Mycobacterium smegmatis Maintains Genomic Stability Under First-Line Antitubercular Drug Stress

Core Concepts

Prolonged exposure to clinically used antibiotics does not lead to de novo adaptive mutagenesis in Mycobacterium smegmatis. The activation of DNA repair pathways preserves genomic integrity, while non-genetic factors convey quick adaptation for stress conditions.

Abstract

The study systematically investigated the effects of first-line antitubercular drugs (isoniazid, rifampicin, ethambutol, pyrazinamide) and a second-line drug (ciprofloxacin) on the genome stability, DNA repair system activation, and dNTP pool of the non-pathogenic mycobacterial model organism Mycobacterium smegmatis.
Key highlights:

Whole-genome sequencing revealed no significant increase in mutation rates after prolonged antibiotic exposure, except for the positive control UV treatment.
The activation of DNA repair pathways, particularly in response to the combination of first-line drugs, maintains genomic integrity.
Alterations in the cellular dNTP pools were observed upon individual drug treatments, but the combination treatment neutralized these effects.
A phenotypic fluctuation assay showed a much higher rate of emergence of antibiotic tolerance compared to the mutation rate, indicating that non-genetic factors drive rapid adaptation to antibiotics.
The results suggest that the mycobacterial genome is not prone to microevolution upon prolonged exposure to the tested antibiotics, and the development of drug resistance is more likely driven by non-genetic factors rather than de novo adaptive mutagenesis.

Stats

The mutation rate of the untreated M. smegmatis mc2155 strain was approximately 5 x 10-11 mutations per base pair per generation.
The mutation rates of the drug-treated lineages ranged from 2.5-20 x 10-11 mutations per base pair per generation, except for the UV treatment which had a significantly higher rate.
The estimated rate of emergence of ciprofloxacin tolerance was three orders of magnitude higher (10-7) than the mutation rate calculated from whole-genome sequencing.

Quotes

"Prolonged exposure to clinically used antibiotics did not lead to de novo adaptive mutagenesis in M. smegmatis under laboratory conditions."
"The activation of DNA repair pathways preserves genomic integrity, while non-genetic factors convey quick adaptation for stress conditions."

Key Insights Distilled From

Genetic Stability of Mycobacterium smegmatis under the Stress of First-Line Antitubercular Agents: Assessing Mutagenic Potential

by Moln... at www.biorxiv.org 02-22-2024

https://www.biorxiv.org/content/10.1101/2024.02.21.581394v2

Deeper Inquiries

How do the findings from this study on M. smegmatis translate to the more clinically relevant Mycobacterium tuberculosis, considering the differences in their pathogenicity and intracellular lifestyle

The findings from this study on M. smegmatis can provide valuable insights into the behavior of Mycobacterium tuberculosis, despite the differences in pathogenicity and intracellular lifestyle between the two species. While M. smegmatis is non-pathogenic and fast-growing, it shares many DNA repair pathways and metabolic processes with M. tuberculosis. Therefore, the molecular mechanisms uncovered in M. smegmatis can be extrapolated to M. tuberculosis to some extent. For example, the study's observation that prolonged exposure to antibiotics did not lead to de novo adaptive mutagenesis in M. smegmatis suggests that M. tuberculosis may also have mechanisms to maintain genomic stability under antibiotic pressure. This could imply that the emergence of drug resistance in M. tuberculosis may be driven more by phenotypic factors and non-genetic adaptations rather than solely through mutational changes. Understanding how M. smegmatis responds to antibiotic stress can provide a foundation for further investigations into the drug resistance mechanisms of M. tuberculosis.

What other cellular mechanisms, beyond DNA repair and dNTP pool regulation, might contribute to the rapid adaptation of mycobacteria to antibiotic stress without inducing de novo mutations

In addition to DNA repair and dNTP pool regulation, several other cellular mechanisms may contribute to the rapid adaptation of mycobacteria to antibiotic stress without inducing de novo mutations. One crucial aspect is the activation of efflux pumps, which can help the bacteria expel antibiotics from the cell and reduce their intracellular concentration, thereby decreasing the drug's effectiveness. Metabolic shifting and changes in cell wall composition are also important adaptive responses that can contribute to antibiotic tolerance. Furthermore, mycobacteria may undergo changes in gene expression, such as the upregulation of stress response genes, to cope with antibiotic-induced stress. Additionally, the formation of biofilms or the development of persister cells, which are dormant and highly tolerant to antibiotics, could play a role in the bacteria's ability to survive antibiotic exposure. These mechanisms collectively enable mycobacteria to adapt quickly to antibiotic stress and survive in challenging environments.

Could the insights from this study on the role of non-genetic factors in driving antibiotic tolerance be leveraged to develop novel treatment strategies that target these adaptive mechanisms and prevent the emergence of drug resistance in tuberculosis

The insights gained from this study on the role of non-genetic factors in driving antibiotic tolerance in mycobacteria could be leveraged to develop novel treatment strategies that target these adaptive mechanisms and prevent the emergence of drug resistance in tuberculosis. By understanding the pathways and processes that enable mycobacteria to survive antibiotic stress without inducing mutagenesis, researchers can identify potential drug targets to disrupt these adaptive responses. For example, developing inhibitors that target efflux pumps or disrupt metabolic shifting in response to antibiotics could enhance the efficacy of existing treatments. Additionally, strategies that prevent the formation of persister cells or biofilms could help eliminate antibiotic-tolerant bacterial populations. By focusing on disrupting these non-genetic adaptive mechanisms, novel treatment approaches could be developed to combat drug resistance in tuberculosis more effectively.