PONE-D-20-27802

Structural and quantitative alterations of gut microbiota in experimental small bowel obstruction

PLOS ONE

Dear Dr. Zhang,

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 Dec 25 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

The authors' manuscript has been reviewed by myself and two experts. As the reviewers note, clarification of several methodological details is required. In addition, it is suggested the authors expand on the potential clinical relevance of their results.

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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: Partly

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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: This study characterized the gut microbiota changes in a surgically constructed small bowel obstruction rat model. The authors observed the post-surgical change of relative and absolute abundance of different microbial species. In addition, the author observed increased expression of pro0inflammatory markers including claudin-1 mRNA and serum IgA. Although it is the first time that the microbiota shifts have been reported in this experimental rat model, not much insights were drawn from these observations. Moreover, the clinical relevance of the rat model or the observation is unclear. Thus, revisions would be necessary to draw scientific or clinical insights of these observations reported.

Some specific concerns:

The study reported the change of different microbial species after surgically placing a polyvinyl chloride clip around the terminal ileum. The change of microbiota, per se, is expected and not surprising. As we know, the microbiota is shaped by environmental factors including nutrient, oxygen level, P.H., host inflammation, etc. The surgery would have caused turbulence in all these factors, and as a result, it is inevitable that the microbiota also changes. The important questions here are whether these changes have any impact on the progress or recovery of the disease, and which of the environmental factors play the most important role in reshaping microbiota. These questions remain unanswered in the manuscript.

In addition, the authors need to address how the observations in this study translate to clinical situations. Are similar changes observed in clinical studies of small bowel observation? Without such information, the changes of microbiota in this study could very well be attributed to experimental artifact caused by the physical clipping. Alternatively, more biomarkers on the host side could address the clinical relevance of such an experimental setup. Serum IgA and claudin A are not SBO specific markers; they are rather general pro-inflammatory markers. The elevated level could be due to the mucosal damage caused by the surgical processes. Some more specific host markers should be used.

Technical concern:

Bedding and cage effects are important factors to control for microbiome study. The authors need to show that prior to the surgery, all the four groups have similar microbiota. This could be done by including a PCoA analysis of prior surgical fecal sample. In addition, the authors should include more details on mix-bedding and cage arrangements of the cohorts.

Minor points:

In the introduction, there are bold claims without evidence. For example, Line 45: “Gut microbiota dysbiosis is a prominent cause of both local and systemic pathological changes in SBO.” And line 50: “Microbiota dysbiosis is considered a major pathogenic factor in small bowel obstruction.” I don’t think the cited articles or the current study can prove such bold claims of causality between microbiota composition and SBO. If the statements were true, then the authors didn’t need to use surgical clips to create a rat SBO model.

Some sentences in the introduction and discussion sessions are unnecessarily long and cause confusion. For example, line 60: “Although sequencing consequences of entire microbial communities are very informative, they may not be able to capture the essence of the problem, which means that it is the absolute quantity and not the relative abundance of microbes that really matters.” This long sentence needs rewriting as the message is not clear.

Please define all the abbreviations. For example, does sIgA stand for serum IgA or secretary IgA in this context?

Reviewer #2: This study examines the microbiota change in rats with complete obstruction. Not surprisingly, there changes that evolved over time in both the tissue and the bacteria. Several points need to be addressed.

1. The rat model should be described in more detail as the references given are different from these rats. What gross changes happened in these rats - weight, intestinal diameter, food and water intake, etc.?

2. The quantify the total bacterial content, it is necessary to consider the intestinal dilation that occurs with bowel obstruction. The enteric content must have increased with obstruction. The total volume of the content should be given in these rats and considered in the calculations.

3. With prolonged obstruction, these rats must be getting significantly dehydrated - how is that taken into account in the data interpretation? Could the observed changes occur due to dehydration rather than obstruction?

4. What is the food intake during the experiments? That might also impact the results of the study.

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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.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-27802R1

Structural and quantitative alterations of gut microbiota in experimental small bowel obstruction

PLOS ONE

Dear Dr. Zhang,

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 Apr 18 2021 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,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

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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 #1: All comments have been addressed

Reviewer #2: (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 #1: Yes

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: No

**********

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 #1: Yes

Reviewer #2: 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 #1: The authors have addressed all my comments in the previous review. The new data and figures are appropriate. I don't have any more questions.

Reviewer #2: In the revised manuscript, the authors did not give adequate responses to the previous reviews. Many confounding factors such as altered food intake, hydration status, and enteric volume make the studies hard to interpret.

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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.

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: 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.]

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-20-27802R2

Structural and quantitative alterations of gut microbiota in experimental small bowel obstruction

PLOS ONE

Dear Dr. Zhang,

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.

Additional minor clarifications are requested, as noted by the reviewer.

Please submit your revised manuscript by Jul 02 2021 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. 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.

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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: (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

**********

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

Reviewer #3: 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

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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

**********

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: This is an interesting manuscript on the impact of small bowel obstruction on the composition of the gut microbiota and its potential impacts on the gut barrier function.

The methodology is clear, and the results are well presented. The findings provide an interesting and novel overview of changes in the gut microbiota in the days following SBO.

The authors have addressed to the best of their ability the comments raised by previous reviewers and made many improvements to the manuscript.

Nonetheless, several issues require attention.

1. In the introduction, the mortality rate associated with SBO (reported to be 13%) seems high compared to the literature. Other sources report much lower rates, and this value should be nuanced (suggested reference: Paulson EK, Thompson WM. Review of small-bowel obstruction: the diagnosis and when to worry. Radiology. 2015 May;275(2):332-42. doi: 10.1148/radiol.15131519. PMID: 25906301).

2. Since SBO may not be understandable to all readers, I would rephrase the following sentence (line 52): “Microbiota dysbiosis is considered a major pathophysiological process in small bowel obstruction” to make clearer the fact that such dysbiosis is a consequence of the SBO process.

3. It is mentioned that the after surgery, the content of the gut above the obstruction point was collected for analysis. Was the content of the same segment collected in rats in the control (sham) group? This is important to specify since the comparison with control rats would be impossible otherwise.

4. Figure 6 - the treatment groups should be specified (sham, S1, S2, S3).

5. Overall, it is expected that a SBO is an acute event that would alter the gut microbiota composition, but the relevant conclusion from this work is a potential deleterious and durable effect that may affect the host’s health beyond the few days where SBO is present. I understand that this is not the purpose of the manuscript, but I would discuss the necessity of conducting long-term experiments in the discussion to investigate possible sustained “dysbiosis” and/or barrier dysfunction.

6. The following sentence needs attention: “A small loop of ileum with mesentery approximately 1 cm distal to the ileocecal valve”. The ileum is proximal to the ileocecal valve anatomically and the word “distal” is inaccurate should be corrected.

7. The following sentence requires attention as well: “A 2 cm long ileal segment 2 cm oral to the obstruction”. Does the word “oral” means proximal or cranial? It should be modified for clarity.

8. In the discussion, the authors suggest that the findings may orient the choice of antibiotics in SBO. However, SBO is not an infectious phenomenon, and no antibiotics are administered routinely, unless a complication (ischemia or perforation) occurs, in which case the used antibiotics are large spectrum already.

9. The main limitation of this study is the message it conveys. The findings are interesting and are worth sharing with the scientific community. These findings however should be seen as the short-term alteration in the gut microbiota during SBO. They do not provide however information on long-term alterations or health risks that SBO may induce. This is especially true as SBO does not seem in practice to induce any long-term diseases in patients. In the discussion, the message should be centered on the effect of SBO on gut microbiota in an acute setting only, as long-term alterations cannot be described or even extrapolated with the present study.

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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.

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

[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.

Structural and quantitative alterations of gut microbiota in experimental small bowel obstruction

PONE-D-20-27802R3

Dear Dr. Zhang,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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

**********

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

**********

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

Reviewer #3: 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

**********

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

**********

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 raised comments were addressed by the authors. The discussion was also nuanced to account for the limitations of the study. The present version of the manuscript seems suitable for publication in its present form.

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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.

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: Yes: Manuela Santos