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Both Reviewers have concluded their concerns have been sufficiently addressed.
[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
It includes a clear desicription of backgroud and context.
The experiment was designed well and this research is meaningful.
Authors have provided all data and the conclusion are well stated.
Authors have resolved my questions.
The author has taken care of all issues raised by me in this section. Thanks.
By rewriting some parts of the manuscript the scope of the study now becomes clearer and the context now is correct.
Thanks to the author for the explanation regarding the nature of the peptides.
Thanks for the clarification. Indeed, from the section written in the materials and methods I, mistakenly, assumed that diluted MHB was used for the experiments.
I would suggest that the concentration of MHB is ether given as amounts of each of the components (2.0 g beef extract, 17.5 g casein hydrolysate, 1.5 g starch), or as total amount of medium powder (21 g/L) instead of %. This will avoid confusion.
All points raised during review have been addressed. Many thanks!
You will see that, both reviewers agreed that the manuscript could become technically sound with revision and they have raised points that need to be addressed by a major revision. I therefore invite you to revise and resubmit your manuscript, taking into account the points raised.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
This article was written clearly, including sufficient background, intact structure/tables/data and good results.
Activity of three defensins against clinical S. aureus and E. coli and their combination with antibitocis have been conducted rigorously; the methods in this study were described in detail.
The data in this study will provide a good guidance for clinical application of defensins.
Author detected the antimicrobial activity of three human defensins (HNP-1, hBD-1, 274 and hBD-3) against clinical 27 S. aureus and 24 E. coli isolates. The combination of three defensins with rifampicin or amikacin against these clinical isolates was assessed by FICI. It is very interesting work; similar to antibiotics, some AMPs also develop bacterial resistance. So, please consider the resistance of AMPs although AMPs may be one of alternatives to antibiotics in the future. However, some questions need to resolve before publishing:
Please give the resources of 27 S. aureus isolates and 24 E. coli isolates if it is possible.
Table 1: please give the unit of length.
Table 5: please indicate “-”; “since MIC values of …were > 64 mg/L or since MIC of …was > 64 mg/L” in legend.
What about the resistance of three defensins?
Line 116: It is uncompleted sentence “…modified according to…”
Other minors:
Line 120-131: lowercase ml and μl
Table 4, 5: numbers in bold in column 3-5
Line 293-295, 315-316, 320-321: the formats of references.
The structure of the article adheres to the PeerJ guidelines, however there is an imbalance in the information content of the sections. While the introduction and the results are formulated concisely, the discussion section is rather lengthy and contains discussion about concept that are far beyond the research question that has been addressed by the study. For example, the author discussed the disadvantages of peptide stability during human usage over two paragraphs and provides an example concept on how to solve this (cyclic AMPs), however, peptide stability was neither monitored in this study nor was the study designed in such a way that peptide instability due to human peptidases would have been an issue. I highly recommend to use the results of the study as a fundament for the discussion and thus write the discussion as concise as the rest of the article.
Apart from that, the article is nicely written, well-structured and formulated in a clear English. All arguments presented by the author can be easily followed. However, there are some minor mistakes (e.g. usage of wrong words as in line 121: I assume the author wanted to use the word "diluted" not "dissolved"). I would therefore recommend to proof-read the article by a native speaker before final publication.
The author provided a supplementary file containing the MIC data (that also have been shown in the main result tables) as well as the results from the checkerboard assays and the calculation of the FICI. This data is labeled as “raw data”. While the dataset seems to be complete, it is not real “raw data” but has been processed already. For example, the MIC values have been provided in a consolidated form using a non-disclosed method instead of providing the value for each independent MIC experiment. I would recommend to add the single measurement values for each of the experiments (“raw data”) in addition to the consolidated values (e.g. mean) as they can be helpful for the readers to judge variation of the assay.
The concept of the study and the experimental design the author has chosen is puzzling: In the introduction the author justifies the importance of his work with increasing antibiotic resistance (39ff) and suggest the usage of AMPs together with conventional antibiotics as potential solution to antibiotic resistance (91f). In the discussion the author furthermore connects the data obtained in this study to a potential new strategy for overcoming antibiotic resistance (186ff). The message that is transported with this argumentation is: the use of AMPs together with antibiotics might help to reduce the impact of antibiotic resistance (i.e. makes resistance strains susceptible to the antibiotic again). While this has been postulated and shown by others (e.g. DOI: 10.1128/IAI.71.7.3730-3739.2003) the experimental design cannot support this hypothesis: The author has selected the antibiotics rifampicin for S. aureus and amikacin for E. coli and showed synergistic effects with AMPs, however, none of the strains was resistance against the antibiotic tested. The question if the usage of AMPs together with the antibiotic is really capable of pushing MIC values of resistant strains below the clinical breakpoints (and thus makes the antibiotic useful for the treatment of such strains again) remains therefore unanswered. In case of the selected antibiotics such an observation would be highly relevant as resistance against both molecules is typically based on target modification or compound inactivation. Interestingly enough, the author did include strains with various antibiotic resistant phenotypes in the experiment e.g. the S. aureus strains SA9 to SA27 are amikacin or methicillin resistant. Even though the author did use amikacin in his study, he did only determine FICI values of AMPs and amikacin against E. coli (which are all amikacin sensitive) and not against the resistant S. aureus strains such that a real benefit of co-treatment of AMP and antibiotic against a specifically resistant strain has not been shown. Using this setup, the claim to contribute to a potential solution to the antibiotic resistance crisis cannot be made. I would either urge the author to perform an additional experiment where e.g. the AMN or MRSA phenotype strains are indeed tested together with AMP + amikacin or methicillin (i.e. using an antibiotic where the strain has really acquired resistance to), or avoid making the claim that the cotreatment presented in this study could help to overcome antibiotic resistance.
Minor issue: The author referred to the peptides as recombinantly produced (99f) by an external provider (Cloud-Clone, USA). The author furthermore listed the sequences of the peptides in table 1. When checking the peptides at the catalog of the provider I realized that those are his-tagged. However, the author did not add the his-tag in the sequence table. If a his-tag is indeed present, it would be good to list the tag in the table and mention this in the text clearly as this might have an impact on the activity of the peptide.
Antibiotic susceptibility is tested under standardized conditions as defined in CLSI or EUCAST guidelines. This includes the usage of Mueller-Hinton broth (MHB) as medium for cultivation of the strains. While the author used EUCAST guidelines to test antibiotic susceptibility of the clinical isolates against standard antibiotics (108), the protocol was changed fundamentally for testing synergism with AMPs (121ff). Here the author uses 48-fold diluted MHB (2.1%) for testing. Such a dilution changes salt concentration and osmolarity of the medium dramatically, which are know to have a direct effect on the integrity of the cell membrane of bacteria. The AMPs used in this study are membrane-active and the author (and others) postulate that the synergistic effects of AMPs with antibiotics are due to increased permeability of the membrane for antibiotics (i.e. the antibiotic reaches the target more efficiently). Testing synergism under such a highly atypical condition raises serious concerns about the validity of the findings described here. As the synergistic effect is the key finding of this study, I would highly urge the author to repeat the experiment under conditions following EUCAST guidelines or give a clear indication why such conditions have been used for testing and how this would translate to a potential application.
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