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

Reviewer #1 (Public Review):

In this manuscript, Molnar, Suranyi and colleagues have probed the genomic stability of Mycobacterium smegmatis in response to several anti-tuberculosis drugs as monotherapy and in combination. Unlike the study by Nyinoh and McFaddden http://dx.doi.org/10.1002/ddr.21497 (which should be cited), the authors use a sub-lethal dose of antibiotic. While this is motivated by sound technical considerations, the biological and therapeutic rationale could be further elaborated. The results the authors obtain are in line with papers examining the genomic mutation rate in vitro and from patient samples in Mycobacterium tuberculosis, in vitro in Mycobacterium smegmatis and in vitro in Mycobacterium tuberculosis (although the study by HL David (PMID: 4991927) is not cited). The results are confirmatory of previous studies. It is therefore puzzling why the authors propose the opposite hypothesis in the paper (i.e antibiotic exposure should increase mutation rates) merely to tear it down later. This straw-man style is entirely unnecessary. The results on the nucleotide pools are interesting, but the statistically significant data is difficult to identify as presented, and therefore the new biological insights are unclear. Finally, the authors show that a fluctuation assay generates mutations with higher frequencies that the genetic stability assays, confirming the well-known effect of phenotypic antibiotic resistance.

Reviewer #2 (Public Review):

In this study, the authors assess whether selective pressure from drug chemotherapy influences the emergence of drug resistance through the acquisition of genetic mutations or phenotypic tolerance. I commend the authors on their approach of utilizing the mutation accumulation (MA) assay as a means to answer this and whole genome sequencing of clones from the assay convincingly demonstrates low mutation rates in Mycobacteria when exposed to sub-inhibitory concentrations of antibiotics. Also, quantitative PCR highlighted the upregulation of DNA repair genes in Mycobacteria following drug treatment, implying the preservation of genomic integrity via specific repair pathways.

Even though the findings stem from M. smegmatis exposure to antibiotics under in vitro conditions, this is still relevant in the context of the development of drug resistance so I can see where the authors' train of thought was heading in exploring this. However, I think important experiments to perform to more fully support the conclusion that resistance is largely associated with phenotypic rather than genetic factors would have been to either sequence clones from the ciprofloxacin tolerance assay (to show absence/ minimal genetic mutations) or to have tested the MIC of clones from the MA assay (to show an increase in MIC). There seems to be a disconnect between making these conclusions from experiments conducted under different conditions, or perhaps the authors can clarify why this was done. With regards to the sub-inhibitory drug concentration applied, there is significant variation in the viability as calculated by CFUs following the different treatments and there is evidence that cell death greatly affects the calculation of mutation rate (PMCID: PMC5966242). For instance, the COMBO treatment led to 6% viability whilst the INH treatment led to 80% cell viability. Are there any adjustments made to take this into account? It would also be useful to the reader to include a supplementary table of the SNPs detected from the lineages of each treatment - to determine if at any point rifampicin treatment led to mutations in rpoB, isoniazid to katG mutations, etc. Overall, while this study is tantalizingly suggestive of phenotypic tolerance playing a leading role in drug resistance (and perhaps genetic mutations a sub-ordinate role) a more substantial link is needed to clarify this.

Reviewer #3 (Public Review):

Summary:

This manuscript describes how antibiotics influence genetic stability and survival in Mycobacterium smegmatis. Prolonged treatment with first-line antibiotics did not significantly impact mutation rates. Instead, adaptation to these drugs appears to be mediated by upregulation of DNA repair enzymes. While this study offers robust data, findings remain correlative and fall short of providing mechanistic insights.

Strengths:

The strength of this study is the use of genome-wide approaches to address the specific question of whether or not mycobacteria induce mutagenic potential upon antibiotic exposure.

Weaknesses:

The authors suggest that the upregulation of DNA repair enzymes ensures a low mutation rate under drug pressure. However, this suggestion is based on correlative data, and there is no mechanistic validation of their speculations in this study.

Furthermore, as detailed below, some of the statements made by the authors are not substantiated by the data presented in the manuscript.

Finally, some clarifications are needed for the methodologies employed in this study. Most importantly, reduced colony growth should be demonstrated on agar plates to indicate that the drug concentrations calculated from liquid culture growth can be applied to agar surface growth. Without such validations, the lack of induced mutation could simply be due to the fact that the drug concentrations used in this study were insufficient.

Author response:

Reviewer #1:

The phenomenon of stress-inducible mutagenesis in bacterial evolution remains a topic of heated debate. Consequently, the emergence of genetically encoded resistance may stem from either microevolution or the dissemination of pre-existing variants from polyclonal infections under drug pressure. We believe that the Introduction presents both of these hypotheses in a balanced manner to elucidate the rationale behind our mutation accumulation investigations.

While we acknowledge the well-known existence of phenotypic antibiotic resistance, it's worth noting that conclusions regarding mutation rates are often drawn from fluctuation assays without confirmation of genetic-level changes. This discrepancy persists despite fluctuation assays accounting for both phenotypic and genotypic alterations. Combining genome sequencing with fluctuation assays underscores the importance of making this distinction.

Thank you for the suggestion regarding improving the figures; we will incorporate these changes accordingly in the revised version. Additionally, we will address the rationale for using sub-lethal doses of antibiotics and compare our results with the referenced papers.

Reviewer #2:

Thank you for acknowledging the values of the manuscript and for the insightful suggestions for improvement. We agree on the necessity to directly connect the mutation accumulation experiments with the tolerance assay, and we have already initiated additional experiments to integrate into a revised version.

We also agree with and have been aware of the notion that cell death affects the calculation of the mutation rate. However, the error in the estimation of the generation time leads to an overestimation of the mutation rate, which, in our case, reinforces the conclusion that no discernible increase in mutation rate occurs in our mutation accumulation experiment. In the revised version, we aim to address i) the source of variation in cell death degree and ii) its influence on calculations.

The SNPs identified from the lineages of each treatment are compiled in the "unique muts.xls" file within the Figshare document bundle we included with the manuscript. We regret not providing a detailed reference to this in the manuscript; instead, the Figshare files were merely mentioned under the Data Availability section (No. 6) without specifics. As advised, we will create a supplementary table containing this data.

Reviewer #3:

Thank you for appreciating the manuscript's merits and for the instructive suggestions (also articulated in the specific comments). We agree that we should show the data on reduced colony growth on agar plates to demonstrate that the drug concentrations used in the study are relevant. We will include this in the revised version, as well as changes in response to all specific comments.

We acknowledge that the observed upregulation of DNA repair enzymes and the low mutation rates under drug pressure represent correlative data. Therefore, we opted against presenting the qPCR results as a mechanistic explanation. In the manuscript, we carefully stated: "The observed upregulation of the relevant DNA repair enzymes might account for the low mutation rate even under drug pressure." We did not establish a mechanistic link or emphasize the repair activation in the title, abstract, or discussion. We recognize the necessity for a new series of targeted experiments to provide mechanistic explanations. In this paper, our aim is to convincingly demonstrate that antibiotic pressure did not induce the occurrence of new adaptive mutations.