PONE-D-21-14932

Gut microbiota features associated with Clostridioides difficile colonization in dairy calves

PLOS ONE

Dear Dr. Redding,

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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Academic Editor

PLOS ONE

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3. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works.

- https://doi.org/10.1016/j.anaerobe.2021.102353

- https://doi.org/10.1371/journal.pone.0215497

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Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

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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: Yes

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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: No

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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

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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 is a very well written manuscript that presents interesting findings regarding C. diff in dairy calves and I only have minor comments to be addressed.

L62: Please provide additional details for the "manual" sample collection as this is not widely known by PlosOne readers (or provide a reference). Please include the amount of material that was collected, were samples processed the same day, shipping conditions, time in transport, etc. You state calves were less than 2-wk-of-age, but Figure 1 shows data from calves aged 21-22 days. Please clarify. Does the animal protocol number need to be included in the text?

Reviewer #2: Overall, the authors present a study with pertinence to the dairy industry and the results have potential implications for advancing our understanding of the fecal microbiota for benefiting calf health.

In reference to the technical soundness of this manuscript, the sequencing and analysis methods appear adequate; however, details are missing that are required for evaluation of this manuscript. Positive and negative controls should be listed for the DNA extraction kit and PCR; additionally, PCR specification details should be included. The authors should also substitute "microbiota" for "microbiome" throughout the manuscript, as they did not include any whole genome sequencing data. Also, the authors should comment on the accuracy of the Xpert kit used for C diff diagnosis with appropriate references included. In terms of the conclusions being supported by the data included, more clarity in the results section is needed. For example, one of the major findings is that farm location explained most of the variation of the fecal microbiota composition (ln 141-143). Figure 3 and 4 are both referenced in ln 141-143; however, Fig 3 doesn't illustrate this result of farm variability, as Bray-Curtis and unweighted UniFrac are shown for all C. diff + and C. diff - calves. The y axes for Figure 3a and 3b should also be labeled to improve clarity in this figure. Figure 4 should have a legend explaining the breakdown of colors and farm ID; clustering by farm should also be indicated on the PCA plot.

In terms of the methods/statistical analysis, sample size calculations should be included. Additionally, the method for selecting "healthy" calves should be included, specifically whether there were exclusion criteria. The authors should also be specific regarding whether they are referring to "relative abundance" when they indicate "abundance" or "levels" of taxa throughout the manuscript. Additionally, in line 127 the authors mention calculation of odds ratios for whether calf age or feeding waste milk was associated with the likelihood of detecting C. diff. This was not included in the methods section and this was not listed as a study objective. Also, the authors mention a mixed-effects ANOVA in line 136-141 that was not explained in the methods/statistical analysis section.

The authors did not include whether the raw sequence data had been submitted to NCBI SRA. If not available publicly, the raw abundance data should be listed as supplementary information for the manuscript.

Overall, the manuscript is written clearly; however, some errors need to be corrected. Please see individual line comments:

Ln 57: classify the study type (cross-sectional is stated in the discussion)

Ln 125: Consider revising to: "There were no farms where all samples were qPCR-negative and the pooled sample from the farm was positive". (“but” makes it sound like the pooled samples were positive)

Ln 127: Both factors (age and waste milk feeding) are not illustrated in Figure 1; consider revising

Ln 130: The authors should explain why 87 are listed instead of 92

Ln 133: Denominator is 80 for the calves that were sequenced and Figure 1 has 92 calves. Consider removing the “(Fig 1)” reference

Ln 136-141: move to methods section

Ln 141 and throughout: microbiota should be substituted for microbiota

Ln 142,143,146: revise R2 formatting to R^2

Ln 152-154: should be in a results/discussion section or moved to the discussion section

Ln 167-168: should be in the discussion or in a results/discussion section

Ln 169: Please clarify whether “all levels of abundance” refers to the resolution of taxa levels.

Ln 186-187 and throughout: Consider explaining why the fecal microbiota is representative of the gut microbiota or indicate why the complexity of the gut microbiota may not be captured with one fecal sample. Reference 31 evaluated the rumen microbiota, which may be distinct from findings in the fecal microbiota.

Ln 205: The distinction between disrupted and dysbiotic microbiota should be clarified.

Ln 223-255: Please add a citation/explanation as to why LEfSe analysis is weaker than ANCOM

Ln 239: The use of "they" pronoun is confusing. Consider revising to "it is unclear why the relative abundance of Butyricioccus was higher in..."

Ln 240-243: Please revise run-on sentence

Ln 245: genuses should be revised to genera

Ln 436: Microbiota should be substituted for microbiome. The results section indicates that 80 calves were sequenced. The correct denominator should be included with the breakdown of positive and negative C diff statuses in the figure legend. Whether 80 or 86 calves were sequenced should be consistent in all of the figure legends.

Ln 439: Same comments as above. The Y axis for Fig 3a and 3b is not labeled. Additionally, the number of positive C. diff and negative C. diff calves should be consistent in 3a and 3b. If not the authors should explain why they are different.

Ln 442: Explain in the figure legend how each color represents a different farm sampling location.

Figure 7: If there are only the taxa with mean RA > 1% included in the heat map, shouldn’t only 19 be listed based on Ln 160?. 21 are listed for C diff negative and 20 are listed for C diff positive calves. Please clarify.

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Reviewer #1: No

Reviewer #2: No

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Gut microbiota features associated with Clostridioides difficile colonization in dairy calves

PONE-D-21-14932R1

Dear Dr. Redding,

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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Kind regards,

Juan J Loor

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 have addressed my concerns.

There is still one minor inconsistency: Line 27 of the abstract should be corrected and changed to less than 3 weeks of age. Once this has been done the paper is acceptable for publication.

Reviewer #2: The authors adequately answered all of the reviewer comments and made the necessary revisions. I think the manuscript is well-written in its current 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 #1: No

Reviewer #2: No