Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli to multiple bactericidal antibiotics

  1. Sarah A. Revitt-Mills
  2. Elizabeth K. Wright
  3. Madaline Vereker
  4. Callum O’Flaherty
  5. Fairley McPherson
  6. Catherine Dawson
  7. Antoine M. van Oijen
  8. Andrew Robinson

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Abstract

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus re-sensitise bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant re-sensitisation towards five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full re-sensitisation. For ciprofloxacin and nitrofurantoin, sensitisation manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the pro-mutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could re-sensitise antibiotic-resistant bacteria to multiple classes of existing antibiotics and may supress the development of de novo antibiotic-resistance mutations.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6786690.

    The importance of gaining a deeper understanding on antibiotic resistance and developing tools to circumvent and prevent it can hardly be overstated. This study attempts at exploring the possibility of controlling Double-Stranded Break Repair (DSBR) as a way of increasing antibiotic effectiveness and reducing the risk of de-novo emergence of antibiotic resistance.

     

    For studying the effects of inhibited DSBR, a set of candidate genes are selected and mutations on the same have been used as a way of disrupting DSBR. Colonies with bacteria that have mutations on these genes show a marked decrease in the Minimum Inhibitory Concentration (MIC) over a host of antibiotic drugs. (Fig. 1)

     

    However, for antibiotic drugs that affect the ribosome and folate biosynthesis (kanamycin and trimethoprim), the mutants show significant decrease in cell viability only when treated with very high concentrations (10x MIC) of antibiotics. One wonders how strong this effect is across antibiotics and how general can the effect of DSBR will be for various other antibiotics that are not studied in this work.

     

    One way in which antimicrobial resistance might emerge in an accelerated manner in bacterial populations is by a marked increase in mutagenesis (SOS response). This study also shows a definite link between inhibition of DSBR and decreased probability of an SOS response. Combined, these results show that inhibiting DSBR may not just allow one to work with antibiotic resistant strains as and when they emerge, but also perhaps avoid the emergence of new resistant strains. This indeed looks very promising.

     

    However, a major caveat, as previously mentioned, is how generally applicable could this be? The study does show a marked decrease in MIC for many antibiotics, which clearly shows that this is an important direction to be pursued. But it is needed that we are careful about when and how does the usefulness of DSBR inhibition stop, and that we are acutely aware of the limitations.

     

    Another aspect where more exploration is required is a more detailed, mechanistic understanding on how the SOS response also gets induced along with DNA repair pathways. Both of these, in retrospect, seem to be processes that are physiologically at odds with each other. It would therefore be interesting to look at them, and the underlying mechanistic connection, from an evolutionary point of view.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6415406.

    It has been almost 35 years that a major class of antibiotics have been discovered and bacterial are gaining resistance from numerous antibiotics at a much faster rate. Although we continue to ignore this problem, in near future we need to start mitigating the antibiotic resistance. This study may have far reaching effect in the field for this reason. Since bacteria exposed to antibiotic concentration less than MIC can activate SOS response which causes mutagenesis that in turn gives rise to resistant strains, the authors hypothesised that targeting the SOS signal and inhibiting the DSBR process may re-sensitise the bacterial cell to the antibiotic. The study shows how mutations in the SOS protein (recA) and DSBR protein (recB) result in the resistant strain of  E. coli to become sensitive to the respective antibiotic. They have also used two small molecule inhibitors for DSBR to check their effect on re-sensitisation.

     

    Some concerns/comments about the study:

     

    1. The study could have included screening of drugs from other class of antibiotics. Macrolides, carbapenems and cephalosporins are frequently used classes of antibiotics which many pathogens have attained resistance from, and it would be useful to investigate if DSBR can cause re-sensitisation for these drugs as well. 
    2. The mutants used in study could have been characterised or discussed in more detail for a better understanding of the assays and the results.
    3. Although the RecA mutants did have a significant effect on the antibiotic sensitivity, result from other mutants of SSGR pathway weren't that conclusive. But SSGR can also activate SOS response and that may cause mutagenesis even though double strand breaks are not repaired by the cells.
    4. The target proteins for re-sensitisation have been characterised quite in detail through mutants but the small molecule inhibitor has off-target effects and future research is needed for developing a specific molecule for targeting the DSBR pathway for clinical use. The authors have also raise the concern about the two small molecules used.

     

    The study provides a foundation for further research in the field and provides a good solution to tackle anti-microbial resistance. The data does indicate role of double stand breaks in mutagenesis and resistance although more extensive research is needed for getting a clinical outcome from this.

  3. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6334820.

    We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

    Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli to multiple bactericidal antibiotics.

    Sarah A. Revitt-Mills, Elizabeth K. Wright, Madaline Vereker, Callum O'Flaherty, Fairley McPherson, Catherine Dawson, Antoine M. van Oijen, Andrew Robinson. bioRxiv 2022.01.24.477632; doi: https://doi.org/10.1101/2022.01.24.477632

    We will adhere to the Universal Principled (UP) Review guidelines proposed in:

    Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

     

    SUMMARY: Building from previous literature, which demonstrated that inactivation of DNA repair pathways can re-sensitize bacteria to different antibiotics, the authors aimed to identify which DNA repair pathways are important for this re-sensitization and if this can be applied to additional classes of antibiotics. They utilized a collection of E. coli mutants that lacked important members of DNA repair pathways, including deletion of recAor recB genes involved in double-strand break repair (DSBR), or deletion of recF, recO, or recR genes involved in single-strand gap repair (SSGR), or deletion of mutT, which is important for the nucleotide pool sanitation (NPS) pathway. They then analyzed the resistance of these strains to various antibiotics using MIC test strips and spot plate assays. They found that disruption of the DSBR pathway enhanced the effects of ciprofloxacin and nitrofurantoin and re-sensitized resistant E. coli to these antibiotics. Further, they found that DSBR was important for bacterial survival during high concentrations of kanamycin treatment as they observed increased clearance of bacteria in E. coli strains lacking recA or recBcompared to wild type. This observation also repeated for kanamycin-resistant strains. In addition, deletion of recA, recB, or recO increased sensitivity to trimethoprim in both antibiotic-sensitive and antibiotic-resistant strains while deletion of recR only increased trimethoprim sensitivity in resistant strains. They next tested resistance to ampicillin and found no significant differences in sensitivity of DNA repair-deficient E. coli compared to wild-type parental strains. However, they observed a significant reduction in bacteria regrowth following ampicillin treatment in recA or recB-deficient cells, indicating that DSBR is important for bacteria tolerance to ampicillin. Furthermore, they examined induction of the SOS response in bacteria using an agar plate-based SOS reporter assay. They found that induction of the SOS response following ciprofloxacin or nitrofurantoin treatment required recB for both antibiotic-sensitive and -resistant strains, indicated by the decrease in fluorescence following antibiotic treatment. Lastly, the authors analyzed the sensitivity of DSBR-deficient E. coli to ML328 and IMP-1700, two DSBR inhibitors. They found that deletion of recAor recB increased sensitivity to these compounds and induction of the SOS response by these compounds is dependent on recA and recB. They also identified that IMP-1700 primarily targets topoisomerase IV while ML328 targets both topoisomerase IV and DNA gyrase, indicating that the DSBR pathway is not the only target of these compounds. In conclusion, the authors found that inhibition of DSBR can re-sensitize resistant E. colito various antibiotics.

     

    OVERALL ASSESSMENT: 

     

    STRENGTHS: This preprint is very well written and provides ample relevant background to aid general-interest readers. We particularly appreciated how the literature was re-addressed in the Discussion in light of new findings, providing context for future planned studies and potential applications of these discoveries.  

     

    WEAKNESSES: While there were many strengths in this pre-print, we also identified a few weaknesses we would like to address. One of our major concerns was the lack of discussion about the differences between wild-type E. coli and mutant strains. We wanted to know if there were differences between the growth rates of these strains in the absence of antibiotic challenge, to establish a baseline for these strains which could then be compared during antibiotic treatment. Additionally, we worried that changing assay formats from liquid cultures to solid plates between experiments might affect the findings. Indeed, changing how the bacteria are grown can introduce environmental differences which may affect growth rates, stress responses, and antibiotic effectiveness, all criteria which were measured in this pre-print. Some discussion of the strengths and weaknesses of the different assay formats is warranted.

     

    DETAILED U.P. ASSESSMENT:

    OBJECTIVE CRITERIA (QUALITY)

    1.  Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2

    ●      Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

    o   For Figure 1, we questioned why there were a different number of replicates between conditions in panels A and D. We recommend having the same number of replicates shown per condition.

    o   For Figure 2, we found the data to be inconsistent between panel B and panel C. We didn't understand why there was a significant increase in the ZOI from wild-type to the RecA or RecB mutant, shown in panel B, while there was no significant increase between these strains with vehicle-control in panel C. In addition, in reference to panel D, it was stated, "however at 10× MIC (Figure 2d) there was significant killing of both ΔrecA and ΔrecB mutants," yet no statistics were performed. While there is an obvious decrease in cfu/ml for the mutant strains, we think it is appropriate to use the word significant when statistics are used. We recommend either choosing another word or adding statistics to this graph to reinforce that the difference is significant. Lastly, we think the inclusion of the time-kill survival assay without antibiotic treatment would be a helpful control in panel D.

    o   For Figure 3 and Figure 4, we had no critiques and thought they were well conducted and explained. 

    o   For Figure 5, we had concerns about the intensity of the fluorescent signal with the MIC test strips. The inferences provided were helpful to highlight the qualitative differences between the wild-type and mutant strains. However, quantification of this fluorescence intensity would be helpful to highlight quantitative differences between the activation of the SOS response in these strains. We also noticed a difference in the background intensity of the MIC strip itself in some of these images, specifically in panels C, D, and E. We questioned if this was a biproduct of the assay or if this was due to differences in camera settings or the acquisition of the images. Such differences could undermine the integrity of the assay.  

    o   For Figure 6, we wondered why there was a switch from use of the MIC test strips to use of the diffusion assays to assess SOS response activation with the DSBR inhibitors in panel G and H. An additional comment to explain the switch would be helpful for the reader.

     

    ●      Are specific analyses performed using methods that are consistent with answering the specific question?

    o   No, because there are changes in the methodology without explanation.

     

    ●      Is there appropriate technical expertise in the collection and analysis of data presented?

    o   Yes, however we recommend additional explanation of the methods used. 

     

    ●      Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

    o   Yes, except we recommend that the P-values from pairwise tests within the same experimental condition be controlled for family wise error rate.

     

    ●      Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

    o   Yes.

     

     

    2. Quality: Completeness (1–3 scale) SCORE = 2.5

    ●      Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

    o   No, because there is a disconnect between the data and the proposed major conclusions. The major conclusions stated in the title and the abstract suggest that targeting the DSBR repair pathway allows re-sensitization of these bacterial strains to various antibiotics. However, the data suggests that it is deletion of RecB specifically that confers this re-sensitization. We suggest that the major conclusions be adjusted to specify that RecB is responsible for this re-sensitization as opposed to the DSBR pathway in general. This is largely because the only other DSBR pathway component that was analyzed was RecA, and RecA is also involved in the SOS response pathway. If additional experiments analyzing other components of the DSBR pathway also showed re-sensitization to these antibiotics, then the proposed major conclusions would be justified. 

     

    ●      Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

    o   Yes, there are two experiments that if performed could disprove the conclusion. One experiment would be looking at other DSBR repair components, like RecC, RecD, among others, to see of their deletion would have similar effects as deletion of RecB. This would confirm that interference with the DSBR pathway is important for re-sensitization of these antibiotics as opposed to deletion of RecB specifically. Additionally, it would be important to see if the results would be consistent between growth in liquid culture and growth in solid media. This experiment would confirm that differences in antibiotic sensitivity are due to interference with the DSBR pathway and not differences in growth environments. 

     

    3. Quality: Reproducibility (1–3 scale) SCORE = 2.5

    ●      Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?'

    o   As mentioned above, the number of biological replicates performed for different experiments varied considerably throughout the figures for no clear reason. In addition, and as mentioned above, there appeared to be differences in the camara settings and picture quality between images taken for the SOS reporter assay. Consistent camera settings are required to truly make quantitative comparisons. 

     

    ●      Is there sufficient raw data presented to assess rigor of the analysis?'

    o   Yes.

     

    ●      Are methods for experimentation and analysis adequately outlined to permit reproducibility?

    o   Yes.

     

    ●      If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed?

    o   N/A.

     

    4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1

    ●       Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

    o    Yes.

     

    ●       Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

    o    Yes.

     

    ●       Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

    o    One comment we have is about the color scheme used for the bar graphs. Some of the lighter shades used are very light and, at times, indistinguishable from the neighboring bar in the graph. This is a minor point but changing the color shade or including an outline around the bar would be easier for the reader to see the data. 

     

    MORE SUBJECTIVE CRITERIA (IMPACT)

    1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 3

    ●      A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.]

     

    ●      How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

    o   The idea that antibiotics induce DNA damage has been well documented in the field and there are multiple papers that address the DNA-damaging properties of the antibiotics mentioned in this pre-print. The novelty of this pre-print is in the idea of interfering with bacterial DNA repair mechanisms to render pathogenic bacteria susceptible to antibiotics, specifically by targeting RecB in the DSBR pathway. The proposed major conclusions of this preprint suggest that the defect of the DSBR pathway is the dominant cause of the antibiotic-sensitizing effects. If so, then the novelty of this preprint is lacking. Remodeling of the major conclusions to specify defects in RecB to be the major cause of the antibiotic-sensitizing effects, a target that has not been previously investigated to our knowledge, would greatly increase the novelty of this pre-print.

     

    2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A

    ●  Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

    o    N/A

     

    ●  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

    o    N/A

     

  4. Version published to 10.1101/2022.01.24.477632v1 on bioRxiv