PONE-D-20-20970

Comparison of the Human Microbiome in Adults and Children with Chronic Rhinosinusitis

PLOS ONE

Dear Dr. Hong,

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 by both reviewers during the review process.

Please submit your revised manuscript by Sep 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.

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,

Alkis James Psaltis, PhD, MBBS(HONS), FRACS

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. 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.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Additional Editor Comments (if provided):

Thank you for your submission on an important topic. Although studies on the sinonasal microbiome in adult patients with CRS are increasing, little work has been published on the microbiome of paediatric CRS.

Both reviewers raise relevant concerns which need to be addressed prior to the study being suitable for publication.

[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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: 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 manuscript does represent a description of a novel comparison between adult and pediatric CRS that to date has not been presented in the literature. I believe this descriptive study is a useful foundation for exploration of the differences between the two diseases at a metagenomic level. The authors' limitations are clearly stated, namely that the patient numbers are small, and it is difficult to make causative conclusions based on the data presented here. The authors make appropriate conclusions based on the data presented. There are several papers looking at acute URIs and sinusitis in children with relation to baseline microbiome composition and microbiome-based interventions, but there is little in the literature looking specifically at pediatric CRS in otherwise healthy children, so this paper is unique.

It would be additionally useful to include more discussion of what the beta diversity findings mean--ie that children's microbial communities are more similar to each other than they are to adults. This would appeal to a broader audience without as much experience in microbiome research.

There were a few changes I would make within the text:

Abstract:

"The bacterial 16S rRNA gene was pyrosequenced to compare the microbiota of the middle meatal." Would change to read middle meatus

Methods:

How was pediatric CRS defined? It would be useful to establish which symptoms/consensus statement definition was used for pediatric CRS, since these differ slightly compared to the adult European Position paper

Figure 4: Legend mentions S. epidermidis but this is not represented in the figure

Results: Page 11-12, lines 195-197

"While Corynebacterium showed a significantly higher relative abundance in the adult group, there was no difference in the abundance of Haemophilus in the adult group."

This is a little unclear. In comparison to the pediatric group?

Discussion: page 13, lines 226-235

Important to clarify that these studies were done in adult CRS patients

Discussion: page 13, lines 240-241 Similar and very recently published paper

Stapleton AL, Shaffer AD, Morris A, Li K, Fitch A, Methé BA. The microbiome of pediatric patients with chronic rhinosinusitis. InInternational Forum of Allergy & Rhinology 2020 Apr 29.

https://doi.org/10.1002/alr.22597

Throughout the discussion, there are several conclusions about the relative abundance of specific species in the adult and pediatric populations, but this data is not presented or discussed in the results. It would be useful to present this data in the results section since so much of the discussion focuses on these data

Discussion: page 14, lines 265-268

This sentence is split into two with ‘and’ between limitations 2 and 3 and a period before limitation 4. Would combine into one full sentence.

Reviewer #2: The manuscript appears to be technically sound and is well written and logical. The subject is pertinent and important in describing the microbiome in chronic rhinosinusitis (CRS) in adults and children. The methods appear to be well described. On its face, the results appear to be valid.

There are several features that bear revision.

Major Issues:

In the first sentence of the abstract, the authors state "Clinical features of pediatric CRS differ significantly from those of adult CRS", but this is not explained. Further, in the introduction on page 3 line 22, the same assertion is made. The study that is quoted (Silviu-Dan) contains a table of "Symptoms of Pediatric CRS" lists nasal blockage/obstruction/congestion; nasal discharge/cough/facial pressure, which are identical to adults. These should be explained or changed.

In the abstract, it is stated that "we hypothesized that differences in the microbiome could be a cause of substantial differences in clinical features of the adult and pediatric populations." I do not see this as an hypothesis for the manuscript. It is a subject that may be addressed in the discussion and needs to be explained as above.

There is no overt hypothesis stated. On page 4 lines 28-31, The hypothesis of differences in adults and pediatric CRS being caused by microbiome differences is made, but this is not the subject addressed in the manuscript. The authors go on to "start testing our hypothesis" by characterizing differences. "Characterizing differences" is not an hypothesis. An hypothesis should be stated.

As a limitation, the use of unsheathed swabs (even those "collected carefully to avoid contamination") should be listed. It is a weakness and calls into question the microbiologic results eg the presence of staph epidermidis which may be a contaminant from the nasal vestibule.

I personally do not believe that a conclusion should begin with a claim that was not a subject of the manuscript eg "This is the first...". The conclusion should contain information that was shown to be true in the study.

It is also my personal opinion that claims of first discovery should not be made in a scientific manuscript.

Additionally, the statement in the conclusion that "we expect the results of our study to be helpful in explaining the differences in the pathogenesis of sinusitis in children and adults" appears to be an offhand comment not elucidated in the discussion section nor substantially addressed in the manuscript.

Minor Issues

The authors use the term "nasal cavity" at times which appears to refer to the site of culture. If the site of culture is in fact the middle meatus/anterior ethmoid, this is not the nasal cavity. Terminology should be consistent and meaningful and should be standardized.

"Uncultiveability" is used on page 12 which is not a word i am familiar with. It likely refers to "uncluturability" and should be reviewed and corrected.

All data was not presented in the manuscript or in an appendix, as far as could be seen ("data not shown").

In materials and methods, subject characteristics are listed (19 patients, males/females, age, etc). This information belongs in the Results section.

**********

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.]

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.

Comparison of the Human Microbiome in Adults and Children with Chronic Rhinosinusitis

PONE-D-20-20970R1

Dear Dr. Hong,

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,

Alkis James Psaltis, PhD, MBBS(HONS), FRACS

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your revised version of your initial manuscript "Comparison of the Human Microbiome in Adults and Children with Chronic Rhinosinusitis"

I applaud your efforts for addressing the concerns of the reviewers and believe that they have all been addressed

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: 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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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: Yes

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: 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: (No Response)

Reviewer #2: Comments were nicely addressed. The manuscript is technically sound, well written, and intelligible.

**********

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: Yes: Andrew N. Goldberg, MD, MSCE