Integron activity accelerates the evolution of antibiotic resistance

  1. Célia Souque
  2. José Antonio Escudero
  3. R Craig MacLean

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    Summary: In this manuscript, the authors test the long-standing "evolution-on-demand" hypothesis of integrons. Using a combination of genetic construction work, experimental evolution, and WGS the authors present a convincing body of work favoring the presented hypothesis. They introduce three antibiotic gene cassettes into an integron and use an "evolutionary ramp" approach with gentamicin and demonstrate that the gentamicin resistance cassette shuffles towards the start of the integron. This provides compelling evidence favoring the evolutionary effects of an active class 1 integrase.

    The paper is clear, well written and demonstrates neatly the benefits of integron shuffling. The authors should also be given credit for including experimental data from an integron containing a clinical plasmid including resistance cassettes to the last resort antibiotics carbapenems. This is largely missing in the field.

    Our overall assessment of the manuscript is positive. However, a number of questions have been raised regarding the mechanistic aspects and conclusions of the paper. We are therefore suggesting additional assays to measure phenotypic effects of evolved integrons, and possibly data analyses on the negative controls. If these are not possible to perform, the main conclusions could be slightly altered instead to focus more on the genomics. Finally, we provide some suggestions on making the discussion more balanced and in clarifying the role of chromosomal mutations in the integron-facilitated evolution.

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Abstract

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to ‘evolve on demand’ in response to antibiotic pressure. To test this hypothesis, we inserted a custom three-cassette integron into Pseudomonas aeruginosa and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

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  1. Version published to 10.7554/elife.62474 on eLife
  2. eLife

    Reviewer #3:

    In this manuscript, the authors test the long-standing and long overdue "evolution-on-demand" hypothesis of integrons. Using a combination of genetic construction work, experimental evolution, and WGS the authors present a convincing body of work favoring the presented hypothesis. The paper is clear, well written and the authors should be given credit for including experimental data from an integron containing clinical plasmid including resistance cassettes to the last resort antibiotics carbapenems. This is largely missing in the field.

    My overall assessment of the manuscript is very positive. The "evolutionary ramp" approach is an elegant way to test the "evolution on demand" hypothesis and the authors provide compelling evidence favoring the evolutionary effects of an active class 1 integrase. However, reading through the manuscript I have three major questions/comments regarding the mechanistic aspects and conclusions of the paper. Regarding the last two points, I believe a slightly more balanced discussion including other possible explanations (such as experimental conditions) would add more balance to the Conclusion chapter and improve the manuscript.

    Major Comments:

    1. Based on WGS the authors characterize evolved populations and claim to demonstrate extensive integrase driven rearrangements in combination with chromosomal mutations underpinning the adaptations towards both constant sub-MIC and 2- fold increments of gentamicin concentrations.

    My first concern regards the crucial control in Figure S2 where control PCRs confirm data from Illumina short read sequencing on whole populations. It is hard for me to follow and understand this figure. I suggest that a schematic figure of each combination of cassettes, primer positions, and expected band length combined with proper lane descriptions should be prepared.

    1. Surprisingly, and contrasting integron structures from environmental and clinical samples, the authors provide evidence for a strong predominance of "copy and paste" as opposed to the emblematic "cut and paste" insertions of the gentamicin resistance cassette during experimental evolution. They argue that their data suggest that intI1 has a bias towards "copy and paste" cassette rearrangements.

    First, I find the term "copy and paste" somewhat confusing. I cannot see that the underlying mechanism of cassette excision differs between the two outcomes in integron structure. The cassette is in both cases excised (cut) from the ancestral integron before it is inserted (paste) into either arrays. I may have missed something here- but why "copy" and how is this novel?

    Second, I am not convinced that the presented evidence provides sufficient support for the proposed "copy and paste" bias of IntI1. As the authors discuss thoroughly, the presence of multiple copies of the ancestral structure provides more "ancestral" integration targets for the excised cassettes. The authors exclude the alternative hypothesis that a second copy of aadB increased fitness as compared to a single copy (as expected from copy and paste). Fitness effects of different arrays are discussed solely on the basis of retrospective analyses of populations that did not go extinct. I would have been more convinced if this was backed by some measure of fitness, for example MIC values of integron arrays containing two aadB cassettes. From Fig 1C it is not unlikely that it could be increased.

    1. The authors highlight in the abstract and in the Conclusion section that they found no evidence of deleterious off-target integrase effects. They suggest that integrase activity, rather purge deleterious chromosomal mutations and enable more targeted beneficial adaptive responses.

    The authors present cases where likely beneficial off target recombination events occurred. To what extent do the authors think the absence of deleterious off target effects is due to the experimental conditions (continuous increments in gentamicin concentrations combined with strong bottlenecks)?

  3. eLife

    Reviewer #2:

    This manuscript addresses the evolutionary benefits of integrase activity using experimental evolution of integrons in the presence of antibiotics. The authors demonstrate that activity increases survival of populations at high gentamicin concentrations, by shuffling a gentamicin resistance cassette towards the start of the integron.

    The paper is very well written and interesting, and demonstrates neatly the benefits of integron shuffling. I am suggesting a few additional assays, in order to measure phenotypic effects of evolved integrons. However, if these are not possible to perform , the main conclusions could be slightly altered instead to focus more on the genomics.

    Major Comments:

    1. The paper would benefit from MIC assays (or any other resistance measure) using evolved clones, to properly demonstrate and quantify the evolution of increased resistance associated with the different integron arrays. For now, the only phenotypic data measured from the evolution experiment is survival of populations during the experiment itself. I was first going to say that this is a minor comment, as the genetic / genomic data is very interesting and solid on its own, but the paper is still framed around evolution of increased antibiotic resistance, which is not directly quantified. Survival of populations might be influenced by other factors, including the chromosomal mutations described in the manuscript but also non-genetic effects, for instance population density effects, with populations that grow slightly more at a given time point then having a higher inoculum for the next step.

    2. MIC assays could even be done with no need for further sequencing, using clones from the populations in which integrons are not polymorphic (Fig 3B). Comparing resistance levels for the aadB-blaVEB-1-dfrA5-aadB array, and the aadB-aadB and aadB arrays with the ancestral array would allow the authors to link genotype and phenotype, and to demonstrate more directly the selective advantages (or absence of, for some of the arrays) that they suggest. Effects of plasmid evolution could also be separated relatively easily from chromosomal mutations that contribute to gentamicin resistance by transferring evolved plasmids to an unevolved host.

    3. I don't actually think anything else is happening than the evolution of increased resistance via shuffling that the authors are suggesting - and they are very careful in stating clearly that increased resistance is only 'suggested' whenever they discuss the genomic results directly. But I am still a bit uneasy about drawing conclusions of increased antibiotic resistance (in the title, end of introduction, and conclusion) when the only phenotypic data is survival at the population level. Alternatively, this text could be reformulated to focus clearly on the genetics and not on phenotypic resistance.

  4. eLife

    Reviewer #1:

    The manuscript 'Integron activity accelerates the evolution of antibiotic resistance' by Souque et al. investigates the genetic variations created by a class 1 integron during antibiotic exposure. In the study, the authors examine the evolution of an integron encoded on a R388 plasmid; they introduce three antibiotic gene cassettes into the integron and follow its evolution in the presence of one corresponding antibiotic - here gentamicin. They find that antibiotic exposure leads to a rapid re-shuffling of the integron cassette. The re-shuffling favors the aadB gene in the first position downstream of the integron promoter while mainly keeping the (original) last position in the integron. The study represents an interesting example of rapid adaptation to increasing concentrations of an antibiotic that is facilitated by mobile elements. While the experiments are overall interesting and very well designed, the study lacks a certain depth. In the sense that their results might be as well explained by random mutations (genetic diversity). In addition, the two parts of the experiments (integron analysis & chromosomal evolution) need to be connected as it is so far unclear what role the chromosomal mutations have in the integron-facilitated evolution.

    Major Comments:

    1. The authors don't mention whether they detected re-arrangements in the negative control that was evolved without antibiotics. Furthermore, re-arrangements might appear but at a very low frequency. What is the sequence coverage used in the study? How can the authors ensure they don't miss a low frequency of re-arrangements? It might be possible that random re-arrangements appear at a very low frequency that are only fixed under changing conditions (similar to mutations). The authors should clarify this point.

    2. Did the authors measure the Integrase expression levels? This could ensure that there is no expression without stress to the cell.

    3. Regarding the mutational analysis: Is there any sign of a cost to the integrase activity? The authors conduct an intensive analysis on chromosomal and plasmid mutations. Nonetheless, it is unclear how these mutations are generally connected to the integrase activity (and not only to the AB treatment).

    4. The authors call the integrase activity 'adaptation on demand'. It would be interesting to know how fast a potential reversal would appear in the integron in the populations. Is there any evidence for a deletion of the duplication of the aadB gene after removal of the antibiotic? In the same line of thought, do the authors expect the other AB resistance genes to follow the same path when incubated in the corresponding antibiotic? It would be interesting to know how antibiotic 'type' dependent the experimental result might be.

  5. eLife

    Summary: In this manuscript, the authors test the long-standing "evolution-on-demand" hypothesis of integrons. Using a combination of genetic construction work, experimental evolution, and WGS the authors present a convincing body of work favoring the presented hypothesis. They introduce three antibiotic gene cassettes into an integron and use an "evolutionary ramp" approach with gentamicin and demonstrate that the gentamicin resistance cassette shuffles towards the start of the integron. This provides compelling evidence favoring the evolutionary effects of an active class 1 integrase.

    The paper is clear, well written and demonstrates neatly the benefits of integron shuffling. The authors should also be given credit for including experimental data from an integron containing a clinical plasmid including resistance cassettes to the last resort antibiotics carbapenems. This is largely missing in the field.

    Our overall assessment of the manuscript is positive. However, a number of questions have been raised regarding the mechanistic aspects and conclusions of the paper. We are therefore suggesting additional assays to measure phenotypic effects of evolved integrons, and possibly data analyses on the negative controls. If these are not possible to perform, the main conclusions could be slightly altered instead to focus more on the genomics. Finally, we provide some suggestions on making the discussion more balanced and in clarifying the role of chromosomal mutations in the integron-facilitated evolution.

  6. Version published to 10.1101/2020.08.07.237602v2 on bioRxiv
  7. Version published to 10.1101/2020.08.07.237602v1 on bioRxiv