Objective: To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure.
Methods: Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks.
Results: The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group (P < 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (Firmicutes, Bacteroidetes, Actinobacteria, showed that the intestinal flora in and proteobacteria) and 4 genera (finegoldia, Prevotella, Streptococcus, and Corynebacterium_1), all showing no significant differences among the 3 groups (P > 0.05) with the exception of Streptococcus genus, which differed significantly among the 3 groups (P < 0.05). The 16S v3-v4 region in the genus Alloscardovia, Bacteroides, Barnesiella, Butyricicoccus, Facklamia, Faecalibacterium, Lachnospiraceae_FCS020_group, Lachnospiraceae_ND3007_group, Lachnospiraceae_UCG-001, Lachnospirace, Lachnospirace, Ruminococcaceae_UCG-002, Streptococcus and Coprococcus_1 differed significantly among the 3 groups (P < 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups (P < 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera Barnesiellaccae, Aerococcaceae, Family-XIII, Bacteroidaceae, Lachnospiraceae_UCG-001, Barnesiella, Facklamia, Alloscardovia, Faecalibacterium and Bacteroides, as compared with the genera Streptococcaceae, Streptococcus, Coprococcus_1, Ruminococcaceae_ucg-002, Lachnospiraceae_FCS020_group, Lachnospiraceae_ucg-004, Lachnospiraceae_ND3007_group, Lachnospiraceae_ucg-008 and Butyricicoccus in the control group.
Conclusions: Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in Streptococcu genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.
目的: 探索原发性干燥综合征（pSS）与健康人之间肠道菌群是否存在差异。
方法: 收取18例符合纳入标准的女性pSS患者的粪便样本，其中9例为病情活动组（A组）、9例为病情不活动或低活动组（B组），另外收取10例健康人的粪便（C组）。收集每份粪便样品的细菌总DNA，PCR扩增，针对16SrDNA基因的V3~V4区进行illumina hiseq 2500高通量测序，得到3组的肠道菌群生物信息数据，随后结合文献智库信息挖掘进行组间OTU分析、样品多样性分析、样品组间显著性差异分析及LEfSe分析。
结果: 肠道菌群多样性比较：α多样性（Alpha diversity）指数分析，Shannon指数在A组和B组间、Simpson指数在A组和B组间及A组和C组间差异存在统计学意义（P < 0.05）；菌群结构比较：将3组分别进行序列分析，在门水平上3组均主要有4个菌门组成，分别是：厚壁菌门（Firmicutes），拟杆菌门（Bacteroidetes），放线菌门（Actinobacteria），变形菌门（Proteobacteria），在属水平上3组均主要有4个菌属组成，分别是：大芬戈尔德菌属（Finegoldia），普雷沃菌属（Prevotella），链球菌属（Streptococcus），棒状杆菌属（Corynebacterium_1），在门水平和属水平上分析显示3组肠道菌群主要菌门和主要菌属两两比较差异无统计学意义（P > 0.05），但Streptococcus属在3组之间比较，差异具有统计学意义（P < 0.05）；肠道菌群结构的差异分析：肠道菌群16S V3~V4区组间显著性差异分析，在属水平上，3组间相比，在Alloscardovia属、Bacteroides属、Barnesiella属、Butyricicoccus属、Facklamia属、Faecalibacterium属、Lachnospiraceae_FCS020_group属、Lachnospiraceae_ND3007_group属、Lachnospiraceae_UCG-001属、Lachnospiraceae_UCG-004属、Lachnospiraceae_UCG-008属、Ruminococcaceae_UCG-002属、Streptococcus属之间存在统计学差异（P < 0.05）；在Coprococcus_1属存在显著性差异（P= 0.005，P < 0.01）。3组间肠道微生物进行高维生物标识和揭示基因组特征的分析，差异存在统计学意义（P < 0.05）。
结论: pSS患者与健康人之间的肠道菌群多样性存在一定差异，在主要菌门水平和菌属水平上虽无显著性差异，但在Streptococcus属等一些特定细菌比例有显著差异；3组肠道菌群在16S V3~V4区组间差异具有统计学意义，提示菌群结构的差异在pSS的发病中起一定作用。
Keywords: 16S rDNA; high-throughput sequencing; intestinal flora; primary Sj?gren syndrome.