1. Author Response:

    Reviewer #1:

    In this manuscript, Ma, Hung and colleagues rewind the tape to explore the genetic landscape that precedes carbapenem resistance of Klebsiella pneumoniae strains. The importance of this work is underscored by the paucity of new drugs to treat CPO (carbapenemase producing organisms). 'Given the need for 35 greater antibiotic stewardship, these findings argue that in addition to considering the current 36 efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future 37 efficacy are also important.' And so I would say the major weakness of the paper is the aspirational nature of how this work could be used by clinicians in antibiotic selection or treatment of the patient.

    We consider this study as a first step towards recognizing the need to develop more comprehensive diagnostics and more sophisticated antibiotic stewardship programs. This study suggests that factors besides MICs could inform clinical antibiotic selection, including that specific lineages have higher propensity to develop resistance (i.e., ST258), stepping-stone mutations that facilitate the evolution of resistance (i.e., mutations in rseA and ompK36), and antibiotics that have high level resistance barriers (i.e., meropenem). We have now added language to both the introduction and discussion to note that next steps are needed to extend these findings into the clinic, including more extensive whole genome sequencing of isolates and tracking of these strains in the clinic, associated patient outcome and strain evolution data, to understand the full impact of these mutational events in CREs.

    The strains selected for these experiments and the evolutionary in vitro models are both well considered. One idea that has stuck with me from the figures of a review article by Kishony (https://pubmed.ncbi.nlm.nih.gov/23419278/, figure 4) is the concept of constraining the evolutionary pathways or fitness landscape for antibiotic resistance. Are there any peaks that a microbial strain reaches that optimize resistance to one AbX but basically leave it inherently unable to evolve resistance to another AbX? This could have application for dual drug therapy or pulsed therapy.

    This is a good evolutionary question that might be suggested by Kishony’s work. In our particular study however, because the majority of isolates used that are carbapenem susceptible are already resistant to many other antibiotics, we cannot measure their resistance frequencies to other clinically relevant antibiotics. It does suggest that such a strategy would have to be implemented early enough before strains have already acquired significant resistance and cannot be used to manage currently existing resistance.

    When you sequence the isolates that have increased their MIC do you find 'unrelated' mutations in genes that would control protein synthesis or other functions that might be compensatory mutations. Developing a clearer understanding of the rewiring of the bacterium's basic processes might also elucidate both integrated functions and potential weaknesses. You mention mutations in wzc, ompA, resA, bamD.

    Yes. We found some strains had acquired multiple mutations in multiple genes. Please refer to supplementary file 12. In some cases, we found additional mutations of unclear significance; for example, we identified two mutations in Mut86. We tested these two mutations separately and found that only the mutation in ompA affects the susceptibility of the mutant. However, this does not exclude the possibility that the other mutation might have other compensatory functions versus just being a random passenger mutation; this will require further investigation.

    On the other hand, in some cases, we indeed found mutations that affect the fitness of the isolates when cultured in LB medium or M9, e.g., mutations in rseA. Some mutations affect fitness only in LB medium but not M9, e.g., mutations in ompK36. Some mutations do not significantly affect the fitness in either LB or M9, e.g., duplication of blaSHV-12. We are performing RNA sequencing on these mutants to further understand the “rewiring of the bacterium's basic processes.”

    Point of discussion. Classic ST258 carries blaKPC on pKpQIL plasmid. Your ST258 strain (UCI38) carries blaSHV-12 on pESBL. Am I to assume that pESBL is in lieu of pKpQIL?

    Indeed, pESBL encodes an ESBL in UCI38 and may obviate the need for another classical KPC-carrying plasmid such as pKpQIL. However, pESBL and pKpQIL are not incompatible and so it is not clear that anything is precluding UCI38 from picking up pKpQIL.

    Transformation of CPO have many variables and in vitro data does not always mirror what is observed in vivo. So the findings of Fig 2f might need to be considered under different laboratory conditions (substrate, temperature) [https://pubmed.ncbi.nlm.nih.gov/27270289/].

    We revised the statement in the revision and pointed out that the results in Fig. 2F were limited to our assay condition.

    Reviewer #2:

    In this manuscript Ma et al., sought to investigate the breadth of genetic mechanisms available across various lineages of clinical isolates of Klebsiella pneumoniae, with a specific focus on carbapenem resistance evolution. The authors systematically evaluated how different carbapenems and genetic backgrounds affect the rate of evolution by measuring mutation frequencies. The authors found three major observations: First, that a higher mutational frequency is dependent on genetic background and high-level transposon activity affecting porins associated to carbapenem resistance. Importantly transposon activity was not only higher than SNP acquisition rates in distinct backgrounds, but was also reversible, thus emphasizing that resistance evolution via this mechanism might impart less of a cost than by the accumulation of mutations in other genetic backgrounds. Second, that CRISPR-cas systems have the potential to restrict the horizontal acquisition of resistance elements. Importantly, determining the presence or absence of such systems alone is not enough to determine wether a strain is "resistant" to certain foreign elements, but specific sequences within the different spacers can be more informative of the exact range of plasmids or genetic elements to which the system is restrictive. Third, pre-selection with ertapenem increases the likelihood of resistance evolution against other carbapenems both via de novo mutation and HGT.

    Altogether, these results emphasize the importance of additional factors, other than MIC values, such as genetic background, plasmid/transposon activity, and drug identity and choice in determining the rate at which resistance can evolve in K. pneumoniae. I consider that the data generally supports the authors conclusions and provides relevant observations to the field. I do not have any major concern and think the authors have done a very complete and systematic evaluation of the data necessary to answer their questions.

    My only minor concern is regarding the authors emphasis in their introduction and discussion on how these kind of data is relevant for clinical decision making. It remains unclear to me exactly how. While I completely agree that genomic information and drug choice play a major role in the evolution of antibiotic resistance, it is unclear to me how to efficiently and promptly translate all of this information at the bedside. Genome sequencing, however economical it has become in the recent years, is still not affordable to be implemented at the scales needed for diagnosis at the clinic. Perhaps the authors could expand on how they envision this could be implemented?

    We consider this study as a first step towards the development of more comprehensive diagnostics and more sophisticated antibiotic stewardship. Indeed, as current diagnostics exist, it would be difficult to implement. However, we hope that as studies such as these grow, it will usher in a new era of diagnostics that can indeed take such factors into account. We have now added such a discussion to the introduction and discussion in the revised manuscript.

    Read the original source
    Was this evaluation helpful?
  2. Reviewer #2 (Public Review):

    In this manuscript Ma et al., sought to investigate the breadth of genetic mechanisms available across various lineages of clinical isolates of Klebsiella pneumoniae, with a specific focus on carbapenem resistance evolution. The authors systematically evaluated how different carbapenems and genetic backgrounds affect the rate of evolution by measuring mutation frequencies. The authors found three major observations: First, that a higher mutational frequency is dependent on genetic background and high-level transposon activity affecting porins associated to carbapenem resistance. Importantly transposon activity was not only higher than SNP acquisition rates in distinct backgrounds, but was also reversible, thus emphasizing that resistance evolution via this mechanism might impart less of a cost than by the accumulation of mutations in other genetic backgrounds. Second, that CRISPR-cas systems have the potential to restrict the horizontal acquisition of resistance elements. Importantly, determining the presence or absence of such systems alone is not enough to determine wether a strain is "resistant" to certain foreign elements, but specific sequences within the different spacers can be more informative of the exact range of plasmids or genetic elements to which the system is restrictive. Third, pre-selection with ertapenem increases the likelihood of resistance evolution against other carbapenems both via de novo mutation and HGT.

    Altogether, these results emphasize the importance of additional factors, other than MIC values, such as genetic background, plasmid/transposon activity, and drug identity and choice in determining the rate at which resistance can evolve in K. pneumoniae. I consider that the data generally supports the authors conclusions and provides relevant observations to the field. I do not have any major concern and think the authors have done a very complete and systematic evaluation of the data necessary to answer their questions.

    My only minor concern is regarding the authors emphasis in their introduction and discussion on how these kind of data is relevant for clinical decision making. It remains unclear to me exactly how. While I completely agree that genomic information and drug choice play a major role in the evolution of antibiotic resistance, it is unclear to me how to efficiently and promptly translate all of this information at the bedside. Genome sequencing, however economical it has become in the recent years, is still not affordable to be implemented at the scales needed for diagnosis at the clinic. Perhaps the authors could expand on how they envision this could be implemented?

    Read the original source
    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    In this manuscript, Ma, Hung and colleagues rewind the tape to explore the genetic landscape that precedes carbapenem resistance of Klebsiella pneumoniae strains. The importance of this work is underscored by the paucity of new drugs to treat CPO (carbapenemase producing organisms). 'Given the need for 35 greater antibiotic stewardship, these findings argue that in addition to considering the current 36 efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future 37 efficacy are also important.' And so I would say the major weakness of the paper is the aspirational nature of how this work could be used by clinicians in antibiotic selection or treatment of the patient.

    The strains selected for these experiments and the evolutionary in vitro models are both well considered. One idea that has stuck with me from the figures of a review article by Kishony (https://pubmed.ncbi.nlm.nih.gov/23419278/, figure 4) is the concept of constraining the evolutionary pathways or fitness landscape for antibiotic resistance. Are there any peaks that a microbial strain reaches that optimize resistance to one AbX but basically leave it inherently unable to evolve resistance to another AbX? This could have application for dual drug therapy or pulsed therapy. When you sequence the isolates that have increased their MIC do you find 'unrelated' mutations in genes that would control protein synthesis or other functions that might be compensatory mutations. Developing a clearer understanding of the rewiring of the bacterium's basic processes might also elucidate both integrated functions and potential weaknesses. You mention mutations in wzc, ompA, resA, bamD.

    Point of discussion. Classic ST258 carries blaKPC on pKpQIL plasmid. Your ST258 strain (UCI38) carries blaSHV-12 on pESBL. Am I to assume that pESBL is in lieu of pKpQIL? Transformation of CPO have many variables and in vitro data does not always mirror what is observed in vivo. So the findings of Fig 2f might need to be considered under different laboratory conditions (substrate, temperature) [https://pubmed.ncbi.nlm.nih.gov/27270289/].

    Read the original source
    Was this evaluation helpful?
  4. Evaluation Summary:

    This manuscript is of interest to several fields in biology and medicine including evolutionary genomics and antibiotic stewardship. Ma et al. sought to investigate the breadth of genetic mechanisms for evolution of carbapenem resistance across various lineages of the bacterial pathogen Klebsiella pneumoniae. The authors performed systematic and thorough bioinformatic and genetic analyses to identify how transposon activity and CRISPR-Cas systems facilitate the evolution of antibiotic resistance and restriction of horizontally acquired genetic elements, respectively. The study's results emphasize the importance of additional factors, other than MIC values, such as genetic background, plasmid/transposon activity, and drug identity and choice in determining the rate at which resistance can evolve in K. pneumoniae.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

    Read the original source
    Was this evaluation helpful?