Characteristic Analysis Of Gut Microbiota In Patients With Initial Pulmonary Tuberculosis Based On High-throughput Sequencing

Objective: The clinical symptoms of patients with initial smear-positive and smear-negative pulmonary tuberculosis,liver function,and imaging-related indexes were analyzed to investigate the differences in gut microbiota composition and phenotype between patients with smear-positive and smear-negative pulmonary tuberculosis and the healthy population,providing new ideas for early prevention and control,differential diagnosis and treatment of pulmonary tuberculosis.Methods: 1.178 patients admitted with initial pulmonary tuberculosis from April 2020 to December 2021 at Affiliated Changsha Central Hospital of University of South China,were collected and classified into81 cases of smear-positive pulmonary tuberculosis(Smear-positive pulmonary tuberculosis,SP-PTB)and 97 cases of smear-negative pulmonary tuberculosis(Smear-PTB)according to the clinical laboratory diagnosis.Univariate and regression of binary logistic analyses were used to analyze clinical data,liver function,CT imaging performance and risk factors for the development of sputum smear positivity in patients with pulmonary tuberculosis.2.Gut microbiota analysis was performed on a cohort of tuberculosis patients from method 1.A total of 119 volunteer’s stool specimens were included according to the inclusion and exclusion criteria and the results of gut microbiota sequencing,including 38 cases in the SP-PTB group,40 cases in the SN-PTB group,and 41 cases in the healthy control(HC)group.The PE150(pair-end 2×150 bp)sequencing was performed on the16 S r RNA gene V4 region of fresh stool samples,and the sequencing data were analyzed by bioinformatics to explore the differences of gut microbiota in different populations.Results:1.Variables that were significantly associated(P<0.05)with positive Mycobacterium smears in univariate analysis were included as covariates in binary logistic regression analysis,and pulmonary cavitation(OR =2.977,CI: 1.412-6.278,P = 0.004)and tracheobronchial tuberculosis(OR= 6.494,CI: 2.256-18.691,P = 0.001)were independent risk factors for positive sputum Mycobacterium smears in patients with pulmonary tuberculosis.2.Gut microbiota sequencing results: a total of 754 OTUs were obtained in the SP-PTB group,840 OTUs in the SN-PTB group,and 1212 OTUs in the HC group.3.The results of species annotation of gut microbiota: 21 species at the phylum level,30 species at the class level,72 species at the order level,135 species at the family level,and 366 species at the genus level.4.Analysis of gut microbiota diversity: compared with the HC group,theα diversity index(Shannon index,Simpson index,Chao1 index,ACE index)significantly decreased in both the SP-PTB and SN-PTB groups(SP-PTB group vs.HC group,P <0.05;SN-PTB group vs.HC group,P<0.05).And the difference between the SP-PTB and SN-PTB groups showed no statistically significant differences in α-diversity index(P >0.05).β diversity analysis showed no statistical significance between the SP-PTB and SN-PTB groups(P > 0.05),and both the SP-PTB and SN-PTB groups differed significantly compared with the HC group,respectively(SP-PTB vs HC group,P < 0.05;SN-PTB vs HC group,P <0.05).5.Analysis of gut microbiota composition: the relative abundance of the thick-walled phylum decreased in both the SP-PTB and SN-PTB groups compared with the HC group(SP-PTB vs HC group,P < 0.05;SN-PTB vs HC group,P < 0.05),and among the top 20 genera in terms of abundance,there were no group differences between the SP-PTB and SN-PTB groups of genera.There were 11 different genera in the SP-PTB group compared to the HC group,of which 9 belonged to the phylum Thick-walled,1 to the phylum Aspergillus and 1 to the phylum Bacteroidetes.9 different genera were found in the SP-PTB group compared to the HC group,of which 8 belonged to the phylum Thick-walled and 1 to the phylum Bacteroidetes.Changes in the taxonomic level of gut microbiota are mainly caused by changes in the relative abundance of flora at the genus level.6.Screening of biomarkers(Biomarker)between groups: compared to the HC group,Sutterellaceae family,Actinomycetes,Odoribacter,Desulfovibrionia,and Anaerosporobacter.Anaerosporobacter)were the dominant species in the intestine of the SP-PTB group.However,Turicibacter,Anaerosporobacter,Monoglobales,Monoglobaceae and Monoglobus were the predominant microflora in the SN-PTB group.7.Bugbase gut microbiota phenotype prediction: compared with the HC group,the SP-PTB group showed a decrease in the relative abundance of anaerobic and Gram-positive bacteria genes and an increase in the relative abundance of parthenogenic,Gram-negative bacteria and mobile bacteria genes.Compared with the HC group,the SN-PTB group showed a decrease in the relative abundance of Gram-positive bacteria genes and an increase in the relative abundance of Gram-negative bacteria and potentially pathogenic genes.Conclusion:1.Among them,pulmonary cavities and combined bronchial tuberculosis in TB patients are risk factors associated with the susceptibility of TB patients to develop smear-positive tuberculosis.2.No significant difference in the overall composition of the gut microbiota between patients with smear-positive and smear-negative pulmonary tuberculosis was performed,however,both showed decreased gut microbiota richness and homogeneity compared to healthy controls.Among them,the number of short-chain fatty acid-producing genera decreased significantly,and only Erysipelatoclostridium increased in the gut microbiota of TB patients,and Erysipelatoclostridium may be closely related to the occurrence and progression of pulmonary TB.3.The phenotypic analysis of the gut microbiota of the three groups revealed a decrease in the proportion of Gram-positive bacteria,an increase in the proportion of Gram-negative bacteria,and an increase in the proportion of aerobic and facultative bacteria in both groups of TB patients compared to healthy controls,which indicated that there is oxidative stress intolerance and dysbiosis of the gut microbiota in both groups of TB patients.