Reactive Oxygen Detoxification Contributes to Mycobacterium abscessus Antibiotic Survival
Curation statements for this article:-
Curated by eLife
eLife Assessment
Using a transposon sequencing (TN-seq) approach, the authors identified key genetic determinants of drug tolerance in Mycobacterium abscessus. Given that M. abscessus is inherently resistant to multiple antibiotics, this valuable study makes a significant contribution by uncovering how antibiotic tolerance is linked to reactive oxygen species (ROS) in this non-tuberculous mycobacterial (NTM) species. The solid findings further strengthen the growing evidence that ROS play a central role in the mechanism of antibiotic action and tolerance in mycobacteria. However, the use of words persistence or tolerance should follow the consensus definition given in the Balaban 2019 Nat Rev Micro paper.
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
When a population of bacteria is exposed to a bactericidal antibiotic, most cells die rapidly. However, a sub-population of antibiotic-tolerant cells known as “persister cells” can survive for prolonged periods. In addition, antibiotic tolerance can be broadly induced throughout the population by stresses such as nutrient deprivation. However, the pathways required to maintain viability in this setting, and how stress induces antibiotic tolerance are both poorly understood. To identify genetic determinants of antibiotic tolerance in mycobacteria, we carried out transposon mutagenesis insertion sequencing (Tn-Seq) screens in Mycobacterium abscessus (Mabs) exposed to bactericidal translation-inhibiting antibiotics. This analysis identified genes essential for the survival of both spontaneous persister cells as well as for stress-induced tolerance, allowing the first genetic comparison of these states in mycobacteria. Pathway analysis identified multiple genes involved in the detoxification of reactive oxygen species (ROS), including the catalase-peroxidase katG, which contributed to survival in both unstressed and nutrient-starved cells. In addition, we found that endogenous ROS were generated by translation-inhibiting antibiotics, and that hypoxia impaired bacterial killing. KatG specifically contributed to survival following exposure to transcription or translation inhibitors, but not other antibiotic classes tested. Thus, the lethality of some antibiotics is amplified by toxic ROS accumulation, and antibiotic-tolerant cells require detoxification systems to remain viable. These findings further demonstrate that antibiotic-induced ROS plays a broad role in mediating antibiotic lethality across diverse organisms.
Article activity feed
-
eLife Assessment
Using a transposon sequencing (TN-seq) approach, the authors identified key genetic determinants of drug tolerance in Mycobacterium abscessus. Given that M. abscessus is inherently resistant to multiple antibiotics, this valuable study makes a significant contribution by uncovering how antibiotic tolerance is linked to reactive oxygen species (ROS) in this non-tuberculous mycobacterial (NTM) species. The solid findings further strengthen the growing evidence that ROS play a central role in the mechanism of antibiotic action and tolerance in mycobacteria. However, the use of words persistence or tolerance should follow the consensus definition given in the Balaban 2019 Nat Rev Micro paper.
-
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes …
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.
Overall impact:
Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These observations help solidify the field. The work raises an important unanswered question: why are rifampicin and many protein synthesis inhibitors bacteriostatic with E. coli but bactericidal with pathogenic mycobacteria?
Comments on revisions:
I call attention to word choice, because it can indicate how familiar the authors are with the field. An issue that caught my attention was the use of the words persistence and tolerance, because they are not uniformly used in the generally accepted way (see Balaban 2019 Nat Rev Micro). In this consensus statement persistence refers specifically to a subpopulation and as such has survival kinetics that are distinct from those seen with tolerance, a phenomenon that refers to the entire population. I notice that the Balaban paper is not in the reference list. My suggestion is to take a look at the Balaban paper and then examine every use of the words tolerance and persistence in the manuscript to be sure that they fit the Balaban definition.
-
Author response:
The following is the authors’ response to the previous reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported …
Author response:
The following is the authors’ response to the previous reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tnseq was carried out to identify genes involved in spontaneous (nutrient rich) or starvationinduced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a significant persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.
Strengths:
While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.
Weaknesses:
Amongst the 5 shortlisted candidates from the screen, only 2 showed marginal phenotypes which limits the impact of the screening approach.
We appreciate the reviewer’s comments, but we note that 4 out of 5 genes displayed phenotypes concordant with findings of the Tn-Seq data, with katG and pafA, as well as MAB_1456c (during starvation only) and blaR (in rich media only) having decreased survival as shown in Figure 3A-D. We do agree that some of the phenotypes were more modest in a single-mutant context than in the pooled Tn-Seq screen. In addition, several mutants that had modest changes in survival also showed profound defects in resuming growth after removal of antibiotics, with the pafA mutants particularly impaired. (Figure 3 - figure supplement 1).
While the role of KatG mediated detoxification of ROS and involvement of ROS in antibiotic killing was well demonstrated, the lack of replication of this phenotype in some of the clinical isolates limits the significance of these findings.
While the role of katG varied among strains, the antibiotic-induced accumulation of ROS was seen in all three strains (Figure 6A). This suggests that in some strains other ROS-detoxification pathways are able to compensate for the loss of katG.
(Figure 2—figure supplements 1–3)
Figure 1—figure supplement 1.
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.
Weaknesses:
The work can be improved by a more comprehensive treatment of prior work, especially comparison of E. coli work with mycobacterial studies.
Moreover, the work still has some technical issues to fix regarding description of the methods, supplementary material, and reference formating.
See detailed responses below.
Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are fieldsolidifying observations.
Comments on revisions:
The authors have moved this paper along nicely. I have a few general thoughts.
It would be helpful to have more references to specific figures and panels listed in the text to make reading easier.
Text modified to add more figure references.
(1) I would suggest adding a statement about the importance of the work. From my perspective, the work shows the general nature of many statements derived from work with E. coli. This is important. The abstract says this overall, but a final sentence in the abstract would make it clear to all readers.
We appreciate the suggestion and have added a line to the abstract.
(2) The paper describes properties that may be peculiar to mycobacteria. If the authors agree, I would suggest some stress on the differences from E. coli. Also, I would place more stress on novel findings. This might be done in a section called Concluding Remarks. The paper by Shee 2022 AAC could be helpful in phrasing general properties.
We have added mention of this in the discussion (lines 354-356).
(3) Several aspects still need work to be of publication quality. Examples are the materials table and the presentation of supplementary material. Reference formatting also needs attention.
We respond to the specific details below.
Reviewer #3 (Public review):
Summary:
The manuscript demonstrates that starvation induces persister formation in M. abscesses.
They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.
Strengths:
The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.
Weaknesses:
The findings could not be validated in clinical strains.
Comments on revisions: No more comments for the authors.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The authors are strongly encouraged to check the references. There is some systematic error in the citations of references. Started to list but then they were too many.
For example Ln 51, Ref #11 cited, should be #10. Ln 59, #18 is wrongly cited. Should be - Ln 104. Ref #27 wrongly cited.
Ref #26 and #28 identical.
Even in discussion section a lot of references are mis-cited.
We very much appreciate the reviewer catching this issue with the import of our references and we have corrected this.
Reviewer #2 (Recommendations for the authors):
Below I have listed comments on specific issues that I hope are useful during revision.
Line 21 population is singular
Text modified
Line 21 comma after antibiotic (subordinate clause) Line
Text modified
25 is how singular?
Text modified
Impression of abstract: the work seems to confirm and therefore generalize concepts derived from studies with E. coli. If the authors agree, such a statement would be appropriate as a final sentence. I would also look for novel features to stress in the abstract.
Line 41 this challenge is vague
Text modified
Line 43 comma such as (also comma at the end of the parenthetical statement). This type of comma error is common throughout the manuscript and slows reading.
Text modified
Line 60 paradoxically. Is this the best concept? Or is it the natural effect of evolution (assuming that mycobacteria or their ancestors were exposed to environmental antibiotics)?
It is certainly problematic for clearing infection.
Text not modified.
Line 63 highlighted uncertainties ... meaning is unclear especially since you may have changed what "model" is referring to.
Text modified
Line 66 models.... Do you really mean systems? Models of what?
This refers to mechanistic models. Text not modified.
Line 67 arrest cell division. This is written as if it were true. Does the evidence point specifically to cell division or perhaps more accurately suppression of metabolism (see Ye et al 2025 mBio).
Both have been postulated as important. Text modified to add concept of metabolism
... targeted by antibiotics non-essential... Do you think that antibiotics work by inactivating essential targets? That seems overly simplistic, as lethal action is more likely the metabolic response to the damage caused. By the end of the paragraph you come around to this view, but you have already misdirected the reader. The reader is not sure what to believe. Line 70 note that there are many inhibitors of transcription and translation that only block growth, they do not rapidly kill cells
There can be both direct, and indirect secondary killing mechanisms. We devote a significant portion of the Discussion section to this topic.
Line 71 debate. There was indeed a debate, but reference 22 is not a valid citation for this. I think you mislead the reader by not accurately describing the debate. It was basically about the inability of Kim Lewis and James Imlay to reproduce the work of ref. 22. A great deal of prior work and then subsequent work showed that the challenge to ref. 22 lacked substance.
(1) Text modified to fix an error in the citation number related to direct β-lactam-mediated lysis.
(2) We agree that there is a great deal of data supporting antibiotic-induced ROS as important for bactericidal activity in many circumstances and do not argue otherwise. This sentence points out that over the years the paradigm for how antibiotics kill bacteria has evolved.
Line 80. It seems you are starting a new topic here. What about beginning a new paragraph?
The paragraph introduces mycobacteria of which Mabs is one. Text not modified.
Line 85 delete the comma: it implies a compound sentence that is not delivered.
Text modified.
Line 109 screen singular
Text modified.
Line 156 these conditions is imprecise and vague
Conditions were described in paragraph above in the manuscript. Text not modified.
Fig 2 it would be helpful to more clearly define the meaning of the coordinates
Text modified.
Line 230 and throughout please indicate the location of the data being cited for rapid reader reference
Text modified.
Lines 315-323 You could use this paragraph as the first of the Discussion. Some readers prefer to read the Discussion before the results. For them, a summary at the beginning of the Discussion is useful.
Text modified.
Line 328 without underlying mechanism... for E. coli refer to Zeng PNAS 2022. Depending on when the final version of this paper happens, there should be a figure in a Zhao Zhu mLife paper on purA that will have been published. Since it is not yet available, it cannot be cited.
We agree that the Zeng et al study is interesting and have added this reference to our discussion. However, these findings related to broad Crp-regulated tolerance actually underscore the point that we are making: that there are multiple factors (Crp, RelA, Lon, TisB, MazE, others) that mediate antibiotic tolerance.
Line 339 where are the data?
These data are in Figure 5, panels C, D. We have clarified the text to indicate that only a single agent from each of these classes was tested.
Line 346 here you are summarizing evidence for ROS in killing mycobacteria. You should include the moxifloxacin study by Shee et al 2022 AAC.
Reference added.
Line 348 refer to James Collins' work with E. coli in which his lab examined agents with a variety of mechanisms. There seems to be a fundamental difference between E. coli and mycobacteria with respect to rifampicin, a strictly static agent in E. coli but clearly lethal in mycobacteria. Note that chloramphenicol is static in E. coli and blocks ROS production. What does it do in mycobacteria? A brief discussion of this difference might be relevant at line 362
Text modified.
Lines 364-368 Here the idea might be simply that there are two modes of killing, one that is a direct extension of class-specific damage (chromosome fragmentation with fluoroquinolones, for example, or cell lysis by beta-lactams) and a second that is a metabolic response to the antibiotic damage (ROS accumulation). The second type is not class specific. Within this context, the mycobacterial killing by rifampicin might be a class-specific extension of inhibition of transcription that does not occur in E. coli.
Agreed, text modified to include this.
Line 400 The Key Resource table is not of publication quality. Precision and repeatability can be improved by spelling out the name of the vendor and its location (City, Country). In the present case, use of BD is lab jargon.
We appreciate the reviewer’s precision. However, this is actually not lab jargon. Becton, Dickinson and Company now refers to itself as BD (see https://www.bd.com/en-us), and the American Type Culture Collection now refers to itself as ATCC (see https://www.atcc.org/about-us/who-we-are).
Line 639 It would be good to have experienced colleagues critically review the manuscript, especially for English usage. Listing those persons here adds to the credibility of the work
Text not changed.
References: please refer to the journal style. Here you use italic for titles and scientific names, thereby obscuring the scientific names. Normally article titles are not italic and scientific names are ALWAYS italic unless prohibited by journal style.
Our reference format is concordant with eLife submission guidelines, and all references are reformatted by the journal at the time of final publication (see https://elifesciences.org/insideelife/a43f95ca/elife-references-yes-we-take-any-format-no-we-re-not-rekeying).
Supplemental Material: Please refer to journal style. Normally this is a stand-alone document that includes a title page and carefully crafted figure legends. Supplemental figures would be numbered as 1, 2, ... A professional appearing Supplemental Material section shows author publication experience not obvious in other parts of the paper. The text indicated MIC determinations. I would like to see a table of MIC values.
(1) MIC table added as Supplemental Table 5.
(2) The Supplemental figures are submitted and named in accordance with eLife instructions. Please note that for eLife, there is not a stand-alone supplementary figure section with a title page as you are requesting, but instead the figure supplements for each figure are provided as online files linked to each figure.
-
-
-
-
eLife Assessment
Using a transposon sequencing (TN-seq) approach, the authors identified key genetic determinants of drug tolerance in Mycobacterium abscessus. Given that M. abscessus is inherently resistant to multiple antibiotics, this valuable study makes a significant contribution by uncovering how antibiotic tolerance is linked to reactive oxygen species (ROS) in this non-tuberculous mycobacterial (NTM) species. The solid findings further strengthen the growing evidence that ROS play a central role in the mechanism of antibiotic action and tolerance in mycobacteria. However, the manuscript would benefit from improved clarity of presentation and corrections in the reference section.
-
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for …
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tn-seq was carried out to identify genes involved in spontaneous (nutrient rich) or starvation-induced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a significant persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.
Strengths:
While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.
Weaknesses:
Amongst the 5 shortlisted candidates from the screen, only 2 showed marginal phenotypes which limits the impact of the screening approach.
While the role of KatG mediated detoxification of ROS and involvement of ROS in antibiotic killing was well demonstrated, the lack of replication of this phenotype in some of the clinical isolates limits the significance of these findings.
-
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes …
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.
Weaknesses:
The work can be improved by a more comprehensive treatment of prior work, especially comparison of E. coli work with mycobacterial studies.
Moreover, the work still has some technical issues to fix regarding description of the methods, supplementary material, and reference formating.Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are field-solidifying observations.
Comments on revisions:
The authors have moved this paper along nicely. I have a few general thoughts.
(1) It would be helpful to have more references to specific figures and panels listed in the text to make reading easier.
(2) I would suggest adding a statement about the importance of the work. From my perspective, the work shows the general nature of many statements derived from work with E. coli. This is important. The abstract says this overall, but a final sentence in the abstract would make it clear to all readers.
(3) The paper describes properties that may be peculiar to mycobacteria. If the authors agree, I would suggest some stress on the differences from E. coli. Also, I would place more stress on novel findings. This might be done in a section called Concluding Remarks. The paper by Shee 2022 AAC could be helpful in phrasing general properties.
(4) Several aspects still need work to be of publication quality. Examples are the materials table and the presentation of supplementary material. Reference formatting also needs attention.
-
Reviewer #3 (Public review):
Summary:
The manuscript demonstrates that starvation induces persister formation in M. abscesses. They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.
Strengths:
The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.
Weaknesses:
The findings could not be validated in clinical strains.
Comments on revisions: No more comments for the authors.
-
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Weaknesses:
Only 1 gene (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype,
We have now carried out more detailed validation studies on the Tn-Seq, with analysis of timedependent killing over 14 d. This more comprehensive analysis shows that 4 of 5 genes analyzed do indeed have antibiotic tolerance defects under the conditions that Tn-Seq predicted a survival defect (Revised Figure 3). In addition, we found that even before actual cell death, several mutants had delayed resumption of growth after antibiotic removal (Figure 3 Supplemental).
Fig 3 - Why is there such a huge difference in the extent of …
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Weaknesses:
Only 1 gene (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype,
We have now carried out more detailed validation studies on the Tn-Seq, with analysis of timedependent killing over 14 d. This more comprehensive analysis shows that 4 of 5 genes analyzed do indeed have antibiotic tolerance defects under the conditions that Tn-Seq predicted a survival defect (Revised Figure 3). In addition, we found that even before actual cell death, several mutants had delayed resumption of growth after antibiotic removal (Figure 3 Supplemental).
Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.
We agree with the reviewer that there is variability in the timing and extent of cell death from experiment to experiment. As noted by the reviewer, in Figure 1C the largest decrement in survival is between day 1 - day 3 (also seen in Figure 6A). As they noted in Figure 4 the largest decrement is between day 3 – day 6 (also seen in Figure 3A, Figure 5F). In each experiment with katG mutants we carefully compare the mutant vs. the control strain within that experiment, which is more accurate than comparing the behavior of mutant in one experiment to a control in another experiment.
Reviewer #2 (Public review):
Weaknesses:
.First, word-choice decisions could better conform to the published literature. Alternatively, novel definitions could be included. In particular, the data support the concept of phenotypic tolerance, not persistence.
We appreciate the reviewers comments, text modified.
Second, two of the novel observations could be explored more extensively to provide mechanistic explanations for the phenomena.
We have added several additional experiments, these are detailed below in response to specific comments.
Reviewer #3 (Public review):
Weaknesses:
The findings could not be validated in clinical strains.
We understand the reviewer’s concern that the katG phenotype was only observed in one of the two clinical strains we studied. We feel that our findings are relevant beyond the ATCC 19977 strain for two reasons
(1) We have performed additional analyses of the two clinical isolates and indeed find significant accumulation of ROS following antibiotic exposure in both of these strains (revised Figure 6A).
(2) We do in fact see a role for katG in starvation-induced antibiotic tolerance in Mabs clinical strain-2. It is not surprising that different strains from a particular species may have some different responses to stresses – for example, there is wide strain-specific variability in susceptibility to different phages within a species based on which particular phage defense modules a given strain carries (for example PMID: 37160116). We speculate that different Mabs strains may express varying levels of other antioxidant factors and note that the genes encoding several such factors were identified by our Tn-Seq screen including the peroxidases ahpC, ahpD, and ahpE. Our analysis of the genetic interactions between katG and these other factors is ongoing.
Comments/Suggestions
(1) In Fig1E, the authors show no difference in killing Mtb with or without adaptation in PBS. These data are contrary to the data presented in Figure 1B. These also do not align with the data of M. smegmatis and M. abscesses. Please discuss these observations in light of the Duncan model of persistence (Mol Microbiol. 2002 Feb;43(3):717-31.).’
The above referenced Duncan laboratory study found tolerance after prolonged starvation but did not actually examine tolerance at early time points. While some of the transcriptional and metabolic changes seen by Duncan and others are slow, other groups have described starvation responses in Mtb that are quite rapid. For example, the stringent response mediator ppGpp accumulates within a few hours after onset of starvation in Mtb (PMID: 30906866). We suspect that a rapid signaling response such as this underlies the phenotype we observe. Regarding the difference between Mtb and other mycobacterial species we also find it surprising that Mtb had a much more rapid starvation response. This is a clear species-specific difference that may reflect an adaptation of Mtb to the nutrient-limited physiologic niche within host macrophages.
(2) Line 151, the authors state that they have used an M. abscesses Tn mutant library of ~ 55,000 mutant strains. The manuscript will benefit from the description of the coverage of total TA sites covered by the mutants.
Text modified to add this detail. There are 91,559 TA sites in the abscessus genome. Thus, our Tn density is ~60%.
(3) Line 155: Please explain how long the cells were kept in an Antibiotic medium.
This technical detail was noted above on line 153 in the original text: “…and then exposed them to TIG/LZD for 6 days”. To clarify the overall conditions, we have also revised the text of the manuscript and added the detail of how long cells were passaged after removal of antibiotics.
(4) Line 201: data not shown. Delayed resumption of growth after removal of antibiotic would be helpful in indicating drug resilience. This data could enhance the manuscript.
Data now provided in Figure 3 Supplemental
(5) Figures 4C and 4F represent the kill curve. It will be good to show the date with CFU against the drug concentration in place of OD600. CFU rather than OD600 best reflects growth inhibition.
Figures 4C and 4F are measuring the minimum inhibitory concentration (MIC) to stop the overall growth of the bacterial population. While we agree that CFU could be analyzed, this would be measuring a different outcome – cell death and the minimum bactericidal concentration (MBC). In these experiments we sought to specifically examine the MIC so as to separate growth inhibition from cell death. For this we used the standard method employed by clinical microbiology laboratories for MIC, which is optical density of the culture (PMID: 10325306).
(6) Figure 5C. The authors shall show the effect of TIG/LZD on M. abscesses ROS production without the PBS adaptation. It is important to conclude that TIG/LZD induces ROS in cells. Authors should utilize ROS scavengers such as Thiourea, DFO, etc., to conclude ROS's contribution to bacterial killing following inhibition of transcription and translation.
New data added (revised Figure 5 and Figure 5 Supplemental)
(7) Line 303. Remove "note".
Text revised. We thank the reviewer for identifying this typographical error.
(8) The introduction and Discussion are very similar, and several lines are repeated.
Text revised with overlapping content removed.
Reviewer #1 (Recommendations for the authors):
It appears that the same datasets for PBS adapted cultures were plotted in A-C and D-F. Either this should be specifically mentioned in the legend or it might be better to integrate the non-adapted plots into A-C which would also allow easier comparison.
Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.
This manuscript is focused on M. abs and the antibiotics TIG/LZD, so the Mtb data or data using the antibiotics INH/RIF/EMB and serves more as a distraction and can be removed
We appreciate the reviewer’s perspective. However, we wish to include these data to show the similarities (and differences) in starvation-induced tolerance between the three organisms.
Fig 3 -As mentioned for Fig. 1, it appears that the same dataset was used for the control in all the figures A-E. This should be explicitly stated in the Figure legend.
Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.
The divergent results from the clinical strains are extremely interesting. It would be helpful to determine the oxidative stress levels (similar to the cellROX data shown in 5E), to tease out if the difference in katG role is because of lack of ROS induction in these strains or due to expression of alternate anti-oxidative stress defense mechanisms.
We have performed additional cellROX analysis as suggested by the reviewer and found that the ROS induction is indeed present across all three Mabs strains, but that katG is only required in one of the two strains (Strain #2). These data are now included in the revised Figure 6.
Reviewer #2 (Recommendations for the authors):
GENERAL COMMENTS
This is a nice piece of work that uses the pathogen Mabs as a test subject.
The work has findings that likely apply generally to antibiotics and mycobacteria: 1) phenotypic tolerance is associated with suppression of ROS, 2) lethal protein synthesis inhibitors act via accumulation of ROS, and 3) levofloxacin behaves in an unexpected way. Each is a new observation. However, I believe that each topic requires more work to be firmly established to be suitable for eLife.
Phenotypic tolerance: Association with suppression of ROS is important but expected. I would solidify the conclusion by performing several additional experiments. For example, confirm the lethal effect of ROS by reducing it with an iron chelator and a radical scavenger. There is a large literature on effects of iron uptake, levels, etc. on antibiotic lethality that could be applied to this question. In 2013 Imlay argued against the validity of fluorescent probes. Perhaps getting the same results with another probe would strengthen the conclusion.
We have carried out additional experiments with both an iron chelator and small molecule ROS scavengers to further test this idea but note that these experiments have several inherent limitations: 1) These compounds have highly pleiotropic effects. For example while N-acetyl cysteine (NAC) is an antioxidant it also increases mycobacterial respiration and was shown to paradoxically decrease antibiotic tolerance in M. tuberculosis (PMID: 28396391). 2) It has been shown by the Imlay group that small-molecule antioxidants are often ineffective in quenching ROS in bacteria (PMID: 388893820), making negative results difficult to interpret. Nonetheless, we present new experimental data showing that iron chelation does indeed improve the survival of antibiotic-treated Mabs (revised Figure 5). However, small molecule antioxidants such as thiourea do not restore antibiotic tolerance and actually increased bacterial cell death, suggesting that they may be affecting respiration in Mabs in a manner similar to that seen for NAC in Mtb. We also note that our genetic analysis, which identified numerous other genes encoding proteins with antioxidant function (Figure 2) is a strong additional argument in support of the importance of ROS in antibiotic-mediated lethality.
Regarding the concern raised by Imlay about the validity of oxidation-sensitive dyes - this relates to concern bacterial autofluorescence induced by antibiotics that can confound analyses in some species. We have ruled this out in our analyses by using bacteria unstained by cellROX as controls to confirm that there is negligible autofluorescence in Mabs (<0.1%, Figure 5E, Figure 6A).
Protein synthesis inhibitors: At present, this is simply an observation. More work is needed to suggest a mechanism. For example, with E. coli the aminoglycosides are protein synthesis inhibitors that also cause membrane damage. Membrane damage is known to stimulate ROS-mediated killing. Your observation needs to be extended because chloramphenicol, another protein synthesis inhibitor, blocks ROS production. The lethality may be a property of mycobacteria: does it occur with E. coli (note that rifampicin is bacteriostatic with E. coli but lethal to Mtb)?
We agree with the reviewer that the mechanism underlying ROS accumulation following transcription or translational inhibition in Mabs is of significant interest. It is likely to be a mechanism different from E. coli, because in E. coli tetracyclines and rifamycins are both bacteriostatic, whereas in Mabs they are both bactericidal. Determining the mechanism by which translation inhibitors cause ROS accumulation in Mabs is an ongoing effort in our laboratory using proteomics and metabolomics, but is outside the scope of this manuscript.
Levofloxacin: This is also at the observational stage but is unexpected. In other studies, ROS is involved in quinolone-mediated killing of bacteria. Why is this not the case with Mabs? The observation should be solidified by showing the contrast with moxifloxacin, since this compound has been studied with mycobacteria (Shee 2022 AAC). With E. coli, quinolone structure can affect the relative contribution of ROS to killing (Malik 2007 AAC), as is also seen with Mtb (Malik 2006 AAC). What is happening in the present work with levofloxacin, an important anti-tuberculosis drug? Is there a structure explanation (compare with ofloxacin)?
While these are interesting questions, a detailed exploration of the structure-function relationships between different fluoroquinolone antibiotics and their varying activities on Mtb and Mabs is outside the scope of this manuscript.
The writing is generally easy to follow. However, the concept of persistence should be changed to phenotypic tolerance with text changes throughout. I base this suggestion on the definitions of tolerance and persistence as stated in the consensus review (Balaban 2019 Nat Micro Rev). Experimentally, tolerance is seen as a gradual decline in survival following antibiotic addition; the decline is slower than seen with wild-type cells. The data presented in this paper fit that definition. In contrast, persistence refers to a rapid drop in survival followed by a distinct plateau (Balaban 2019 Nat Micro Rev; for example, see Wu Lewis AAC 2012 ). Moreover, to claim persistence, it would be necessary to demonstrate subpopulation status, which is not done. The Balaban review is an attempt to bring order to the field with respect to persistence and tolerance, since the two are commonly used without regard for a consistent definition.
We appreciate the reviewer’s suggestion; text modified in multiple places to clarify.
Another issue requiring clarification is the relationship between resistance and tolerance. Killing by antibiotics is a two-step process, as most clearly seen with quinolones. First a reversible bacteriostatic event occurs. Resistance blocks that bacteriostatic damage. Then a lethal metabolic response to that damage occurs. Tolerance selectively blocks the second, killing event, a distinct process that often involves the accumulation of ROS. Direct antibiotic-mediated damage is an additional mode of killing that also stems from the reversible, bacteriostatic damage created by antibiotics. The authors recognize the distinction but could make it clearer. Take a look at Zheng (JJ Collins) 2020, 2022.
Text modified to clarify this point
Many readers would also like to see a bit more background on Mabs. For example, does it grow rapidly? Are there features that make it a good model for studying mycobacteria or bacteria in general? The more general, the better.
Text modified, background added
Below I have listed specific comments that I hope are useful in bringing the work to publication and making it highly cited.
SPECIFIC COMMENTS
Line 30 unexpectedly. I would delete this word because the result is expected from the ROS work of Shee et al 2022 with mycobacteria. Moreover, Zeng et al 2022 PNAS showed that ROS participates in antimicrobial tolerance, and persistence is a form of tolerance (Balalban et al, 2019, Nat Micro Rev).
Text modified as per review suggestion
Line 39 key goal: this is probably untrue in the general sense stated, since bacteriostatic antibiotics are sufficient to clear infection (Wald-Dickler 2019 Clin Infect Dis). However, it is likely to be the goal for Mtb infections.
We agree with the reviewer that bacteriostatic antibiotics are effective in treating most types of infections and do not claim otherwise in the manuscript. However, from a clinical standpoint, eradication of the pathogen causing the infection is indeed the goal of antibiotic therapy in virtually all circumstances (with the exception of specific scenarios such as cystic fibrosis where it is recognized that the infecting organism cannot be fully eliminated). In most cases, the combination of bacteriostatic antibiotics and the host immune response is sufficient to achieve eradication. We have modified the manuscript text to reflect this nuance noted by the reviewer.
Line 62 several: you list three, but hipAB works via ppGpp, so the sentence needs fixing
Text modified
Line 70 uncertain: this uncertainty is unreferenced. Since everything is uncertain, this vague phrase does not add to the story.
The reviewer makes an interesting philosophical argument. However, we would submit that some aspects of biology, for example the regulation of glycolysis, are understood in great detail. However, other mechanisms, such as the precise mechanisms of lethality for diverse antibiotics in different bacterial species, are far more uncertain and remain a subject of debate (for example PMID: 39910302). Text not modified.
Line 72 somewhat controversial: I would delete this, because the points in the Science papers by Lewis and Imlay have been clarified and in some cases refuted by prior and subsequent work.
Text modified
Line 72 presumed: this suggests that it is wrong and perhaps a different idea has replaced it. Another, and more likely view is that there is an additional mode of killing. I suggest rephrasing to be more in line with the literature.
Text modified for clarity. In this sentence “presume” refers to the historical concept that direct target inhibition was solely responsible for antibiotic lethality. As the reviewer notes, there is now significant literature that ROS (and perhaps other secondary effects) also contribute to bacterial killing.
Line 73 However and the following might also: this phrasing, plus the presumed, misleads the reader from your intent. I suggest rephrasing.
See above re: line 72
Line 75 citations: these are inappropriate and should be changed to fit the statement. I suggest the initial paper by Collins (Kohanski 2007 Cell) a recent paper by Zhao (Zeng PNAS 2022), and a review Drlica Expert Rev Anti-infect Therapy 2021). The present citations are fine if you want to narrow the statement to mycobacteria, but the history is that the E. coli work came first and was then generalized to mycobacteria. A mycobacterial paper for ROS is Shee 2022 AAC.
We thank the reviewer for noticing that we inadvertently omitted several important E. coli-related references. These have been added.
Line 75 and 76: Conversely ... unresolved. Compelling arguments have been made that show major flaws in the two papers cited, and a large body of evidence has now accumulated showing the validity of the idea promoted by the Collins lab, beginning with Kohanski 2007. In addition to many papers by Collins, see Hong 2019 PNAS and Zeng 2022 PNAS). It is fine if you want to counter the arguments against the Lewis and Imlay papers (summarized in Drlica & Zhao 2021 Expert Rev Anti-infect Therapy), but making a blanket statement suggests that the authors are unfamiliar with the literature.
We agree with the reviewer that the weight of the evidence supports a role for antibiotic-induced ROS as an important mechanism for antibiotic lethality under many (though not all) conditions. We have revised the text to better reflect this nuance.
Line 78. Advantages over what?
Text modified
Line 80 exposure: to finish the logic you need to show that E. coli and S. aureus persisters fail to do this.
We thank the reviewer for their suggestion but studying these other organisms is outside the scope of this study.
Line 82 whereas: this misdirects the reader. It would seem that a simple "and" is better
Text modified
Line 89 I think this paragraph is about the need to study Mabs, the subject of the present report. This paragraph could use a more appropriate topic sentence to guide the reader so that no guessing is involved. I suggest rephrasing this paragraph to make the case for studying more compelling.
Text modified
Line 96. I suggest citing several references after subinhibitory concentration of antibiotic.
The references are in the following sentence alongside the key observations.
Line 99. Genetic analysis: how does this phrase fit with the idea of persister cells arising stochastically?
There are two issues: 1) We would argue that persister formation is not completely stochastic, but rather a probability that can be modified both genetically and by environment (for example hipA PMID: 6348026). 2) Even if persister formation were totally stochastic, the survival of these cells may depend on specific genes – as we indeed find in our Tn-Seq analysis of Mabs.
Line 106. In this paragraph you need to define persister. The consensus definition (Balaban 2019 Nat Micro Rev) is a subpopulation of tolerant cells. Tolerance is defined as the slowing or absence of killing while an antibiotic retains its ability to block growth. See Zeng 2022 PNAS for example with rapidly growing cells. Phenotypic tolerance is the absence of killing due to environmental perturbations, most notably nutrient starvation, dormancy, and growth to stationary phase. By extension, phenotypic persistence would be subpopulation status of a phenotypically tolerant cells. If you have a different definition, it is important to state it and emphasize that you disagree with the consensus statement.
Text modified
Line 109 unexpectedly. I would delete this word, because the literature leads the reader to expect this result unless you make a clear case for Mabs being fundamentally different from other bacteria with respect to how antibiotics kill bacteria (this is unlikely, see Shee 2022 AAC). Indeed, lines 111-113 state extensions of E. coli work, although suppression of ROS in phenotypic tolerance and genetic persistence have not been demonstrated.
Text modified
Line 124 you might add, in parentheses and with references, that a property of persisters is crosspersistence to multiple antibiotic classes. This is also true for tolerance, both genetic and phenotypic. An addition will support your approach.
Text modified
Line 128 minimal
Text not modified. We appreciate the reviewer’s preference but both “minimal” and “minimum” are both widely accepted terms. Indeed, the Balaban et al 2019 consensus statement on definitions cited by the author above also uses “minimum” (PMID: 30980069), as do IDSA clinical guidelines (PMID: 39108079).
Line 130 is MIC somehow connected to killing or did you also measure killing? Note that blocking growth and killing cells are mechanistically distinct phenomena, although they are related. By being upstream from killing, blockage of growth will also interfere with killing.
Text modified
Line 133 PBS is undefined
Text modified
Line 134 increase in persisters ... you need to establish that these are not phenotypically tolerant cells. Do they constitute the entire population (tolerance)? Your data would be more indicative of persisters if you saw a distinct plateau with the PBS samples, as such data are often used to document persistence (retardation of killing is a property of tolerance, Balaban 2019). Fig. 1B is clearly phenotypic tolerance, as the entire population grows. Your data suggest that you are not measuring persistence as defined in the literature (Balaban 2019). Line 139 persister should be tolerance •
Text modified
Lines 142, 143, 144. 159, 163, 171, 181, 211, 226, 238, 246, 277, 279,289 persistent should be tolerant
Text modified
Line 146 fig 1E Mtb does not show the adaptation phenomenon and it is clearly tolerant, not persistent. This should be pointed out. As stated, you may be misleading the reader.
Text modified
*Line 169. Please make it clear whether these genes are affecting antibiotic susceptibility (MIC will affect killing because blocking growth is upstream) or if you are dealing with tolerance (no change in MIC). These measurements are essential and should included as a table. By antibiotic response, do you mean that antibiotics change expression levels?
Regarding MICs, the data for MICs in control and katG mutant are presented in Figure 4C and 4F. Regarding ‘response’ we have clarified the text of this sentence.
Line 174 Interestingly should be as expected
Text not modified; tetracyclines do not induce ROS in E. coli and oxazolidinones have not been studied in this regard.
Line 183 you need to include citations. You can cite the ability of chloramphenicol to block ROS-mediated killing of E. coli. That allows you to use the word unexpected
Text modified
Line 199. All of the data in Fig. 3 shows tolerance, not persistence, requiring word changes in this paragraph.
Text modified
Line 226. The MIC experiment is important. You can add that this result solidifies the idea that blocking growth and killing cells are distinct phenomena. You can cite Shee 2022 AAC for a mycobacterial paper
Text modified
Line 241. The result with levofloxacin is unexpected, because the fluoroquinolones are widely reported to induce ROS, even with mycobacteria (see Shee 2022 AAC). You need to point this out and perhaps redo the experiment to make sure it is correct.
We appreciate the reviewer’s interest in this question. All experiments in this paper were repeated multiple times. This particular experiment was repeated 3 times and in all replicates the katG mutant was sensitized to translation inhibitors but not levofloxacin. Shee et al examined Mtb treated with moxifloxacin and found ROS generation, but did not assess whether a Mtb katG mutant had impaired survival. Thus, in addition to differences in: i) the species studied and ii) the particular fluoroquinolone used, the two sets of experiments were designed to address different questions (ROS accumulation vs protection by katG) . A cell might accumulate ROS without a katG mutant having impaired survival if genetic redundancy exists – a result we indeed see in our clinical Mabs strains under some conditions (new data included in revised Figure 6A).
Line 269 Additional controls would bolster the conclusion: use of an antioxidant such as thiourea and an iron chelator (dipyridyl) both should reduce ROS effects.
New experiments performed, revised Figure 5.
Line 276 the word no is singular
Text modified
Line 284 this suggested ... in fact previous work suggested. This summary paragraph might go better as the first paragraph of the Discussion
Text modified to specify that this is in reference to the work in this manuscript
Lines 294-299 Most of this is redundant and should be deleted.
Text modified
Line 299 this species is vague
Text modified
Line 310 Do you want to discuss spoT?
Text not modified
Line 313 paragraph is largely redundant
Text modified
Line 314 controversial. As above, I would delete this, especially since it is not referenced and is unlikely to be true. If you believe it, you have the obligation to show why the ROS-lethality idea is untrue. If you are referring to Lewis and Imlay, there were almost a dozen supporting papers before 2013 and many after. This statement does not make the present work more important, so deletion costs you nothing.
Text modified
Line 314 direct disruption of targets. This is clearly not a general principle, because the quinolones rapidly kill while inhibition of gyrase by temperature-sensitive mutations does not (Kreuzer 1979 J.Bact; Steck 1985). Indeed, formation of drug-gyrase-DNA complexes is reversible: death is not.
Text modified
Line 318 as pointed out above, you have not brought this story up to date. The two papers mainly focused on Kohanski 2007, ignoring other available evidence.’’
Text modified
Line 326 you need to cite Shee 2022 AAC
Text modified
Line 342 the idea of mutants being protective is not novel, as several have been reported with E. coli studies. Thus, there is a general principle involved.
We agree that this suggests a potential general principle
Line 344. It depends on the inhibitor. For example, aminoglycosides are translation inhibitors and they also cause the accumulation of ROS.
We agree that ROS generation depends on the inhibitor, and indeed upon other variables including drug concentration, growth conditions, and bacterial species as well.
Line 347. You need to point out the considerable data showing that the absence of catalase increases killing
Text modified
Line 363 look at Shee 2022 AAC and Jacobs 2021 AAC
Text modified, reference added.
Line 585 I suggest having a colleague provide critical comments on the manuscript and acknowledge that person.
Text not modified
-
-
eLife Assessment
Using a TN-seq based approach, the authors identified the genetic determinants of drug tolerance in M. abscessus. Since M. abscessus is resistant to multiple antibiotics, the study is valuable in generating new knowledge linking antibiotic tolerance with ROS in this non-tuberculosis mycobacterial (NTM) species. However, the study is incomplete due to a need for more validation of the Tn-seq data, inconsistency with the clinical strains, and insufficient experiments confirming the role of ROS detoxification in drug tolerance.
-
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for …
Reviewer #1 (Public review):
Summary:
Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.
To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tn-seq was carried out to identify genes involved in spontaneous (nutrient rich) or starvation-induced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.
Strengths:
While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.
Weaknesses:
- Fig. 3 - 5 of the hits from the transposon screen were reconstructed as clean deletion strains and tested for persistence. However, only 1 (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype, however it was not clear if this difference was really relevant as the mutant exhibited differences at Day 0, prior to the addition of antibiotics. Considering these results from the validation, the conclusion would be that the Tn-seq approach to screen persistence defects is not reliable and is more likely to result in misses than hits.
- Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.
-
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes …
Reviewer #2 (Public review):
Summary:
The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.
Strengths:
The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.
Weaknesses:
The work can be improved in two major ways. First, word-choice decisions could better conform to the published literature. Alternatively, novel definitions could be included. In particular, the data support the concept of phenotypic tolerance, not persistence. Second, two of the novel observations could be explored more extensively to provide mechanistic explanations for the phenomena.
Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are field-solidifying observations.
-
Reviewer #3 (Public review):
Summary:
The manuscript demonstrates that starvation induces persister formation in M. abscesses. They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.
Strengths:
The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.
Weaknesses:
The findings could not be validated in clinical strains.
-
