WGS on antibiotics-challenged Mycobacterium smegmatis

Tuberculosis is still one of the most frequent infectious diseases worldwide, with an estimated 10 million active, 1.7 billion latent cases, and 1.5 million deaths. The sustained success of Mycobacterium tuberculosis as a pathogen is due to its ability to survive inside macrophages for long periods and its highly unresponsive nature to antibiotics. Additionally, there is a high incidence of resistance against the few available antituberculosis drugs. In most bacteria, horizontal gene transfer is the major molecular mechanism driving the evolution of drug resistance. However, Mycobacterium tuberculosis seems to acquire antibiotic resistance merely by single point mutations. The molecular background of stress adaptation and mutability is mostly unknown in Mycobacteria. To gain new insights into the connections between stress adaptation, genetic variability, and the significance of these phenomena in Mycobacterial survival strategies, we systematically investigated the effects of currently used antibiotics on genome stability, the activation of the DNA repair system, and the dNTP pool using Mycobacterium smegmatis, a non-pathogenic model organism for the medically relevant Mycobacterium species. Using whole genome sequencing, we found that following long-term exposure to first-line antibiotics, hardly any increase in the mutation rate can be detected. However, the phenotypic fluctuation assay showed quick adaptation to stress conditions conveyed by nongenetic factors. The increase in the expression of DNA repair genes we measured using qPCR suggests that genomic integrity is maintained by activating specific DNA repair pathways. Our findings, indicating that exposure to antibiotics does not lead to the emergence of de novo adaptive mutagenesis, question the prevailing hypothesis of antibiotic resistance development driven by microevolution.

结核病仍是全球范围内最常见的传染病之一，据估算，当前全球共有1000万活动性结核病例、17亿潜伏性结核感染者，以及150万结核相关死亡病例。结核分枝杆菌（Mycobacterium tuberculosis）作为致病菌的持续致病能力，源于其可长期存活于巨噬细胞内的特性，以及对抗生素极高的不敏感性。此外，现有少数可用抗结核药物的耐药发生率居高不下。在多数细菌中，水平基因转移（horizontal gene transfer）是推动耐药性进化的主要分子机制，但结核分枝杆菌似乎仅通过单点突变（single point mutations）即可获得抗生素耐药性。分枝杆菌的应激适应与突变性的分子背景目前仍未明确。为深入探究应激适应、遗传变异之间的关联，以及这些现象在分枝杆菌生存策略中的重要意义，我们以与医学相关的分枝杆菌属非致病性模式菌株——耻垢分枝杆菌（Mycobacterium smegmatis）为研究对象，系统探究了当前临床使用的抗生素对基因组稳定性、DNA修复系统激活状态，以及脱氧核苷三磷酸（dNTP）池的影响。通过全基因组测序（whole genome sequencing），我们发现长期暴露于一线抗生素后，突变率几乎未出现可检测到的升高。然而，表型波动试验（phenotypic fluctuation assay）结果显示，菌株可通过非遗传因素快速适应应激环境。我们通过定量聚合酶链反应（quantitative PCR，qPCR）检测到DNA修复基因的表达水平上调，这表明菌体可通过激活特定的DNA修复通路维持基因组完整性。我们的研究结果显示，抗生素暴露并不会诱导从头适应性诱变（de novo adaptive mutagenesis）的产生，这对“由微进化驱动抗生素耐药性形成”这一主流假说提出了质疑。