PONE-D-24-06163Lower respiratory tract microbiome in patients with clinically suspected nontuberculous mycobacterial pulmonary disease according to the presence of gastroesophageal refluxPLOS ONE

Dear Dr. Kwon,

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.

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Frederick Quinn

Academic Editor

PLOS ONE

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https://err.ersjournals.com/content/errev/30/160/200299.full.pdf?

https://www.mdpi.com/2079-6382/10/7/766?

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“This study was supported by grants from the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Education, Science, and Technology (grant no. NRF- 2022R1C1C1002741 (Eun Jeong Won) and 2020R1F1A1076570 (Yong-Soo Kwon)). The funding body played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.”

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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: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: 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 paper illustrated the microbial characteristics in the lower respiratory tract due to reflux in NTM-PD patients. While it may not play a direct role in clinical treatment, I believe it is an extremely important study for understanding the pathophysiology of NTM-PD.

Q1) [Line 89-99 "typical findings of the disease on the chest CT] It would be beneficial to provide additional details on the aspects described in lines 89-99.

Q2) [Line 105 "BAL samples] Were BAL samples adequately collected from all patients (retrieve volume and color)? Were there any samples that did not pass quality control due to low biomass? Was there significant variation in valid read values? How was normalization carried out for read count and gene copy number?

Q3) [Line 157 "24 patients were bacteriologically confirmed NTM-PD] Were these patients confirmed to have NTM through BAL fluid culture?

Q4) [Line 166 Table, NTM-PD with reflux] Shouldn't "NTM-PD with reflux" in Table 1 be accurately labeled as "suspected NTM-PD with reflux"?

Q5) [Line 188-190] It would be advisable to mention in the limitations that Pseudomonas aeruginosa can also be found in conditions of severe lung parenchymal damage (not just reflux). Were there any clinical differences between NTM-PD patients without reflux and those with reflux? (e.g., extent of lung involvement or bacterial load).

Reviewer #2: 16s gene amplicon sequencing to characterize the microbiota of lower respiratory and GI tract from clinical samples.

Patients suspected NTM-PD were selected and included cohorts were NTM-PD was confirmed by bacteriological assay, as well as individuals without confirmation. The samples were further divided between those with and without reflux. Bronchoscopy samples were used for DNA extraction and amplicon sequencing. While no significant difference in overall diversity among patients with suspected NTM-PD, lower diversity was observed in the reflux group. Specific bacterial taxa were found to be associated with efflux.

Despite the potential importance of the questions being addressed, the study is lacking in the level of analysis typically seen for studies assessing microbiota structure. Specific points include:

1. Since 16s gene amplicon sequencing was used to identify bacterial taxa, the microbiome, which includes genes and genomes, was not assessed. References to the microbiome should be changed to “microbiota”.

2. Unfortunately, no LRT samples recovered from bronchoscopy not associated with NTM-PD were analyzed. The lung microbiota should be considered from a greater diversity of samples, even if data from public repositories are analyzed.

3. Beyond bacterial taxa, the authors should identify pathways/functions that correlate with the clinical characteristics. This can be accomplished using standard bioinformatic tools.

4. Fig. 3 claims to show the composition of the lower lung microbiota at the species level. This is placing too much confidence in the results of 16s rRNA gene amplicon sequencing and the figure should be adjusted to show more accurate taxonomic assignments.

5. If reflux is a mechanism for “seeding” the lung with microorganisms from the gut,

6. The limited conclusions from the work do not consider the biology of the bacteria whose differences are noted. A more rigorous assessment of how the bacterial taxa could be relevant. For example Fig. 4 lists strict anaerobic bacteria typically associated with the gut microbiota. What are the implications of these observations?

Reviewer #3: This is a well-written paper and addresses an important topic in NTM-LD. I have a few concerns which are as follows:

1) The introduction needs a hypothesis and summary of results.

2) The authors have chosen all subjects with NTM-LD. This means the cohort must be very diverse. Some patients would have bronchiectasis and others would not. This needs to be discussed and in particular how this must be affecting the microbiome.

3) There are no immunological corrollaries in this study. This is a limitation and therefore must be discussed. The paper is descriptive which is fine but should be discussed as a limitation.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-24-06163R1Lower respiratory tract microbiome in patients with clinically suspected nontuberculous mycobacterial pulmonary disease according to the presence of gastroesophageal refluxPLOS ONE

Dear Dr. Kwon,

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 Jul 29 2024 11:59PM. If you will need significantly more time 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,

Frederick Quinn

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: Partly

**********

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

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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 #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 of the paper adequately answered the questions. Thank you for providing the opportunity to read such a good paper.

Reviewer #2: While the revised version is improved, the omission of standard bioinformatics analysis to predict gene function rather than only catalog bacterial taxonomy continues to limit the impact of the manuscript.

Comments

Reviewer number two suggested a more comprehensive analysis of the data to identify features of the samples that are more informative than bacterial taxa alone. For example, PICRUSt (now PICRUSt2, DOI: 10.1038/s41587-020-0548-6), which predicts functional potential of a bacterial community based on marker gene sequencing profiles, is a standard tool for analyzing 16s amplicon sequencing data. The continued lack of marker gene analysis leaves the reader lacking potentially useful information that can better inform future studies.

The following misstatements should be corrected:

1. 16S ribosomal RNA sequencing was not done, rather 16S ribosomal RNA gene amplicon sequencing was performed.

2. 16s amplicon sequencing is used to interrogate the microbiota, not the microbiome, as specific genes and genome are not being characterized. This was noted by original reviewer #2 but was not changed in the title, keywords and abstract.

3. The abstract states that: “These observations confirm that the lower respiratory microbiome is reliably altered by reflux but not in NTM-PD”. The word “reliably” is awkward and should be replaced with “consistently”.

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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

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Lower respiratory tract microbiota in patients with clinically suspected nontuberculous mycobacterial pulmonary disease according to the presence of gastroesophageal reflux

PONE-D-24-06163R2

Dear Dr. Kwon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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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,

Frederick Quinn

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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: 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: The authors responded well to the reviewers' questions. There are no additional questions. This paper is expected to make significant contributions to the study of the microbiome in the field of NTM pulmonary diseases.

Reviewer #2: This is a second revision of a manuscript that describes the use of 16s taxonomic profiling to characterize the lung microbiota from individuals with different NTM-PD status. In particular, the study seeks to understand the potential impact of GE reflux. The manuscript reports the identification of bacterial taxa that are associated with the patients experiencing reflux.

The authors have responded appropriately to the previous reviews of the manuscript.

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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

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