关键词: 结核,肺, 细菌, 16S rRNA测序, 多样性, 表型

Abstract:

Objective To study the differences of composition and phenotypes of intestinal flora between diagnosed pulmonary tuberculosis(PTB) patients and healthy individuals by using high-throughput sequencing technology and bio-informatics analysis. Methods PE 150 (pair-end 2×150 bp) sequencing scheme was used to sequence flora 16S rRNA gene V4 region from fresh stool samples of 29 primary bacteriologically-confirmed PTB patients (PTB group)and 28 healthy volunteers (healthy controls) at Changsha Central Hospital,University of South China from April 14, to December 28, 2020. Operational taxonomic units (OTU) were annotated by aligning with Silva database (Silva 128). QIIME software was used to calculate OTU level, α diversity index, β diversity index and relative abundance at phylum and genus levels, and to generate OTU level abundance grade curve (Ranked abundance). R software package “VennDiagram” was used to generate VennDiagram. R order “ggplot” was used to draw a horizontal bubble diagram. BugBase software was used to classify and compare 7 phenotypes of intestinal flora. Results For the α diversity index, the Shannon index of intestinal flora in the PTB group and the health controls were 4.24±1.71 and 5.64±0.85 respectively (t=3.889, P<0.01), Simpson index were (0.89 (0.80,0.94)) and (0.94 (0.90,0.96)) (Z=-3.256, P<0.01), Chao1 index were 1085.35±659.99 and 1844.04±658.95 (t=4.378, P<0.01), ACE index were 1091.64±666.93 and 1857.34±657.90 (t=4.403, P<0.01). The difference of β diversity between the PTB group and the healthy control group was statistically significant (R²=0.253, P<0.01).Twelve bacterial genera with significant differences between groups were found, which were mainly concentrated in Firmicutes (PTB group: 35.44%, control group: 51.55%, Z=-3.290,P<0.01). Phenotypic analysis showed that the relative abundance of anaerobic gene, intestinal gram-positive bacteria in the intestinal flora of the PTB group were lower than that of the health controls (21.80% vs 26.50%, Z=-3.080, P<0.01; 13.86% vs 17.75%, Z=-2.283, P<0.05). The relative abundance of intestinal gram-negative bacteria, intestinal facultative bacteria, oxidative stress tolerance bacteria and the ability of biofilm formation in the intestinal flora of the PTB group was higher than the health controls (15.14% vs 10.25%, Z=-2.283, P<0.05; 5.00% vs 0.59%, Z=-3.240, P<0.01; 3.10% vs 0.35%, Z=-2.075, P<0.05; 4.94% vs 1.19%, Z=-2.267, P<0.05). Conclusion The PTB group got changes in the composition of intestinal flora and bacterial phenotypes, which were mainly manifested as decrease in the diversity of the intestinal flora. The phenotypic changes were mainly caused by changes in the intestinal oxygen environment.

Key words: Tuberculosis,lung, Bacteria, 16S rRNA sequencing, Diversity, Phenotype