16s rDNA扩增子测序分析慢性乙型肝炎患者肠道菌群的改变

Analysis of intestinal microflora in patients with chronic hepatitis B by 16s rDNA amplicon sequencing

目的应用16s rDNA扩增子测序技术分析慢性乙型肝炎(chronic hepatitis B, CHB)患者和健康人肠道菌群的差异,探讨肠道菌群对CHB患者疾病的影响及可能的作用机制。方法收集2017年1月—2018年12月就诊的CHB患者(60例)、健康对照者(30例)粪便样本,应用16s rDNA扩增子测序技术,进行菌群鉴定及差异性分析。结果与CHB组比较,健康对照组肠道菌群的丰富度、多样性、均匀度更高。在门水平,两组样本菌群均以厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为主;健康对照组出现了独有的浮霉菌门(Planctomycetes)。在纲水平,健康对照组独有Melainabacteria和OM190;芽孢杆菌纲在CHB组较健康对照组多(P=0.000 999)。在目水平,放线菌目、假单胞菌目在CHB组中独有;Gastranaerophilales在健康对照组独有;乳酸菌目在CHB组多于健康对照组(P=0.000 999);柄杆菌目、脱硫弧菌目、根瘤菌目在CHB组少于健康对照组(P=0.000 999、0.045 954、0.000 003 57)。在科水平,放线菌科、丛毛单胞菌科、莫拉氏菌科在CHB组中独有;叶杆菌科、柄杆菌科、氨基酸球菌科在CHB组少于健康对照组(P=0.000 003 57、0.000 999、0.039 96);Defluviitaleaceae、链球菌科、毛螺菌科在CHB组多于健康对照组(P=0.000 067 3、0.000 999、0.018 981)。在属水平,CHB组独有12个属,健康对照组独有5个属;CHB组毛螺旋菌属UCG-010、中慢生根瘤菌属少于健康对照组(P=0.033 966、0.000 003 57);以下菌属在CHB组多于健康对照组:瘤胃球菌属1、伊格尔兹氏菌属、毛螺旋菌属NC2004、毛螺旋菌属FCS020、梭菌属、Defluviitaleaceae UCG-011、颤杆菌克属、Blautia(P=0.009 99、0.004 995、0.030 969、0.011 988、0.027 972、0.000 067 35、0.000 969、0.019 98)。慢乙肝患者不同个体间肠道菌群差异大,健康对照组个体差异小。结论与CHB组比较,健康对照组肠道菌群的丰富度、多样性、均匀度更高。在门、纲、目、科、属等不同分类水平,CHB组和健康对照组肠道菌群有各自独有的菌种和差异菌。

Objective To analyze the intestinal microflora in patients with chronic hepatitis B(CHB)with 16s rDNA amplification sequencing.Methods Sixty CHB patients admitted in the infection and digestive departments of Shanghai Tongji Hospital during 2017—2018,and 30 healthy subjects were enrolled in the study.Feces samples were collected from CHB patients and healthy controls;16s rDNA amplicon sequencing technique was applied to analyze the intestinal microflora and the results were compared between CHB patients and healthy subjects.Results Compared with CHB group,intestinal flora in healthy controls was richer,more diverse and more uniform.At phylum level,the flora were mainly Firmicutes and Bacteroidetes in both groups;while the healthy control group had a unique Planctomycetes.At class level,there were unique Melainabacteria and OM190 in healthy controls;CHB group has much more Bacilli(P=0.000999).At order level,there were unique Actinomycetales and Pseudomonadales in CHB group;and unique Gastranaerophilales in healthy control group;The Lactobacillales in CHB group was more than that in healthy controls(P=0.000999);Caulobacterales,Desulfovibrionales,Rhizobiales in CHB group were less than those in healthy controls(P=0.000999,0.045954,0.00000357).At family level,there were unique Actinomycetaceae,Comamonadaceae and Moraxellaceae in CHB group;Defluviitaleaceae,Streptococcaceae and Lachnospiraceae in CHB group were more than those in healthy controls(P=0.0000673,0.000999,0.018981),while Phyllobacteriaceae,Caulobacteraceae and Acidaminococcaceae in CHB group were less than those in healthy controls(P=0.00000357,0.000999,0.03996).At genus level,there were twelve unique bacterial genera in CHB group and five in healthy controls;Lachnospiraceae UCG-010 and Mesorhizobium in CHB group were less than those in healthy controls(P=0.033966,0.00000357).Following bacteria genera in CHB group were more than those in healthy controls:Ruminococcus l,Eggerthella,Lachnospiraceae NC2004 group,Lachnospiraceae FCS020 group,Clostridium innocuum group,Defluviitaleaceae UCG-011,Oscillibacter,Blautia(P=0.00999,0.004995,0.030969,0.011988,0.027972,0.00006735,0.000969,0.01998).There were significant differences in intestinal flora structure among CHB patients and less differences among the healthy subjectss.Conclusion Compared with CHB group,intestinal flora in healthy controls is richer,more diverse and more uniform.In phylum,class,order,family and genus levels,intestinal flora in CHB group and healthy controls have their unique and different bacteria strains.