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

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.