PONE-D-23-00107Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief reportPLOS ONE

Dear Dr. Sones,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers found the study to be of value; however, additional clarification of the results is necessary. Furthermore, the discussion and conclusions should be edited to match the data generated in this study, as indicated by the reviewers.

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Academic Editor

PLOS ONE

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JLS

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract: line 42, define FBZ.

Please clearly mention which comparisons (gender, medication, before/after medication, obesity) are you targeting. Each result line in mentioned for a different comparison. Please give

Give Bray-Curtis dissimilarity p value. The difference is only between male and female which means treatment has no effect. Please give each p values. Results are not coherent with the conclusions.

I wouldn’t say Lactobacillus as a marker of healthy gut. Lactobacillus at genus level has more complex role.

Methods:

What we mean by Adult (8 weeks-6 months of age)?

Line 86 to 91: break in smaller sentences.

Line 102, 103: were not are

Line 107 : exiting the room, respectively

Line 109: Samples collection:

Line 110: individually in sterile empty cages

Line 101: were placed in sterile tubes

Line 129: Microbial community

Changes at the phyla level in BPH/5 mice with Fenbendazole treatment

Herein just mention treatment effects (FBZ). Or if you want to add gender as well, make a crystal clear difference from the start.

Only mention changes with p value < 0.05 or elsewhere mention p value. Result sentences are not written properly, and each sentence has multiple results with no clear reflection. Sometimes past and present are mixed (164-165).

Either figures are not clear, or p values are not mentioned. For figure 2. C, D, and E, use the same headings. As I can see that the post-male has larger bell size, this may lead to non-significant results. Lines 181 to 184: reflect that the effect is due to treatment which contradicts with the conclusions. Overall, I believe that we shall rewrite results with more coherent text and figures.

179: dissimilarity

Discussion:

Line 203: What we mean by institution?

Figure 2 A and B; also use the similar titles. In PCoA I can see that post treatment clustering is poor, which needs to be justified.

Reviewer #2: You should acknowledge as a limitation that although you found some differences in microbial frequency, you did not evaluate effects on blood pressure or obesity, which are key features of the animal model you studied. Also, 16S rRNA testing is relatively insensitive compared to more complete genomic analyses.

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Reviewer #1: Yes: Muhammad Umar Sohail

Reviewer #2: No

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PONE-D-23-00107R1Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief reportPLOS ONE

Dear Dr. Sones,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 18 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

Following review by two new reviewers, additional minor clarifications are requested. Importantly, sex differences and confounding factors should be accounted for where possible in the analyses, as suggested by the reviewer. Where this is not possible, it should be explained and explored in the text.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: No

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The manuscript by Beckers et al. addresses the effect of FBZ anti-parasitic treatment on stool microbiota in a mouse model of hypertension and obesity. This manuscript is well written and the analyses are straightforward. I reviewed the R1 version of the manuscript which was a response to the comments from 2 other reviewers. My comments do not take into account these earlier comments and I leave it to the editor which of my comments should be addressed. I do have a few comments:

One interesting aspect was not addressed in the discussion. These mice are inbred and are housed and fed in a very controlled environment. Therefore, I would expect (although I am not very experienced in the laboratory mouse stool microbiome) that inter-individual differences are not as high as presented in Fig. 1. If the number of words allows, I would like to see some comments on that in the discussion.

Another interesting aspect that was not clearly addressed. This is longitudinal data. The analyses can make use of these differences before and after treatment for each animal. That can be done for alphas as well as single genera. Was this done in the two way ANOVA analyses? Please comment on this.

Third interesting aspect that was not addressed. Phylum Verrucomicrobiota was higher in males after treatment which was not observed in females. The differential abundance analyses at genus-level did not show what genera were responsible for this. Please discuss why. Further, in humans Verrucomicrobiota (especially genus Akkermansia) are known to be involved in glucose metabolism and obesity. Also, genus Akkermansia is higher in females. This might be interesting in combination with the phenotype of these mice. If the number of words allows, I would like to see some comments on that in the discussion.

Analyses have been performed either at phylum-level or at genus-level, if I am correct. It is not clear how taxa were aggregated to these higher levels and it is not clear at what levels the analyses were performed, i.e., Bray-Curtis dissimilarity and differential abundances. Also, in lines 188 and 189, ASV-level analyses were mentioned but ASVs are basically at strain-level. Please make clear.

Line 115, please indicate if bead beating was part of the protocol.

Line 125, the protocol in the reference is not the manufacturer’s guideline. Illumina has another protocol. Please make sure the correct reference is given.

In line 140 it is stated that normality was confirmed, but most ASVs are not normally distributed. Please explain.

In line 141, I assume that “both p values and FDR-corrected p values <0.05 were considered significant”.

Throughout the text, taxonomic levels are written in plural form which is not always correct, e.g., phyla instead of phylum (lines 146, 149) and genera instead of genus (line 234).

Sentence in line 138 is unclear.

Reviewer #4: In this article, the authors investigated the effects of Fenbendazole on the gut microbiome of mice with hypertension and reported the difference in gut microbiome responses to the treatment between male and female mice.

This article reported an observation without controls over baseline status or any confounding factors, such as BMI, in female and male mice. The main conclusion “there are changes in the gut microbial communities after FBZ use in a sex dependent manner” is not very convincing with current study design or evidence present in the article.

Major issues:

1. Difference between baseline microbiome in male and female mice

According to figure2, there are sex-related differences at baseline. Microbiome components interact between each other. The observation of change in one single component might have multiple components involved. Plus the regression toward the mean effects in statistics. If the baseline microbiome was different between sex, then we will expect the change will be different. The authors need to calibrate for the baseline difference.

2. Lack of controls over confounding factors

As mentioned by the authors, obesity has a great influence on gut microbiome and the treatment may have sex-dependent effects on obesity. Is there a difference in BMI between female and male mice at baseline? Is there a difference in the change of BMI between female and male mice during treatment? These were not reported.

3. Statistical analysis

This study collected fecal samples at two time points from each mice. Using baseline microbiome as reference and comparing within subject changes will be a better choice than current methods, for example difference in difference model. The authors may also consider propensity score matching to remove effects of confounding factors.

4. More details in “Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes”

“Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes” worth more emphasis than “Changes at the phyla level in BPH/5 mice with Fenbendazole treatment”. ASV level is more informative than phyla level in showing microbiome functions.

Minor issues:

1. “Treatment had a significant effect on community composition using PERMANOVA of the Bray-Curtis dissimilarity matrix, with both BPH/5 female and male microbial communities differing post-treatment but not different by sex or interaction (treatment p=0.002, sex p=0.3, interaction p=0.05) (Figure 3b).” For “ sex p=0.3”, what was compared? Female and male samples with both time points pooled together? This analysis is actually showing that treatment had much bigger effects than sex, not supporting the conclusion.

2. The resolution of figures is too low.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief report

PONE-D-23-00107R2

Dear Dr. Sones,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: My comments have been addressed properly, therefore, I recommend to accept this version of the manuscript.

Reviewer #4: The authors have explained thoroughly for the confusions and all the comments have been addressed. Thank you!

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

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