Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics

Round 1

Reviewer 1 Report

This review article is focused on an important topic of the influence of bacteriophages on bacterial antibiotic resistance/sensitivity and tolerance. The paper is well-structured and informative. However, I found several shortcomes which must be corrected.

1. Line 52: for indication that bacteriophages are parasites of bacteria, another, more recent and more accurate paper can be cited (apart from or instead of ref. 15). See https://doi.org/10.33073/pjm-2022-005
2. Line 63: There is a significant problem here, and then in the next chapters. Namely, pseudolysogeny is wrognly defined in this manuscript. According to the literature (including ref. 22, cited by the authors), pseudolysogeny is defined as dormant state of a lytic phage in bacteria which are metabolically inactive (or of a very low activity). This means that during pseudolysogeny, phage replication is blocked or stuck, and can be re-established after the host cell re-initiate metabolic processes. In contrast to this definition, and contrary to what is written by the authors, Inoviruses do not enter pseudolysogeny after infection of their hosts under standard physiological conditions. Although they do not cause classical lysis of the host cell, they can effectively replicate inside the cells, continuously producing progeny phages. This type of phage development is called "permanent infection" or "chronic cycle", but not pseudolysogeny (for recent review, see for example https://doi.org/10.1093/femsre/fuab052). The crucial difference between pseudolysogeny and the chronic cycle is that phages do not replicate during the former process while they replicate intensively during the latter one. Therefore, the authors should re-write parts of the manuscript where pseudolysogeny is mentioned (is most places, it is wrongly interpreted).
3. Page 5: Imprecise excission of prophages occurs only during specialized transduction, not during the generalized transduction.
4. Page 5: It is not true that during lateral transduction, phage capsids package ONLY bacterial DNA. Not ONLY but PREDOMINANTLY.
5. Please, explain all abbreviations which are used in the text. In the current version, there are many unexplained abbreviations.
6. Page 5: "Pf phage produced by P. aeruginosa...". "Produced" is a very inaccurate word in this sentence. Please, re-write.
7. "gram-negative" and "gram-positive" should be "Gram-negative" and "Gram-positive", respectively (the name is after a surname of researcher, thus, it should be capitalized).
8. Pages 5-8: Please, re-consider the paragraph concerning pseudolysogeny (see point 2, above).
9. Figure 2: See points 2 and 8 above.
10. There are many typographical and minor grammar errors which should be corrected.

Author Response

We thank the reviewer for  supportive comments and for time and effort reviewing this manuscript. We have attached our response for revision. 

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript the authors showed a revision of phage therapy to fight AMR.

The review by itself does not show any novelty from several other literature manuscripts (e.g. doi.org/10.3389/fcimb.2019.00022; doi: 10.1038/s41426-018-0169-z; doi: 10.1128/mSystems.00218-21; doi.org/10.2217/fmb-2017-0261; doi.org/10.1016/j.chom.2019.01.014; http://dx.doi.org/10.1016/j.copbio.2021.02.002, etc). This is the main issue related with this manuscript.

Minor issues:

#1- The figures are showed with a very low resolution.

#2- Some references are too old.

#3- The text needs some updates/reformulations (e.g. were 52
discovered independently in 1915 by Frederick Twort, British pathologist [16], and in 1917 by Félix d’Hérelle, a French-Canadian microbiologist..."

too much detail that does not help the reader).

Author Response

Thank you very much for your invaluable comment. We do appreciate all those suggestions during revision.

Reviewer 3 Report

The authors of the manuscript entitled “Bacteriophage Impact on Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics” decide to review a topic of an ongoing debate for many years now. Generally, the manuscript is written in a very comprehensive and understandable way and has a certain point of view, that of the application of lysogenic phages in medicine. The manuscript contains great information but its scope based on its title and the final conclusions are not alligned.

Being said that I am left a little bit confused by the general conclusion authors decide to write about related to the information included in the text.

Point 1

Lines 82-87 and subsequently figure 1. Indeed, susceptible bacteria acquire AMR in multiple ways. One of them is not included in this paragraph. That of transcriptional adaptation of porins or transporters in the membrane of bacteria. This actually is a more common way for bacteria to develop resistance to certain antibiotics, by blocking their entrance into the cytoplasm, and can also be spread through quorum sensing peptides between species. In line 275 there is a small mention of that mechanism but for different purposes. I believe that the authors should rewrite this paragraph and review the figure.

Point 2

This is a general point, after reading the whole manuscript, is like authors mislead the readers by proposing that bacteriophages are the main vehicle for spreading AMRs and that is why the use of bacteriophages should be generally reconsidered. In fact, there are recent publications proposing the exploitation of the lytic nature of even lysogenic phages.

Monteiro, R., Pires, D.P., Costa, A.R. and Azeredo, J., 2019. Phage therapy: going temperate?. Trends in microbiology, 27(4), pp.368-378.

In my opinion, the manuscript should be written in a different way, proposing that the bacteriophages used under clinical applications should be in detail studied (including NGS and in vitro testing) prior to their use. This could ensure the avoidance of spreading AMRs. In fact, easy in vitro tests can show us the dissemination of AMRs from a specific phage nowadays.

Point 3

Only 25% of the references are from the last 6 years. This is creating some confusion because there are many recent publications that propose that the spread of AMRs with bacteriophages in nature is not as common as it was believed. In fact, most recent reviews about phages do not even describe now that the use of bacteriophages is a potential risk for spreading AMRs.

Kaur, R. and Sethi, N., 2022. Phage Therapy as an Alternative Treatment in the Fight Against AMR: Real-World Problems and Possible Futures. In Emerging Modalities in Mitigation of Antimicrobial Resistance (pp. 357-374). Springer, Cham.

Loh, B. and Leptihn, S., 2020. A call for a multidisciplinary future of phage therapy to combat multi-drug resistant bacterial infections. Infectious Microbes & Diseases, 2(1), pp.1-2.

Concluding

There are numerous publications that are involved on how bacteriophages own ARGs and the probabilities of spreading them. What we should have in mind I believe is that there are vast differences between what is happening in nature and what can happen under clinical applications.

 

That is why we should study both the host and the bacteriophage before any application, monitor how they interact, and perhaps how many ARGs are available for spread. On most occasions though (especially in hospitals) AMR is due to transcriptional adaptation of bacteria against antibiotics and the risk of spreading AMRs (especially with the use of lytic phages) is minimized. 

Minor comment

Please ensure the high quality of the figures. 

Author Response

We thank the reviewer for the supportive comments and for time and effort reviewing this manuscript. We have attached our response for revision. 

Author Response File: Author Response.docx

Reviewer 4 Report

Opinion on Chen et al., " Bacteriophage Impact on Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics "

The authors give an extensive review of a topic (indicated in the title) that is, despite its importance, not usually discussed in such detail. The review is easy and relaxing to read, written in good English. The high number of references makes the paper quite informative, but at the same time, the details provided in the text do not overwhelm the reader. I would just recommend a few minor changes prior to acceptance:

Lines 126-128: "Moreover, in ex vivo, transferring phage that isolated from antibiotic treated mice to aerobically culture naïve microbiota can increase AMR in naïve microbiota [53]. " Please rephrase this sentence for improved clarity.

Lines 141-150: This paragraph is overly complicated. It starts with referring to the work of Enault et al., but the actual reference is only provided at the end of the last sentence. In between, several other works are cited, and the connection of the five cited papers is not fully clarified.

Figure 1B should be more explanatory concerning the differences of the three ways of transduction. E.g. add: "The dashed lines delimit the transferred genetic content of the transducing phages." Also, for generalized transduction, the figure misleadingly suggests that only DNA neighboring the integrated phage genome can be transferred. Actually, no integration is required at all, generalized transduction functions great even for hypervirulent phages.

Lines 262-264: the authors argue that certain phages induce antibiotic resistance in Ralstonia solanacearum, at the cost of twitching motility, and cite 9 papers to support this statement. I would recommend being more focused when citing the key references of this single sentence.

 

Line 281: " It would convert bacteria back to being sensitive to antibiotics." This sentence is overly simplified, and could mislead naïve readers. It would sound more correct instead that the phage infection poses a selective pressure for the cells to lose the transporter, and regain sensitivity to antibiotics.

 

Author Response

We thank the reviewer for the supportive comments and for time and effort reviewing this manuscript. We have attached our response for revision.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed my previous comments. I do not have further concerns.

Author Response

The authors thank the reviewer for the recommendations.

Reviewer 2 Report

In the revised version of the manuscript the authors made an effort to improve it. 

The text contains now much more clear sentences and the manuscript figures have the necessary quality.

Although, this review does not contain enough novelty (because there are several reviews in this field) it can be published after some modifications:

i) Section 2 - Please improve the first paragraph as AMR is much more than  MIC quantification;

ii) Please include a few more recent references (last 2-3 years);

iii) Please prepare in greater detail the first paragraph of section 3. Not sure the author´s definition is totally correct. 

iv) Conclusion - "...it is intriguing to imagine that cocktails of phage and conventional antibiotics could have utility against AMR pathogens" in fact there are literature concerning the application of phage to fight AMR. Thus this  reviewer recommends the authors to see this in more detail. 

v) Also, "This approach may have particular utility against biofilm infections or other hard to treat infections". The authors should support this sentence and again they can find several research papers on this topic.

 

Author Response

Please find attached authors' response.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have responded adequately to my comments. 

Author Response

The authors thank the reviewer for the recommendations.

Round 3

Reviewer 2 Report

Authors have addressed my comments adequately.