Analysis Of Gut Microbiota And Metabolic Characteristics Of Tuberculosis Patients And Its Association With Dietary Nutrients

Objective: To explore whether there are changes in the diversity composition and metabolic profile of gut microbiota in population with active pulmonary tuberculosis(PTB)and latent tuberculosis infection(LTBI),and to identify the potential biomarkers of gut microbiota and metabolite for identifying PTB and LTBI.Then explore the functional metabolic pathways that may be involved in different disease states,and try to put forward the hypothesis that by regulating the intake of dietary nutrients to change the gut microbiota and its metabolites to regulate the tuberculosis disease state.Methods:PTB,LTBI and healthy controls(HC)were recruited.Stool samples were collected and semi-quantitative food frequency questionnaire(SQFFQ)was used to investigate.(1)16SrRNA gene sequencing was performed on the fecal samples.The diversity and composition spectrum of gut microbiota of the three groups were analyzed,and to found the differential genus.Random forest algorithm was used to screen out the independent predictor,and the ROC curve was drawn to evaluate its diagnostic efficacy.And functional prediction was performed.(2)In the first part,representative subjects were screened out,and their fecal samples were subjected to liquid chromatography-mass spectrometry(LC-MS/MS)untargeted metabolomics to clarify the changes in metabolic profiles of the three groups,to found differential metabolites,and screen out potential metabolites that can be used as diagnostic biomarkers by lasso regression model,and the diagnostic efficacy was evaluated.Then functional enrichment analysis of differential metabolites was performed.And the correlation between the differential metabolites and the differential bacterial genera was analyzed.(3)The SQFFQ questionnaire was used to obtain the daily intake of dietary nutrients,and logistic regression was used to screen out the dietary nutrients that had an impact on the disease among the three groups.The correlation between these nutrients and the differential bacteria in the first part and the differential metabolites in the second part were analyzed.The association network of dietary nutrients,gut microbiota and metabolites was constructed.Results: A total of 170 subjects were enrolled in this study,including 69 PTB,51 LTBI and 50 HC.(1)Part I:16SrRNA gene sequencing was performed in 170 individuals,1)The genus composition of the three groups changed,with increased relative abundance of Bacteroides,Bifidobacterium and Gemmiger and decreased relative abundance of Oscillospira in the PTB group.The relative abundance of Prevotella and Dialister increased,and Blautia decreased in the LTBI group.The relative abundance of Roseburia and Faecalibacterium increased and Lactobacillus decreased in the HC group.2)The alpha diversity decreased significantly in the PTB group among the three groups,while LTBI did not change;Beta diversity showed differences among the three groups.3)Eleven bacterial genera were screened out as potential microbial markers of PTB,with the area under the curve was85.9%;Six bacterial genera could be used as biomarkers for the progression of LTBI to PTB,and the area under the curve was 86.1%.And 5 bacterial genera could be used as biomarkers distinguish LTBI from HC,with the area under the curve was 77.8%.4)The functional prediction results of the microbiota showed that there were mainly differences between PTB/HC and PTB/LTBI groups,and the main differential metabolic pathways in two comparison groups were metabolism and genetic information processing,respectively.(2)Part II: In this part,30 samples were selected from each group,and a total of 90 samples were detected by LC-MS/MS.1)The metabolic spectrum of the three groups was analyzed by PCA,which showed that the PTB group was quite different from the other two groups,while the LTBI group and the HC group performed similarly.OPLS-DA analysis showed that the three groups could be clearly distinguished.2)According to the screening criteria(VIP>1,P<0.05),there were 218,256 and 80 differential metabolites in PTB/ HC groups,PTB/ LTBI groups and LTBI/HC groups,respectively,mainly lipids and lipid-like molecules.3)4,2 and 7 metabolites were screened for combined diagnosis in PTB/ HC groups,PTB/LTBI groups and LTBI/HC groups,respectively.The AUC values were all greater than 0.96,and the sensitivity and specificity were all above 86%.4)Functional enrichment analysis showed that protein metabolism was the main pathway enriched in PTB/HC and PTB/LTBI groups,and lipid metabolism was the main pathway enriched in LTBI/HC groups.5)Association analysis showed that the bacteria and metabolites with high diagnostic value were also highly correlated with each other;(3)Part III: The SQFFQ survey was conducted for all subjects.1)The dietary nutrients affecting the risk of disease were vitamin C,cholesterol,copper,isoleucine,phenylalanine,tyrosine,threonine,tryptophan,valine,alanine,glutamic acid,glycine and serine in PTB/HC groups;The dietary nutrients affecting the risk of disease progression were riboflavin,cholesterol,calcium,glutamate,folic acid,and saturated fatty acids in PTB/LTBI groups.The dietary nutrients affecting the risk of pathogen infection were carbohydrate,dietary fiber,niacin,vitamin A,vitamin E,potassium,sodium,lysine,alanine and aspartic acid in LTBI/HC groups.2)Association network shows: Ten amino acids were associated with Roseburia in PTB/HC groups.These 10 amino acids and the genus Roseburia were also found to be associated with the metabolites Niacinamide,benzenesul fonamide,Ginsenoside Rh2,Ellipticine and((+)-6-aminopenicillanic acid).PTB/LTBI groups had more pairwise correlation,the key dietary nutrients were Folic acid and calcium,and the key gut microbiota were Lachnospira and Roseburia,and the key metabolites were Genipin and Neoabietic acid.There was no pairwise correlation between LTBI/HC groups.Conclusion: This study clarified the changes in the diversity,composition and metabolic profile of gut microbiota in three groups,screened the gut microbiota and metabolite biomarkers that can be used for diagnosis in different disease states,and found the functional metabolic pathways that may be involved.At the same time,we also put forward the hypothesis that the gut microbiota and its metabolites can be changed by adjusting the intake of dietary nutrients to regulate the disease state of tuberculosis,which can provide theoretical basis for follow-up research and new ideas for the prevention and treatment of tuberculosis in this region.