PONE-D-20-02497

History of breastfeeding but not mode of delivery shapes the gut microbiome in childhood

PLOS ONE

Dear Dr Leve,

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.

We would appreciate receiving your revised manuscript by Apr 20 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Corrie Whisner

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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified, if your study included minors under age 18, whether you obtained consent from parents or guardians.

3. To comply with PLOS ONE submission guidelines please deposit your sequencing data in a publicly available repository (you can find a list of repositories in the link here : https://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories).

4. Please include in your Methods section the date ranges over which you recruited participants to this study.

5. Please ensure that you refer to Figure 4 and 5 in your text as, if accepted, production will need this reference to link the reader to the figure.

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[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: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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

**********

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: Yes

**********

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 paper adds to the literature on the effects of delivery mode and infant feeding on the gut microbiota by examining whether differences in diversity and bacterial composition can be seen in children aged 3-18 years by delivery and breast v formula feeding. While this is an important question, the relatively small sample size, with a wide range of child ages 3-18, and the lack of other control variables limit the generalizability of the study. Further, the authors make several strong claims that do not appear to be supported by the data and/or results.

First, the authors claim that this is the first paper to explore whether the effects of these early childhood exposures persist past age 4 (for example, pg 3 lines 44-45, pg 13, line 271). At least three other papers have examined these associations after age 4: Salminen S, Gibson GR, McCartney AL, et a influence of mode of delivery on gut microbiota composition in seven year old children. Gut 2004;53:1388-1389; Thompson, A. L., Houck, K. M., & Jahnke, J. R. (2019). Pathways linking caesarean delivery to early health in a dual burden context: Immune development and the gut microbiome in infants and children from Galápagos, Ecuador. American Journal of Human Biology, 31(2), e23219; and Nagpal R, Yamashiro Y: Gut Microbiota Composition in Healthy Japanese Infants and Young Adults Born by C-Section. Ann Nutr Metab 2018;73(suppl 3):4-11. doi: 10.1159/000490841

Second, the age range of the participants is quite wide and, while the authors control for age in their multilevel models of the factors associated with Bacteroides abundance it would be helpful to see whether diversity or any other measures differ by age group. Such an analysis would provide more support for their conclusion on page 10 that: “our study suggests that changes in the microbiome occur well into adolescence…” As currently described, this cross-sectional analysis doesn’t provide evidence that the microbiome changes in childhood and adolescence, just that there are differences in the colonization that may be associated with infant feeding practices. A similar wording issue is on pg 12, line 251 where the authors describe the effects as “diminishing over time.”

Similarly, the authors should be more explicit about the limitations/potential bias in their sample data. How likely are maternal reports of child size, breastfeeding and birth type to be accurate given the mixed history of the sample (some biological children, some adoptive children with likely varying ages at adoption)? Further, how likely are other variables to have influenced their findings (i.e. timing of complementary feeding, antibiotic use, etc) given the lack of other infant or childhood exposures?

The authors don’t appear to make use of the adoption-sibling design of the study other than controlling for clustering by household and differing genetic relatedness. This seems like a lost opportunity to make this study unique in the literature by providing an examination of how household environment may interact with early birth/feeding exposures to shape child microbiota.

A few more minor concerns:

Pg 9, line 185: “latent” seems to be the wrong word

Pg 13, lines 276: It is not clear that the authors are referring to breastfeeding initiation in the US (79%) compared to other measures (i.e. duration at 3 months, ebf at 6 months). The fact that the authors only collected “ever breastfed” appears to be the more limiting factor for looking at ebf or duration rather than the lower breastfeeding initiation prevalence than the US population.

Reviewer #2: There are several areas that can be improved with additional clarification.

1. Do not assume that the children that were not breastfed were formula fed, as that gives the impression of commercial formula use. Parents may have used other types of liquids to feed their children, or to have created their own types of 'baby milk'. I recommend terms such as breastfed and non-breastfed.

2. The discussion of the participants is confusing. A table displaying the children who were adopted or biological and reared with birth parents or adoptive parents would be easier to understand than the text. The term 'relatedness' is also not clarified as it could mean 'related through shared genetics' or 'related through being members of the same family'.

3. BMI needs to be stated as BMI percentile. A BMI of 20 does not mean anything to the reader since the children ranged in age from 3 to 18. It would be more helpful to know if the BMI% is at 50th% or 90th%.

4. Knowing the child's typical diet at the age when the fecal sample was collected would be useful. A food frequency questionnaire would have been a beneficial instrument for knowing if the child had a predominantly meat heavy or vegetable heavy diet, which can affect the gut microbiome.

5. It would be helpful to know the age of adoption. Some adoptees may have been initially breastfed. Cannot assume that if adopted, must have not been breastfed.

6. Acknowledge in additional considerations that knowing the current diet (important co-variate) could also contribute to analysis of these results.

**********

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: 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-20-02497R1

History of breastfeeding but not mode of delivery shapes the gut microbiome in childhood

PLOS ONE

Dear Dr. Leve,

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.

In revising your manuscript, please pay special attention to the comments provided by Reviewer 2, especially the need to present body mass index as a percentile instead of in kg/m^2. Please see the bulleted list of important changes I noted when reviewing the paper and the comments from reviewers:

• For children, the World Health Organization suggests that this approach to reporting child size and body weight status are more accurate given that they are in a period of more intense growth. This is a best practice in both clinical and research settings, and should therefore be carried out in this paper before it will be deemed acceptable for publication.
• The paper suggests that formula feeding is fine and may be beneficial given the promotion of gut maturation ia Bacteroides. This message needs to be softened throughout the manuscript as many health agencies support breastfeeding as the gold standard for gut and systemic child health. Please revise the manuscript to ensure that formula feeding is not viewed as equal or superior to breastfeeding.

Please submit your revised manuscript by Jul 11 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,

Corrie Whisner

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 #1: All comments have been addressed

Reviewer #2: (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 #1: (No Response)

Reviewer #2: Partly

**********

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

Reviewer #1: (No Response)

Reviewer #2: 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 #1: (No Response)

Reviewer #2: 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 #1: (No Response)

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: (No Response)

Reviewer #2: I read your rationale why you used BMI only. But it lacks clarity on the predominant body size of the children. Please use BMI% for the BMI of 20 to make sense to readers. I know it's age corrected but it still doesn't tell me of the child's weight status; normal (5% to 85%) over/under weight, (85%-<5th%) obese >95%.

The child's rearing environment is confusing, especially Table 1. Does the box 'sibling genetically related and adoptive home mean that of the 25 adopted children, there were 17 genetically sibs who were reared together in an adoptive home? The the box 'child genetically unrelated to adopted child and biological home mean that in the biological home, 14 of children were there who were not related to the adoptees? For example, step-siblings and not half-siblings?

For feeding practices, did you also measure if the adoptive parents used breast milk from a milk bank or from friends/family/community support breast milk networks? That practice is becoming more common and could have occurred in the younger children in your sample.

Was the delivery mode measured by questions from adoptive or birth mother when records were not available?

In the results section, was the 21% breastfed combined from both genetically related and non-genetically related sibs?

On line 195, add 'gut' between human and microbiome.

Line 222 Mention also that bacteriodes can also cause serious infections if escape outside the gut, potentially leading to septicemia too.

Line 282 The percentage of breastfed varies by age of infant. Do you mean ever breastfed, exclusively breastfed, or breastfed to 6 months? more specificity needed.

Line 292 remove formula; it may be interpreted as 'better than breastfeeding' and that's not the approach supported by WHO or multiple health agencies.

**********

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.

History of breastfeeding but not mode of delivery shapes the gut microbiome in childhood

PONE-D-20-02497R2

Dear Dr. Leve,

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,

Corrie Whisner

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: