上海2~6岁肥胖/超重儿童肠道菌群多样性和物种组成的病例-对照研究

A case-control study on gut microbiota diversity and species composition in obese/overweight children aged 2-6 years in Shanghai

[背景]多项研究表明肠道微生物菌群组成上的变化与肥胖关系密切,但儿童研究较少,且研究结果受到种族、地理位置等因素的影响。[目的]在上海市2~6岁儿童人群中进行肠道微生物菌群多样性分析,观察肥胖/超重与正常体重儿童之间肠道微生物菌群分布特点及物种差异,探讨肥胖/超重与肠道菌群多样性的关联。[方法]采集上海市74例2~6岁儿童粪便样本,其中肥胖/超重18人,男性6人,女性12人(男女比例为1∶2);正常体重56人,男性18人,女性38人(男女比例约为1∶2)。提取粪便样品中细菌的16S rDNA,进行PCR扩增、文库构建和高通量测序。采用Naive Bayes算法对扩增子序列变异(ASV)的代表序列及丰度信息进行物种分类学分析(门、纲、目、科、属、种)、群落多样性(Sobs指数、Shannon指数、Shannoneven指数、Coverage指数、PD指数、主坐标分析)分析等。运用Wilcoxon秩和检验、P值多重检验校正、相似性分析检验等对两组进行差异分析,获得儿童肠道菌群组成分布特点及物种差异信息。[结果]共计完成74个粪便样本的测序并对测序结果进行质控和过滤,获得优化序列4905306条,得到1860个ASVs。对ASVs进行多样性数据分析,得到8个分类学水平的物种注释结果889个。α多样性分析表明肥胖/超重儿童群落丰富度(Sobs指数)、多样性(Shannon指数)、均匀度(Shannoneven指数)、谱系多样性(PD指数)均比正常体重儿童有所上升,但是两组无统计学差异(P>0.05)。β多样性分析显示两组微生物物种组成组间差异不大,未见明显聚类。从门、目、科、属四种分类学水平上对74例样本进行物种组成分析,结果显示两组肠道菌群都存在一致的核心菌群结构,但是菌群组成有差异。两组间菌群组成差异表现在目、科、属这三个分类学水平上,其中厚壁菌门下的丹毒丝菌目丹毒荚膜菌科的丹毒丝菌属UCG003、链型杆菌属在OB_OW组中显著富集,且对肥胖/超重这一表型差异贡献较大[线性判别分析值(LDA)=3.72,P<0.01;LDA=3.29,P<0.05]。变形菌门下的肠杆菌目肠杆菌科未分类肠杆菌属在体重正常组中显著富集,且对体重正常这一表型差异贡献较大(LDA=3.93,P<0.05)。[结论]上海2~6岁肥胖/超重儿童肠道菌群的丰富度和多样性增高,但与正常体重儿童相比无差异。肥胖/超重组和正常体重组存在肠道菌群组成差异。

[Background]Multiple studies have shown a close relationship between changes in gut microbiota composition and obesity,and research results are influenced by factors such as race and geographical location,but there are few studies on children.[Objective]To analyze the diversity of gut microbiota related to obesity in a population of 2-6 years old,observe the distribution characteristics and species differences of gut microbiota between obese/overweight and normal weight groups,and explore the association between obese/overweight and gut microbiota diversity.[Methods]Fecal samples were collected from 74 children aged 2-6 years in Shanghai,including 18 obese/overweight individuals,6 males and 12 females(male to female ratio of 1:2),and 56 normal weight individuals,18 males and 38 females(male to female ratio is nearly 1:2).The 16S rDNA was extracted from bacteria in fecal samples,followed by PCR amplification,cDNA construction,and highthroughput sequencing.Naive Bayes algorithm was used to perform taxonomic analysis(phylum,class,order,family,genus,species)and community diversity analysis(Sobs index,Shannon index,Shannoneven index,Coverage index,PD index,and principal co-ordinates analysis)on representative sequences and abundance of amplicon sequence variants(ASV).Wilcoxon rank sum test,P-value multiple test correction,and analysis of similarities were used to test differences between the two groups to obtain information on the distribution characteristics and species differences of intestinal microbiota in children.[Results]Seventy-four fecal samples were sequenced,and the sequencing results were subjected to quality control and filtering.A total of 4905306 optimized sequences were obtained,resulting in 1860 ASVs.The diversity data analysis of ASVs generated 889 species annotation results at 8 taxonomic levels.The alpha diversity analysis showed that the richness(Sobs index),diversity(Shannon index),evenness(Shannoneven index),and phylogenetic diversity(PD index)of fecal community of the obese/overweight children were increased compared to those of the normal weight children,but there were no statistical differences between the two groups(P>0.05).The beta diversity analysis showed that there was little difference in the composition of microbial species between the two groups,and no significant clustering separation was observed.The results of species composition analysis at phylum,order,family,and genus levels of 74 samples showed a consistent core microbiota structure in the two groups of gut microbiota,but there were differences in microbiota composition.The differences in microbial community composition between the two groups were manifested at the taxonomic levels of order,family,and genus,among which phylum Firmicutes,order Erysipelotrichales,family Erysipelatocyclostridiaceae,genus Erysipelotrichaceae_UCG-003 and genus Catenibacterium were significantly enriched in the obese/overweight group and contributed significantly to the phenotypic difference of obese/overweight[linear discriminant analysis(LDA=3.72,P<0.01;LDA=3.29,P<0.05).Phylum Proteobacteria,order Enterobacterales,family Enterobacteriaceae,genus unclassified was significantly enriched in the normal weight group and contributed significantly to the phenotypic difference of normal body weight(LDA=3.93,P<0.05).[Conclusion]The richness and diversity of gut microbiota in obese/overweight children aged 2-6 years in Shanghai are increased,but there is no difference compared to normal weight children.There is a difference in the composition of gut microbiota between the obese/overweight group and the normal weight group.