PONE-D-19-35822

Succinivibrio and Mitsuokella are Dominant Generas in Fecal Microbiota of Behçet’s Syndrome Patients with Mucocutanous and Uveitis Involvement

PLOS ONE

Dear Dr Kalkanci,

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.

I would like to sincerely apologise for the delay you have incurred with your submission. We have now received two completed reviews; their comments are available below.

Both reviewers have raised several concerns about the selection of patients and the methodology of this study that need to be addressed in a revision. Please revise the manuscript to address all the reviewer's comments in a point-by-point response in order to ensure it is meeting the journal's publication criteria. Please note that the revised manuscript will need to undergo further review, we thus cannot at this point anticipate the outcome of the evaluation process.

We would appreciate receiving your revised manuscript by Jun 26 2020 11:59PM. When you are 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Miquel Vall-llosera Camps

Associate Editor

PLOS ONE

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4. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

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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: No

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: No

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: The authors conducted a metagenomic analysis in patients with BD and mined the species Prevotella copri from the data as a BD characteristic microbe. BD is rare and often difficult to treat even now and the finding may become very important data for the patients and treating physicians in the near future.

Major:

1. In the introduction section, the authors should describe detailed information of the differences in the “immunological pathologies” between BD patients with and without uveitis. We did not understand why the authors selected BD patients with uveitis for this study. If the authors need to use the data of FMF and CD as controls in this study, commonalities and differences in the “immunological pathologies” between BD and FMF, and between BD and CD would be helpful for us to understand more accurately. Discussion of the relationships between previous data of the immunological pathologies in patients with BD and metagenomic data of this study would be also important for the precise understanding.

2. The authors described their metagenomic data using OTU numbers in their figures and tables. Nonetheless, in their statistical analyses, the authors compared the metagenomic data using the taxonomic abundance. In the materials and methods section, the authors described that they used Kruskal-Wallis rank sum test to find significant differences in taxonomic abundance between patients and normal individuals (page 5). In the discussion section, the authors described that OTUs of this study groups were technically homogeneous (page 9). These descriptions were very confusing for us. If we understand it right, the authors used taxonomic abundance data for all statistical analyses of this study. We suggest that the authors utilize relative abundance titers of the metagenomic data for the comparison in the figures and tables with the statistical data. At least the figures and tables need the data of statistical analyses.

3. The authors should provide us with BD patient demographical and clinical data with the statistical analyses. Gender (Front Immunol 2017; 8: 754.), aging (Cell Mol Life Sci 2018; 75: 129-148.), and medications (nonantibiotics, Nature 2018; 555: 623-8.) were reported to associate with gut microbe alteration. If the authors need to use the data of FMF and CD as controls in this study, the authors should provide us with the patients' data.

4. High titers of alpha diversity in gut microbe composition were considered as a healthy state (Hepatology 2017; 65: 451-64.). The authors should describe the alpha diversity index titers of the 4 subgroups in this study with the detailed statistical analyses.

5. We need several references in the next two sentences for the precise understanding.

(1) Page 9: The molecular mimicry based on sequence homology between microbial and human peptides triggers autoimmune responses in BS patients.

(2) Page 10: We found Firmicutes and Proteobacteria were dominant phyla in BS patients presenting inflammatory effects in the gastrointestinal system.

6. The authors should send the manuscript to the natives for revising the English.

Minor:

1. We were wondering if the authors could provide us with the information of their minor revisions of the elusion steps in DNA extractions (Page 4).

2. The authors should spell out the term of Treg in Page 9. Several references in the sentence would be helpful for us.

Reviewer #2: The aim of this study was to investigate the gut microbiota of Behçet’s Syndrome patients with uveitis involvement and compare the gut microbiota of BS with that of Familial Mediterranean Fever and Crohn’s Disease. However, the following issues should be addressed.

1. The disease profiles of BS patients included in this study are unclear. Authors need to prepare the “Table” of patient’s data, such as the patients with BS (oral ulcer, skin lesion, genital ulcer, and so on).

2. Authors should also mention whether the patients were with active or inactive BS. When was the fecal sample obtained relative to the onset of an attack?

3. Did the patients included in this study receive medication before the fecal sample obtained? Immunomodulatory drugs and steroids would impact on the gut microbiota composition.

4. The detailed information of control was also unclear. The human gut microbiota composition will differ from the age, sex, life style, diet and inflammatory status of donors (see PMID: 24705962 and 20498852). Thus, author should provide more profiles of patients and healthy controls, such as their age and sex. And author should mention whether the healthy control were with diabetes, cardiovascular diseases, systemic disease and other inflammatory diseases.

5. The data regarding the abundant taxa at the family level in four groups suggested Succinivibrio and Mitsuokella were dominant and signature families, whereas Bacteroides was absent in BS patients. However, the statistical analysis were without correction. The statistical analysis in the genera and species levels also required correction.

6. The authors are aware of the limitations of this study. However, mono-colonization of dominant species or fecal transplantation from BS patients in germ-free mice would also be informative.

7. The OTU clustering methods need to be clarified. The version of the software and algorithms used also need to be indicated. It is also unclear how the authors performed taxonomic assignment or what database they used.

8. Minor issue, see Page 3, lines 17, "Behçet's syndrome" should be used as abbreviation "BS"

9. Minor issue, see Page 11, lines 1, "Prevotella" should be used in italic.

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Reviewer #1: No

Reviewer #2: No

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-35822R1

Succinivibrio and Mitsuokella are Dominant Generas in Fecal Microbiota of Behçet’s Syndrome Patients with Uveitis

PLOS ONE

Dear Dr. Ayse Kalkanci,

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 see below for the reviwer`s comments.

Please submit your revised manuscript by 30 days. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Noboru Suzuki

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments (if provided):

Major:

1. The authors described that they found significant differences in the bacterial relative abundance of family level with the p values (Fig 3) between diseases and healthy controls.

We did not obtain p values in the data of genus (and species) level.

For the statistical analyses, the authors might utilize value zero (0) for the not detected elements. After the calculation, if the authors cannot obtain significant data, they should describe clearly the findings in the results section of the manuscript and alter the manuscript title.

The manuscript needs detailed description of statistical analysis in the result section.

We think that the data are very precise for the future research of BS.

2. The authors seemed to consider that the laboratory findings of next two sentences were scientifically sound.

The authors should provide direct evidence on the pro-inflammatory effects of the “bacterial and human peptides” and the “microbes of the phyla Firmicutes/Proteobacteia” in the sentences with the findings of their cited reference articles.

1) P12: The molecular mimicry based on sequence homology between microbial and human peptides triggers autoimmune responses in BS patients.

2) P12: we found Firmicutes and Proteobacteria were dominant phyla in BS patients presenting inflammatory effects in the gastrointestinal system.

Minor

1. We found several typographical errors in the manuscript.

Example

P3: Last paragraph needs the name of the disease. In the paragraph, we may recognize double space after the word “enhances”.

P8 in the next generation paragraph: Sequences wereanalyzed by ---- needs space.

P9 in the second paragraph: Groups were of similar age(p = 0.098)-----needs space.

P10 in figure legends of Fig 4: The term “genera” should be corrected.

P11 in the second paragraph: The word “fallows” should be corrected.

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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.

PONE-D-19-35822R2

Succinivibrionaceae is Dominant Family in Fecal Microbiota of Behçet’s Syndrome Patients with Uveitis

PLOS ONE

Dear Dr. Kalkanci,

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 Oct 31 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Noboru Suzuki

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

We found several typographical errors in the manuscript of 2nd revision.

We strongly suggest you to consult with a Native English speaker for the correction of their English.

ie. Top 5 species in all groups were as fallow;

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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.

Succinivibrionaceae is Dominant Family in Fecal Microbiota of Behçet’s Syndrome Patients with Uveitis

PONE-D-19-35822R3

Dear Dr. Kalkanci,

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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Kind regards,

Noboru Suzuki

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: