基于宏基因组测序的肺结核患者下呼吸道微生态菌落分布特点分析

Analysis of the distribution characteristics of microbial communities in the lower respiratory tract of pulmonary tuberculosis patients based on metagenomic sequencing

目的:探讨肺结核、其他病原感染肺炎及非感染性肺部疾病患者下呼吸道微生态菌落分布之间的差异。方法:采用回顾性研究方法,选取2021年3月1日至2023年2月28日于首都医科大学附属北京积水潭医院和北京大学人民医院住院的疑似肺结核患者作为研究对象,共83例。研究对象均痰涂片和结核感染T细胞斑点试验阴性,并按照诊疗常规完善支气管镜检查并进行支气管肺泡灌洗,支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)进行病原学检查(包括宏基因组二代测序、GeneXpert MTB/RIF检测,以及常规细菌、真菌培养)。利用电子病历系统收集研究对象的基本信息及实验室检查结果等数据资料,分析不同患者之间下呼吸道微生物群的特点。结果:83例研究对象根据病原学检查结果分为结核组(28例)、肺炎组(38例)和非感染组(17例)。下呼吸道微生物丰度及多样性分析显示,结核组、肺炎组和非感染组Shannon指数[中位数(四分位数)]分别为1.2864(0.3021,1.9459)、1.1032(0.3725,1.7711)、1.2049(0.6873,1.9974);Simpson指数[中位数(四分位数)]分别为0.5693(0.1346,0.8259)、0.5503(0.1900,0.7533)、0.5141(0.3065,0.7507);Chao1指数[中位数(四分位数)]分别为8.50(3.00,20.75)、7.50(3.25,18.75)、7.00(2.00,22.00),差异均无统计学意义(H值分别为0.130、0.208、0.235,P值分别为0.973、0.901、0.889);对于β多样性,结核组样本分布分散,组内异质性较大,而在肺炎组与非感染组间存在广泛重叠。病原微生物的群落组成分析显示,结核组样本中厚壁菌门、葡萄球菌属和链球菌属丰度较高;肺炎组样本中拟杆菌门、变形菌门、棒状杆菌属、肠球菌属及假单胞菌属丰度较高;非感染组样本中放线菌门、梭杆菌门,以及呼吸道定植菌罗氏菌属及奈瑟菌属等丰度较高。LEfSe线性判别分析发现,结核组中结核分枝杆菌、黏滑罗斯菌、非典型韦荣氏球菌显著富集;而在肺炎组中纹带棒状杆菌、肺炎克雷伯菌、嗜麦芽假单胞菌明显富集;与结核组和肺炎组比较,非感染组则显示出12种微生物富集,菌种之间和上述两组存在明显差异,且更接近上呼吸道微生态菌群。在病原微生物的丰度及多样性方面,结核组和肺炎组间Shannon指数[中位数(四分位数)]分别为0.0260(0.0000,0.5283)和0.2000(0.0000,0.3335);两组Simpson指数[中位数(四分位数)]分别为0.0080(0.0000,0.5609)和0.0980(0.0000,0.4379);两组Chao1指数[中位数(四分位数)]分别为2.00(1.00,2.25)和2.00(1.00,4.00);差异均无统计学意义(Z值分别为-0.794、-1.116、-1.582,P值分别为0.430、0.260、0.110)。两组间的病原微生物菌群β多样性有明显区别,结核组样本中共检测到16种病原体,肺炎组共检出40种病原体。微生物相关性分析显示仅有结核分枝杆菌复合群与大部分微生物之间呈负相关。结论:结核分枝杆菌感染后,会影响患者下呼吸道微生态菌落分布,虽然对微生物菌群的α多样性影响较小,但对微生物菌群β多样性的影响明显高于其他病原体感染所致肺炎患者和非感染性肺部疾病患者。

Objective:To explore the differences in the distribution of microbial communities in the lower respiratory tract among patients with pulmonary tuberculosis,other pathogenic infections,and non-infectious pulmonary diseases.Methods:In this retrospective study,83 patients suspected of having pulmonary tuberculosis,admitted to Beijing Jishuitan Hospital affiliated with Capital Medical University and Peking University People’s Hospital between March 1,2021,and February 28,2023,were selected for analysis.All study subjects were negative for sputum smears and T-SPOT.TB tests,and received bronchoscopy with bronchoalveolar lavage as per standard diagnostic procedures.Pathogen testing on bronchoalveolar lavage fluid(BALF)included metagenomic next-generation sequencing,GeneXpert MTB/RIF assays,and conventional bacterial and fungal cultures.Basic patient information and laboratory test results were collected via the electronic medical record system,enabling the analysis of lower respiratory tract microbiota characteristics across diverse patient groups.Results:Following pathogenetic analysis,the cohort was categorized into tuberculosis(28 patients),pneumonia(38 patients),and non-infection groups(17 patients).Examination of microbial abundance and diversity within the lower respiratory tract revealed median Shannon indices of 1.2864(IQR:0.3021,1.9459),1.1032(IQR:0.3725,1.7711),and 1.2049(IQR:0.6873,1.9974)for the tuberculosis,pneumonia,and non-infection groups respectively.Similarly,median Simpson indices were 0.5693(IQR:0.1346,0.8259),0.5503(IQR:0.1900,0.7533),and 0.5141(IQR:0.3065,0.7507),and Chao1 indices were 8.50(IQR:3.00,20.75),7.50(IQR:3.25,18.75),and 7.00(IQR:2.00,22.00)respectively,with no significant statistical differences noted(H-values of 0.130,0.208,0.235;P-values of 0.973,0.901,and 0.889).β-Diversity analysis showed a dispersed distribution within the tuberculosis group indicating notable within-group heterogeneity,whereas the pneumonia and non-infection groups exhibited extensive overlap.Pathogenic microbial community analysis indicated heightened prevalence of Firmicutes,Staphylococcus,and Streptococcus in tuberculosis group samples;Bacteroidetes,Proteobacteria,Corynebacterium,Enterococcus,and Pseudomonas were notably prevalent in pneumonia group samples;whereas Actinobacteria,Fusobacteria,and respiratory colonizers such as Rothia and Neisseria showed increased abundance in non-infection group samples.LEfSe analysis revealed significant enrichment of Mycobacterium tuberculosis,Streptococcus mucosus,and atypical Veillonella in the tuberculosis group;Corynebacterium striatum,Klebsiella pneumoniae,and Pseudomonas maltophilia were prominently enriched in the pneumonia group;in contrast,the non-infection group exhibited a diversification of 12 microbial species,marking a notable deviation from the pathogenic profiles observed in the tuberculosis and pneumonia groups and bearing closer resemblance to the microbiota of the upper respiratory tract.Conclusion:Infection with Mycobacterium tuberculosis influences the distribution of microbial communities in the lower respiratory tract,exerting minimal effects onαdiversity.Notably,its impact onβdiversity significantly surpasses that observed in pneumonia caused by other pathogens and in non-infectious pulmonary conditions.