Table_3_Effects of Host, Sample, and in vitro Culture on Genomic Diversity of Pathogenic Mycobacteria.xlsx

Mycobacterium tuberculosis (M. tb), an obligate human pathogen and the etiological agent of tuberculosis (TB), remains a major threat to global public health. Comparative genomics has been invaluable for monitoring the emergence and spread of TB and for gaining insight into adaptation of M. tb. Most genomic studies of M. tb are based on single bacterial isolates that have been cultured for several weeks in vitro. However, in its natural human host, M. tb comprises complex, in some cases massive bacterial populations that diversify over the course of infection and cannot be wholly represented by a single genome. Recently, enrichment via hybridization capture has been used as a rapid diagnostic tool for TB, circumventing culturing protocols and enabling the recovery of M. tb genomes directly from sputum. This method has further applicability to the study of M. tb adaptation, as it enables a higher resolution and more direct analysis of M. tb genetic diversity within hosts with TB. Here we analyzed genomic material from M. tb and Mycobacterium bovis populations captured directly from sputum and from cultured samples using metagenomic and Pool-Seq approaches. We identified effects of sampling, patient, and sample type on bacterial genetic diversity. Bacterial genetic diversity was more variable and on average higher in sputum than in culture samples, suggesting that manipulation in the laboratory reshapes the bacterial population. Using outlier analyses, we identified candidate bacterial genetic loci mediating adaptation to these distinct environments. The study of M. tb in its natural human host is a powerful tool for illuminating host pathogen interactions and understanding the bacterial genetic underpinnings of virulence.

结核分枝杆菌（M. tb），一种严格的宿主特异性病原体，也是结核病（TB）的致病因素，始终是全球公共卫生的重大威胁。比较基因组学在监测TB的兴起与传播，以及深入了解M. tb的适应性方面具有无价的价值。大多数关于M. tb的基因组学研究均基于在体外培养数周的单一细菌分离株。然而，在其自然的人类宿主中，M. tb构成复杂，有时庞大的细菌种群，这些种群在感染过程中会多样化，无法完全由单个基因组来表征。近期，杂交捕获技术已被用作快速诊断TB的工具，绕过了培养程序，并允许直接从痰液中恢复M. tb基因组。该方法在研究M. tb适应性方面具有更广泛的应用，因为它能够实现更高分辨率和更直接的分析，从而揭示TB宿主体内M. tb的遗传多样性。在本研究中，我们利用宏基因组学和Pool-Seq方法分析了直接从痰液和培养样本中捕获的M. tb和牛分枝杆菌（M. bovis）的基因组材料。我们确定了采样、患者和样本类型对细菌遗传多样性的影响。细菌遗传多样性在痰液中比在培养样本中更为多变，且平均更高，这表明实验室操作会重塑细菌种群。通过离群值分析，我们确定了介导适应这些不同环境的候选细菌遗传位点。在自然的人类宿主中研究M. tb是一种强大的工具，有助于阐明宿主与病原体之间的相互作用，并理解细菌遗传学在致病性基础中的作用。