis,while the abundance of Saccharomycetes,Saccharomycetales,Saccharomycetaceae,Dothioraceae,Candida and Alternaria was significantly lower in the infected nail group than in the healthy nail group.Conclusion The diversity and abundance of bacterial communities significantly differed between infected nails and healthy nails among patients with onychomycosis,while only the abundance of fungal communities differed between the two groups,and perhaps there was correlations between some bacterial and fungal communities.