WGS on antibiotics-challenged Mycobacterium smegmatis. The roles of phenotypic, and genotypic stress-response of mycobacterial adaptation to first-line antituberculotics

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.