Study On Gut Microbiota In Children With Type 1 Diabetes Mellitus

Objective:The 16SrRNA gene sequencing method was used to explore the characteristics of intestinal flora in T1DM children,and to compare the differences of gut microbiota between T1DM patients and healthy controls.Methods:This study was carried out with 18 children aged 5-14 who were newly diagnosed as type 1 diabetes mellitusin in the Department of Endocrinology and Metabolism,Kunming Children's Hospital from October 2018 to October 2019.And 19 healthy children with similar gender and age were selected as controls.After collecting fresh stool samples from two groups,they were strictly transported to the laboratory of Kunming Jiasang Technology Co.Ltd.as soon as possible under the conditions of low temperature on dry ice.The full amount of bacterial DNA from two sets of samples was extracted using a gene extraction kit,amplified the V3-V4 targeted region of 16SrRNA by PCR,purified the PCR products and constructed the library,and then sequenced by MiSeq platform.The raw datas were optimized and the effective features obtained by QIIME2 analysis were used for feature clustering,microbiota structure composition analysis,Alpha diversity analysis,Beta diversity analysis and LEfSe analysis.Then,the marked differences of gut microbiota among two groups were compared.Finally,the Receiver-Operating Characteristic curve analysis and Area Under The Curve were used to preliminarily assess the value of bacterial biomarkers in potential risk assessment for type 1 diabetes mellitus.Results:1.There were no significantly statistical differences in age,gender,body mass index and birth pattern between children with type 1 diabetes mellitus and healthy children in this study(P>0.05).Compared with healthy controls,T1DM patients of fasting blood glucose,glycolated HemoglobinAlc and glycosylated serum protein were strikingly higher(P<0.01),whereas C-peptide and serum insulin were seriously lower(P<0.01).The results showed that diabetes autoantibodies were positive at least one per child in 18 T1DM patients(100%),while 9 cases were?2 items,accounting for 49.5%.2.A total of 2150550 valid reads were obtained from 37 samples,of which 1203581 were in T1DM group.The average number of sequences was 66865 per sample and the length was 411.26bps;Healthy controls had 946969 valid reads,with an average of 49840 sequences per sample and the length was 415.19bps,and the average of Q20%was over 96%in two groups.3.The 3248 Features with 100%similarity were obtained by using QIIME2 software.4.Most of intestinal bacteria were classified into genus and species after identification and annotation.In community composition analysis,at the level of phylum,the abundance of Actinobacteria and?Bacteroidetes and Cyanobacteria was increased while Proteobacteria and Patescibacteria was decreased in T1DM group(P<0.05);At the genus level,the abundance of Faecalibacterium?Bifidobacterium?Bacteroides and other bacterias was increased in T1DM group(P<0.01),but the abundance of Escherichia_Shigella,Blautia and Enterococcus was reduced(P<0.05).5.Alpha diversity analysis showed that the sequencing depth was sufficient in this study.Moreover,in terms of the gut microbiota richness and diversity,type 1 diabetes mellitus children were lower than those in healthy controls(P<0.05).6.In the analysis of beta diversity,the PCoA image exhibited that the gut microbiota structure not only was different between type 1 diabetes patients and healthy controls,but also was defined as moderate difference by ANOSIM analysis(R=0.51);The Unweighted Pair-Group Method with Arithmetic mean analysis indicated that the samples among two groups showed their own clustering trends,but the samples within the group were highly similar and clustered together.7.LEfSe analysis showed that the abundance of Bifidobacterium?Bifidobacteriaceae?Bifidobacteriales?Actinobacteria?Actinobacteria?Ruminococcaceae?Bifidobacterium_pseudocatenulatum?Bacteroidales?Bacteroidia?Bacteroidetes?Bacteroidaceae?Bacteroides?Faecalibacterium were higher in T1 DM children,yet the abundance of Bacilli?Lactobacillales?Gammaproteobacteria?Proteobacteria?Enterobacteriaceae?Enterobacteriales?Escherichia_Shigella_unclassified?Escherichia_Shigella were reduced in T1DM group,and there were notablely statistical biomarkers at the levels of phylum,class,order,amily,genus and species between T1DM children and health controls.Among them,T1DM group had 13 unique biomarkers yet healthy control group existed 8 biomarkers.8.The Receiver-Operating Characteristic curve analysis demonstrated that Bacteroidetes and Actinobacteria may have certainly assessed value in predicting potential risk for T1DM in this study.Conclusions:1.In the T1DM children,the ecology of gut microbiota was imbalanced.Compared with healthy children,the richness and diversity were notablely lowered in T1DM children as well;2.Children with type 1 diabetes mellitus had unique biomarkers,and the two bacterial biomarkers of Bacteroidetes and Actinobacteria screened in this study may have potential value in risk assessment for T1DM.