PONE-D-23-41767Altered gut microbiota in patients with schizophrenia indicate links between lipid metabolism and cognitive disorderPLOS ONE

Dear Dr. Qi,

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 Mar 29 2024 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:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“This study was supported by grants from the Inner Mongolia Autonomous Region Science and Technology Innovation Guide project (No. 2021CXYD001) and Inner Mongolia Autonomous Region Science and Technology plan project (No. 2021GG0298).”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This study was supported by grants from the Inner Mongolia Autonomous Region Science and Technology Innovation Guide project (No. 2021CXYD001) and Inner Mongolia Autonomous Region Science and Technology plan project (No. 2021GG0298).

The author thanks engineer ZhiHui Mi, TianYang Wen and LuLin Song of Inner Inner Mongolia Di An Feng Xin Medical Technology Co., LTD for providing data analysis services, Meng Han of Hulunbuir Third People's Hospital for project management service.

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This study was supported by grants from the Inner Mongolia Autonomous Region Science and Technology Innovation Guide project (No. 2021CXYD001) and Inner Mongolia Autonomous Region Science and Technology plan project (No. 2021GG0298).”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

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: No

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: No

**********

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: Yes

Reviewer #2: No

**********

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: In this article, Wang et al., aimed to explore the potential association between the gut microbiome and schizophrenia, utilizing 16S rRNA gene sequencing and metagenomic analysis. The study adds value to the gut-brain axis field by providing insights into the potential role of specific gut bacteria, such as Selenomonas and Clostridium, in schizophrenia. Additionally, it highlights alterations in metabolic pathways associated with the condition. This adds depth to our understanding of the complex interplay between the gut microbiome and schizophrenia pathogenesis. However, some comments are given below to consider to improve the quality of the manuscript,

1. The authors have mentioned in the abstract that “The results of the functional analysis revealed a significant correlation between the metabolism of sphingolipid, phosphonates, and phosphinates, as well as glutamine metabolism, and the manifestation and progression of schizophrenia”. However, these results need to be elaborated in the results section. Moreover, the authors must discuss the effect of phosphonates and phosphinates pathways as well. Although some potential pathways are identified, the functional mechanisms linking specific microbes to schizophrenia remain unclear.

2. The study lacks a longitudinal design, which could provide insights into the dynamic changes in the gut microbiome over time in schizophrenia patients. Moreover, very sample size especially for a metagenomics study makes statistical power and generalizability weak and unreliable.

3. The exclusion criteria for patient selection seem comprehensive but could benefit from further elaboration on how these criteria were applied and any potential biases introduced.

4. The methodology section should be more detailed, particularly regarding the data analysis pipelines for both 16S rRNA sequencing and metagenomic analysis. What are the chances of having human DNA contamination when extracted genomic DNA from fecal samples and how it was avoided?

5. The authors have limited discussion on Selenomonas. This bacterium shows significant enrichment in the schizophrenia group, therefore comparison with existing literature is needed.

6. In lines 432-433, the authors mentioned “This investigation demonstrates that patients with

schizophrenia exhibit abnormal glutamate metabolism”, however, abnormal glutamate metabolism is not mentioned in the results section. Elaborate on this point in the results and then mention it in the discussion otherwise, it is a very confusing statement.

7. As the patients were of mixed gender, please state if there is any gender-specific difference in microbiota and if is related to some specific pathway.

8. Line 449-450: Please explain how your findings provide a potential mechanism explanation for the observed sequencing data while no mechanisms have been explained.

9. Add high-quality pictures because the text in the Figures is not readable in the current format.

10. In lines 355-358, the aim of the study does not match the aim mentioned in the abstract and the objective mentioned in lines 118-119. The statement could be modified by saying that “one of the aims of this study was to investigate…..”

11. The authors have focused more on microbiota comparison. Overall, metagenomics analysis needs to be elaborated and correlated with pathways of the microbiome that respond to SZ in more detail to present clear results of your findings.

12. The article demonstrates proficiency in English language usage, with coherent sentences and technical terminology. However, there are some minor grammar issues and typographical errors that could be corrected for clarity and precision. For example, line 300: "worthing" should be "worth mentioning," and "differentially abundance" should be "differentially abundant." Line 69: “exerts a significant burden on the global population” could be “exerts a significant burden on the global health sector”. Line 210: delete one “control”; line 237: revealed or showed, delete one word; lines 428-429: Previous research??

10. All references are not in the same format. There are no details for reference numbers 6 and 7.

Reviewer #2: The authors studied gut microbiota of patients with schizophrenia using both 16s rRNA gene sequencing and metagenome, and compare it with normal control. Though interesting, the manuscript is not well organized, and many mistakes in the manuscript made it hard to follow.

Major concerns:

1. In introduction, the authors did not cite any original studies and review articles on the microbiota study of schizophrenia, which is misleading. The author should present and give credit to previous pertinent work, before highlighting what their data adds to the existing knowledge.

2. The sequencing reads number of each sample in 16s rRNA gene sequencing should be presented. And was the data normalized before analysis? Since you identified 30 unique genera in patients or control (Line 238-245), the sequencing depth must be enough to support your findings.

3. In Line 262-269, the authors compared the two groups using LEfSe. Then in Line 270-276, the authors used Wilcoxon rank-sum test to compare them again. It’s un-necessary. Same problem existed in metagenomic analysis. I suggest the authors use multiple wilcoxon ranked sign tests to compare the microbial abundances between the groups with calculation of FDR.

4. There are logical issues with metagenomic analysis. I don’t think 16s rRNA gene analysis result should be validated by metagenomic analysis. And the authors did not compare the taxa profiles obtained by this two different methods.

5. The results should be separated into several parts to make it clear and readable.

6. The title must be changed. Indeed the authors only studied gut microbiota, which can not link lipid metabolism and cognitive disorder.

Minor concerns:

Abstract

1. Line 31, ‘the of’ is wrong.

2. Change ‘16S rRNA’ to 16S rRNA gene’ throughout the manuscript.

Introduction

3. Please separate the 1st paragraph into different parts.

4. Microflora should be no longer used in the literature, please change it into microbiota.

Methods

5. Line 186, 2x250?

6. Line 193, details of the 12 participants should be given, such as xx controls and xx patients.

7. The analysis method of 16s rRNA gene sequencing is absent.

8. More details about metagenomic analysis, such as pathway prediction, should be presented.

9. The raw sequencing data should be deposited in a publicly available database.

Results

10. Line 209-210, is this a new cohort, or samples selected from the 59 volunteers?

11. Line 237, ‘revealed showed’?

12. Line 249, ’30 genera’ is not correct. It’s 198 in Figure 2A.

13. Line 251, right parenthesis is absent.

14. Line 262-265, move into method part.

15. Line 291, ‘Suoliviridae’ is not a genus name but family.

16. Line 300-302, it’s not suitable in Results, please delete it or move into Discussion.

17. Line 302-305, ‘Clostridium may exert a significant influence on the etiology and progression of schizophrenia’ is not supported by your data.

18. Line 319-324, PICRUSt is based on 16s rRNA gene prediction. But you should also present your pathway analysis based on metagenomics, which would provide strong support to this prediction.

19. Figure 1, change ‘16s rDNA gene’ into ‘16s rRNA gene’. Line 225, change ‘16S rDNA’ into ‘16s rRNA gene sequencing’. Line 226, ‘Clean reads without human sequences were blasted to NR databases.’ seems weird, please re-write this part.

20. Table 2, it’s genus, not OTU.

Discussion

1. Line 347-355, what you stated is not the truth. Please revise this part.

**********

6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #1: Yes: Nayla Munawar

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.]

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-23-41767R1Perturbations in gut microbiota composition in schizophreniaPLOS ONE

Dear Dr. Qi,

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 Jun 28 2024 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

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #4: All comments have been addressed

**********

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: Partly

**********

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

Reviewer #3: Yes

Reviewer #4: No

**********

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

**********

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: No

**********

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: General comments:

The authors unitized 16S rRNA gene sequencing and metagenomics approach to profile the gut microbiota between schizophrenia patients and normal people. The paper has been reviewed by other reviewers and the authors have revised it, I only have several minor concerns.

Specific comments/questions:

L 41-42: based on your results, we can only conclude a relationship between gut microbiota with schizophrenia, rather than a crucial role.

L 101: collected what? This is an incomplete sentence.

L 216: why you only analysis the function of metagenomics, the diversity and composition of microbial community, even the resistome can also be obtained.

figures: the resolution of figures was low.

Reviewer #4: In the manuscript, the authors compared the microbial diversities, compositions, and functions between schizophrenia patients and control groups by using 16S rRNA gene sequencing and whole-genome shotgun metagenomic sequencing. They concluded that gut microbiome might exert a crucial influence on individuals with schizophrenia.

This is a carefully done study and the findings are interesting. However, some points need clarifying and certain statements require further modification. My detailed comments are given below:

1.In abstract, the authors summarized that “These findings suggest that gut microbiome may exert a crucial influence on individuals with schizophrenia” . I think more important results should be described to support this conclusion, not just “minimal variations in alpha and beta diversity” or “higher abundances of Clostridium and Megasphaera”.

2.In introduction, authors simply stated “Several studies have investigated the microbiota in schizophrenia, revealed its relationships to the onset and progression of schizophrenia”.The detailed findings of previous study are supposed to be listed.

3.In the exclusion criteria, schizophrenia patients with chronic gastrointestinal illness were not ruled out, which might affect the study results.

4.Figures in the manuscript were fuzzy. Please provide clearer figures.

5.In fig 2A, authors compared the microbial compositions between group SZ and group NC, and in abstract, authors found that “The schizophrenia patients showed higher abundances of Clostridium and Megasphaera” , however, whether the data were statistically significant were not mentioned.

6.In discussion, authors didn’t highlight the important findings of the study, analyze the reasons for observed data or compare the research findings with previous studies.

7.Limitations were not mentioned in the discussion.

8.The current manuscript needs to be polished by a native English speaker or a professional language editing service.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

**********

[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.

Perturbations in gut microbiota composition in schizophrenia

PONE-D-23-41767R2

Dear Dr. Qi,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

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: (No Response)

**********

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

**********

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

**********

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: (No Response)

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: The authors have addressed my concerns, and I think this manuscript is ready for publication in PLoS One.

Reviewer #4: (No Response)

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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: Yes: Yafeng Li

**********