The evolution of colistin resistance increases bacterial resistance to host antimicrobial peptides and virulence

  1. Pramod K Jangir
  2. Lois Ogunlana
  3. Petra Szili
  4. Marton Czikkely
  5. Liam P Shaw
  6. Emily J Stevens
  7. Yang Yu
  8. Qiue Yang
  9. Yang Wang
  10. Csaba Pál
  11. Timothy R Walsh
  12. Craig R MacLean

  • Curated by eLife

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    Antimicrobial peptides (AMP) are a class of antibiotics that are inspired by natural components of innate immunity, which raises the specter of bacteria becoming resistant to both. The author this important study test this idea and find compelling evidence that a plasmid that encodes resistance to the AMP colistin also increases resistance to AMPS produced by humans, pigs, and chickens, enables the bacteria to grow better in low levels of AMP, and increases bacterial virulence in an insect model of infection. The study will be of interest to both evolutionary biologists and microbiologists focused on antimicrobial therapy and suggests that the evolution of resistance to these compounds can have collateral effects on immune evasion as well.

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Abstract

Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here, we show that MCR provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and suggest that MCR may be difficult to eradicate, even if colistin use is withdrawn.

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  1. Version published to 10.7554/elife.84395 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The authors of this manuscript aimed to systematically evaluate the pleiotropic effects of MCR-1-mediated colistin resistance. They evaluated the effect of MCR-1 and MCR-3 carried on different plasmids on antimicrobial peptides (AMPs) and assessed their ultimate effect on virulence. The authors find that MCR-1-mediated colistin resistance correlates with increased resistance against some host AMPs, but also increased sensitivity to others. The authors also find that MCR-1 alone is associated with resistance to human serum and to elements of the complement system. This highlights a potential selective advantage for MCR-1-mediated resistance to host immune factors and a potential for enhanced virulence.

    The methods have been well established before and adequately support their main findings. While determining the role of MCR-1 in a single genetic background is important to better understand its potential pleiotropic effects against a diversity of AMPs and in a variety of scenarios, the impact and significance of the results are partially ameliorated because different genetic backgrounds, particularly those most relevant to a clinical (or agricultural) context were not considered. The results depicted here are still a necessary and important step towards a more comprehensive understanding of the pleiotropic effects of MCR-1. But, interactions between plasmids and host genomes and their co-evolution can have important effects more generally. The authors do mention this in the discussion and suggest it to be an important avenue for future work. However, given the objective of the study and the clinical and agricultural context in which the authors have framed their work, it seems more relevant to include those distinct genetic backgrounds already here.

    The conclusions stemming from the results found in Figure 3, and Figures 4c and d seem too overreaching to me. The associated resistance to AMPs from pigs seems to be only strong enough against one of the five tested AMPs and hence concluding that these impose a strong selective pressure in the pig's gut seems unsubstantiated. Similarly, the difference in survival probability within their in vivo system, though statistically significant, seems to be very ild between their MCR-1 and empty vector control.

    Thank you for the comment. We agree on the effect of MCR-MOR on AMP susceptibility and have edited the paragraph by removing the lines on strong selective pressure in the pig gut. As regards the 4c and 4d results (4e and 4f in the revised version), it is interesting and statistically convincing that MCR increases bacterial virulence despite the cost of MCR expression. And importantly, this effect is even stronger in the case of LPS treatment where the immune system is stimulated, expressing diverse host AMPs (PMID: 19897755). This shows MCR-mediated advantages to bacteria in the complex host environment.

    Reviewer #2 (Public Review):

    Jangir et al test the hypothesis that resistance to the antimicrobial peptide (AMP) colistin can simultaneously increase resistance to other AMPS with related modes of action. Because AMPS comprise part of innate immunity, their central concern is that colistin resistance may compromise host defenses and thereby increase bacterial virulence. Their results show that MCR-1, whether expressed from naturally circulating or synthetic plasmids, can increase the MIC to AMPS from humans, pigs, and chickens, and impart fitness benefits at sub-MIC concentrations. In addition, they find that MCR-1-containing strains have increased survival in human plasma and are more lethal in an insect infection model.

    The conclusions of the paper are generally well supported by the results, but some aspects could be clearer and better defended with a few small additional experiments.

    Strengths:

    Using both synthetic and natural plasmids makes it possible to cleanly separate the effects of MCR-1 from the effects of other plasmid-borne genes or plasmid copy numbers. This helps confirm the causal role of MCR-1 on altered AMP susceptibility.

    Testing the survival of transformed isolates in human serum and in insects points to relevance in the more immunologically complex host environment where cells are exposed to a suite of factors that reduce bacterial survival.

    Thank you!

    Weaknesses/suggestions:

    Although increases in MIC are evident for different AMPS, the effects are generally modest. To address this, it might be helpful to use pairwise competition assays, as in Figure 1, to establish that even small changes to MIC are associated with clear selective benefits.

    Thank you for the suggestion. We agree that in some cases the change in MIC is modest, however, we would like to highlight that small-level changes in resistance have important clinical implications. For example, resistance mutations conferring a small change in MIC can ensure the survival of pathogenic bacteria in antibiotic-treated hosts (PMID: 30131514). Additionally, a comparison between competition assays (Fig 1) and MICs (Fig 2) clearly shows that small changes in MIC are associated with substantial fitness benefits. For example, for pSEVA:MCR-1, the fold change in MIC of CATH2 (chicken), PMAP23 (pig), and LL37 (human) ranges between 1.05 and 1.5, however, the competitive fitness ranges from 10% to 17%. This issue is discussed in the revised manuscript (lines 306-317, page 13)

    ….This would be especially helpful in assays with human serum and in Galleria where the concentrations of AMPS or other immune components are unknown.

    It is clear that MCR-1 increases resistance to serum and virulence (Figure 4). However, we agree with the reviewer that the selective benefits of MCR-1 in complex host environments are not known (i.e., serum or Galleria). We have revised the final paragraph of the discussion to reflect this limitation of our study (lines 370-382, page 15).

    Assays using human serum are interesting but challenging to interpret given the diverse causes of bacterial killing, including complement. Although this was partly addressed in Supplementary Figure 6, I found the predictions of these experiments unclear. First, I think these experiments are too central to be relegated to the supplemental materials; they belong in the main text. Secondly, it is important to explicitly spell out the expectations of using heat-killed serum (which will degrade any heat-labile components) or complement-deficient serum. It should be clearer under which conditions MCR-1-containing strains are predicted to do better or worse than controls.

    We have addressed this in the revised version. We have moved Supplementary Fig 6 to the main text, and have edited the text, clarifying the model prediction (lines 245-257, page 10).

    Galleria is a useful infection model for virulence, but it is unclear what drives differences between strains. First, bacterial numbers aren't measured in this assay, so it isn't known if increased virulence is due to increased bacterial growth or decreased bacterial clearance. As above, I think these assays would be stronger using the competition-based approach in Figure 1. This would indicate bacterial numbers through time and directly show the selective benefit associated with MCR-1. Second, it would be useful to elaborate on why MCR-1 increases virulence, especially any known similarities between Galleria AMPS and those tested in Figures 1 and 2. Overall, it would help if Galleria were less of a black box.

    We agree that the mechanism underlying increased virulence remains to be explored and thus, we have already discussed this in the discussion as a limitation (lines, 370-382, page 15). However, elucidating the mechanisms by which MCR-1 increases virulence would clearly be an interesting line of research moving forward.

  3. eLife

    eLife assessment

    Antimicrobial peptides (AMP) are a class of antibiotics that are inspired by natural components of innate immunity, which raises the specter of bacteria becoming resistant to both. The author this important study test this idea and find compelling evidence that a plasmid that encodes resistance to the AMP colistin also increases resistance to AMPS produced by humans, pigs, and chickens, enables the bacteria to grow better in low levels of AMP, and increases bacterial virulence in an insect model of infection. The study will be of interest to both evolutionary biologists and microbiologists focused on antimicrobial therapy and suggests that the evolution of resistance to these compounds can have collateral effects on immune evasion as well.

  4. eLife

    Reviewer #1 (Public Review):

    The authors of this manuscript aimed to systematically evaluate the pleiotropic effects of MCR-1-mediated colistin resistance. They evaluated the effect of MCR-1 and MCR-3 carried on different plasmids on antimicrobial peptides (AMPs) and assessed their ultimate effect on virulence. The authors find that MCR-1-mediated colistin resistance correlates with increased resistance against some host AMPs, but also increased sensitivity to others. The authors also find that MCR-1 alone is associated with resistance to human serum and to elements of the complement system. This highlights a potential selective advantage for MCR-1-mediated resistance to host immune factors and a potential for enhanced virulence.

    The methods have been well established before and adequately support their main findings. While determining the role of MCR-1 in a single genetic background is important to better understand its potential pleiotropic effects against a diversity of AMPs and in a variety of scenarios, the impact and significance of the results are partially ameliorated because different genetic backgrounds, particularly those most relevant to a clinical (or agricultural) context were not considered. The results depicted here are still a necessary and important step towards a more comprehensive understanding of the pleiotropic effects of MCR-1. But, interactions between plasmids and host genomes and their co-evolution can have important effects more generally. The authors do mention this in the discussion and suggest it to be an important avenue for future work. However, given the objective of the study and the clinical and agricultural context in which the authors have framed their work, it seems more relevant to include those distinct genetic backgrounds already here.

    The conclusions stemming from the results found in Figure 3, and Figures 4c and d seem too overreaching to me. The associated resistance to AMPs from pigs seems to be only strong enough against one of the five tested AMPs and hence concluding that these impose a strong selective pressure in the pig's gut seems unsubstantiated. Similarly, the difference in survival probability within their in vivo system, though statistically significant, seems to be very ild between their MCR-1 and empty vector control.

  5. eLife

    Reviewer #2 (Public Review):

    Jangir et al test the hypothesis that resistance to the antimicrobial peptide (AMP) colistin can simultaneously increase resistance to other AMPS with related modes of action. Because AMPS comprise part of innate immunity, their central concern is that colistin resistance may compromise host defenses and thereby increase bacterial virulence. Their results show that MCR-1, whether expressed from naturally circulating or synthetic plasmids, can increase the MIC to AMPS from humans, pigs, and chickens, and impart fitness benefits at sub-MIC concentrations. In addition, they find that MCR-1-containing strains have increased survival in human plasma and are more lethal in an insect infection model.

    The conclusions of the paper are generally well supported by the results, but some aspects could be clearer and better defended with a few small additional experiments.

    Strengths:
    Using both synthetic and natural plasmids makes it possible to cleanly separate the effects of MCR-1 from the effects of other plasmid-borne genes or plasmid copy numbers. This helps confirm the causal role of MCR-1 on altered AMP susceptibility.

    Testing the survival of transformed isolates in human serum and in insects points to relevance in the more immunologically complex host environment where cells are exposed to a suite of factors that reduce bacterial survival.

    Weaknesses/suggestions:
    Although increases in MIC are evident for different AMPS, the effects are generally modest. To address this, it might be helpful to use pairwise competition assays, as in Figure 1, to establish that even small changes to MIC are associated with clear selective benefits. This would be especially helpful in assays with human serum and in Galleria where the concentrations of AMPS or other immune components are unknown.

    Assays using human serum are interesting but challenging to interpret given the diverse causes of bacterial killing, including complement. Although this was partly addressed in Supplementary Figure 6, I found the predictions of these experiments unclear. First, I think these experiments are too central to be relegated to the supplemental materials; they belong in the main text. Secondly, it is important to explicitly spell out the expectations of using heat-killed serum (which will degrade any heat-labile components) or complement-deficient serum. It should be clearer under which conditions MCR-1-containing strains are predicted to do better or worse than controls.

    Galleria is a useful infection model for virulence, but it is unclear what drives differences between strains. First, bacterial numbers aren't measured in this assay, so it isn't known if increased virulence is due to increased bacterial growth or decreased bacterial clearance. As above, I think these assays would be stronger using the competition-based approach in Figure 1. This would indicate bacterial numbers through time and directly show the selective benefit associated with MCR-1. Second, it would be useful to elaborate on why MCR-1 increases virulence, especially any known similarities between Galleria AMPS and those tested in Figures 1 and 2. Overall, it would help if Galleria were less of a black box.

  6. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6386692.

    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:

    The Evolution of Colistin Resistance Increases Bacterial Resistance to Host Antimicrobial Peptides and Virulence

    Jangir P. K., Ogunlana L., Szili P., Czikkely M., Stevens E. J., Yang Y., Yang Q., Wang Y., Pál C., Walsh T. R., & MacLean C. BioRxiv 2022.02.12.480185; https://doi.org/10.1101/2022.02.12.480185

    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: Antimicrobial peptides (AMPs) are small molecules with potent, broad antimicrobial activity that are produced by the innate immune systems of eukaryotic and prokaryotic organisms. Emergence of resistance to AMPs is low, which makes them good alternatives to chemical antibiotics for therapeutic use. The authors suggest that bacterial resistance to therapeutic AMPs could facilitate cross-resistance to host AMPs, because therapeutic and naturally occurring AMPs share structural and mechanistic similarities. To investigate if resistance to the bacterial-derived therapeutic AMP colistin can confer protection against known mammalian AMPs, the authors assessed competitive growth of E. coli strain J53 carrying mobile colistin resistance genes that encode MCR-1, a phosphoethanolamine transferase that decreases the bacterial negative membrane charge and confers protection to the cationic peptide colistin, in the presence of various AMPs from human, pig, and chicken. The authors showed that E. coli expressing naturally occurring MCR-1 and MCR-3 plasmids or expressing MCR-1 from a non-conjugative pSEVA121 vector (pSEVA:MCR-1), displayed increased resistance to colistin and mammalian AMPs compared to parental control E. coli, but also displayed increased sensibility to two of the AMPs tested. The authors also showed that MCR-1 from the pig commensal Moraxella only conferred a small resistance to colistin and no resistance to the mammalian AMPs tested but had similar effects on bacterial membrane charge compared to MCR-1. Furthermore, the authors tested if MCR-1 could confer resistance to a complex mixture of antimicrobials by measuring the growth of MCR-1-carrying E. coli in the presence of human serum. In this experiment, MCR-1 expressing E. coli grew in the presence of higher concentrations of serum than parental E. coli, which suggests that MCR-1 confers resistance to human serum. Lastly, the authors investigated whether MCR-1 expression increases bacterial virulence. For this purpose, the authors deployed a Galleria mellonella infection model and showed that larvae infected with E. coli carrying pSEVA:MCR-1 displayed lower survival rates than larvae infected with control E. coli. The increased virulence of MCR-1-expressing E. coli was not attenuated by LPS-mediated stimulation of host innate immunity. From these experiments, the authors concluded that colistin resistance through expression of MCR-1 also facilitates resistance to AMPs from humans, pigs, and chickens. As such, the authors highlight the importance of assessing cross-resistance to natural AMPs for the design of therapeutic AMPs that are going to be used in clinical and agricultural settings. 

     

    OVERALL ASSESSMENT: The manuscript tackles an interesting topic that relates to the use of therapeutic AMPs in healthcare and agricultural settings and draws attention to the risk of the emergence of cross-resistance to natural AMPs. The manuscript is well written, and the methods presented are described in great detail, but we thought that additional information on the host AMPs examined and the G. mellonella infection model would make the manuscript stronger. Data presentation was consistent throughout the study and easy to understand, and we only had a few suggestions to improve the figures further.

     

    STRENGTHS:

    -  The manuscript is well written, and the methods were thoroughly described to allow for reproducibility.

    -  The authors properly describe their rationale and background for studying therapeutic AMPs and cross-resistance to natural AMPs with a focus on the health and agricultural sectors.

     

    WEAKNESSES:

    -  The manuscript would benefit from additional background information on the natural mammalian AMPs the authors chose to investigate. We would have liked the authors to provide the reader with additional information such as similarities between the different AMPs based on mechanism of action, structure, amino acid identity, etc. These properties could be listed in Table 1. 

    -  The authors repeatedly draw the conclusion that evolution of resistance to AMPs can facilitate cross-resistance to AMPs of host innate immunity but did not perform any experiments to test this directly. This was a good opportunity to address the hypothesis with an experimental evolution study.

    -  We agree that E. coli strains are diverse and that using a single strain as the recipient of the MCR-1-encoding plasmids is not representative of all E. coli strains. We would appreciate if the authors would consider investigating multiple different E. coli strains for future studies. 

     

    DETAILED U.P. ASSESSMENT:

    OBJECTIVE CRITERIA (QUALITY)

    1.  Quality: Experiments (1–3 scale, where 1 is highest and 3 is lowest) SCORE = 1.5

    ● 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.]

    Figure 2B: We think that the bar graphs, especially the horizontal one, made understanding Figure 2B more difficult for the reader. We suggest grouping the data presented in the horizontal bar graph in a different order (i.e., by magnitude of average resistance conferred by each plasmid), or by using a different representation method altogether. We also suggest the authors group the mammalian AMPs based on common structural features or based on relative changes in MIC as shown in their vertical bar graph. 

    Figure 3A-D: The Y axes display relative growth, but in their results, the authors compare resistance conferred by MCR-1 and MCR-MOR as "changes in log2MIC". We suggest the authors compare relative growth in their results or display the data shown in Figure 3 as fold changes in MIC. In their results, the authors also note that "In line with previous work, MCR-MOR expression provided a small increase (5.9-fold) in colistin resistance as compared to MCR-1 (13.2-fold)" (lines 188-190). We would like the authors to clarify whether "previous work" refers to their own work or that of a different group. 

    Figure 4A: While the data is interesting, the authors do not provide an explanation as to why MCR-1 facilitates resistance to human serum. We suggest the authors comment on what constituent(s) of the serum they think MCR-1 might confer resistance to. In addition, the authors could propose follow-up experiments, such as using dialyzed human serum that excludes small molecules, including AMPs, to narrow down which molecules MCR-1 confers protection against. 

    Figure 4D: The authors stimulated the innate immunity of G. mellonella larvae with LPS but did not investigate whether immune responses were stimulated. We do not think that probability of survival of LPS-treated, E. coli-infected larvae is an appropriate measure of immune stimulation. We suggest the authors clarify whether assessing probability of survival of G. mellonella treated with LPS prior to infection provides an indication of innate immune stimulation. Alternatively, we suggest the authors re-interpret the data or re-visit their experiment and assess immune stimulation by different means (i.e., the authors could perform RT-qPCR on the larvae and probe for markers of immunity). Overall, this aspect of the study was underdeveloped. 

     

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

    o   Yes, but we would again like to highlight that we do not believe probability of survival of LPS pre-treated, E. coli-infected G. mellonellais an appropriate indication of immune stimulation. 

     

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

    o   Yes

     

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

    o   Yes, but we would like the authors to unambiguously clarify how many G. mellonella larvae were used per biological replicate and/or in total.

     

    ● 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, methods are standard for this field.

     

     

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

    ● 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   We do not believe that "evolution of colistin resistance" and "accidental evolution of resistance" should be used in the title and abstract of the manuscript. The authors transformed E. coli with naturally occurring plasmids instead of performing evolution experiments through which initially susceptible bacteria would develop resistance to colistin. The use of the word "evolution" is not supported by the data presented in the manuscript and we therefore suggest the authors revise the title. 

     

    ● 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   No

     

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

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

    o   Yes, experiments were performed with an n of 3, 6, or 9.

     

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

    o   Yes, but we have some concerns about the value and reproducibility of the G. mellonella infection model for assessing virulence of MCR-1-carrying E. coli. Specifically, we would like to know how the authors controlled for other bacterial determinants that could affect results (i.e., were the larvae pathogen-free? What commensal bacteria were the larvae colonized with?). 

     

    ● Are the 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 = 2

    ● 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 be significant bases for decisions.

    o   We thought the reader would benefit from additional information on the mammalian AMPs the authors investigated in their study (i.e., do they share structural and mechanistical similarities?).

    o   The authors explain the mechanism of action of MCR-1, but not that of colistin. We suggest the authors include a short description of the function of colistin for completeness. 

    o   We wondered how the 62% "identity" between MCR and MCR-MOR was determined (line 188). Does "identity" refer to DNA sequence identity or amino acid identity? We would appreciate if the authors could clarify this in their results.

     

    MORE SUBJECTIVE CRITERIA (IMPACT)

    1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2

    ● 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.

    o   The authors show that widespread, mobile colistin resistance genes that encode MCR-1 confer cross-resistance to AMPs of the innate immune system of humans, chickens, and pigs. E. coli expressing MCR-1 also display increased resistance to human serum and may be more virulent.

     

    ● 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 discovery that therapeutic AMPs confer cross-resistance to AMPs of the innate immunity is not novel and has been explored before (Kintses et al., 2019. doi:10.1038/s41467-019-13618-z). Likewise, MCR-1-mediated cross-resistance to AMPs other than colistin has also been explored (Xu et al., 2018. doi: https://doi.org/10.1128/mSphere.00411-18). As such, we do not consider the findings of the study to be a significant advance in the field, but we agree that investigating cross-resistance to AMPs of host innate immunity is of significant interest to healthcare and agricultural settings where AMP antibiotics are commonly used, and we believe that the data the authors collected is valuable and adds to the growing evidence that cross-resistance is a major concern.

     

    2.Impact: Extensibility (1–4 or N/A scale) SCORE = 3

    ● 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   The study compares mammalian and bacterial AMPs in a variety of assays and tests them in a model organism (G. mellonella larvae) but this aspect of the study is underdeveloped and there is no further extension into higher organisms. 

     

    ● 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. We think that the scope of this study is largely sufficient and extension to higher organisms like a mouse model is not required. 

     

     

  7. Version published to 10.1101/2022.02.12.480185v1 on bioRxiv