Dear Dr. Beall,

Thank you very much for submitting your manuscript "Fungi and Bacteria Occupy Distinct Spatial Niches within Carious Dentin" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jason E Stajich, Ph.D.

Guest Editor

PLOS Pathogens

Alex Andrianopoulos

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Overall, this study is an important contribution to our understanding of caries.

Reviewer #2: This is an interesting and well-performed study which examines the microbiome and spatial distribution in dentin from Early Childhood Caries (ECC). S. mutans and Veillonella were associated with caries and there was also a higher ratio of fungi to bacteria in more severe lesions. The most notable finding was that fungi and streptococci primarily occupied separate dentinal tubules.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Concerns:

1. First, plaque was collected from the surface of exposed dentin. It was not collected from within a tubule. Second, the histology omitted gram-negative bacteria. Consequently, we do not have a complete picture (taxonomic profile) within the tubules and fungi and bacteria may co-colonize tubules more frequently than reported. The environment within a tubule is likely distinct from the surface of dentin. For example, it could be more anaerobic. This would select for different bacteria. This distinction should be made clear. Specifically, that the dentin taxonomic profiles presented do not necessarily reflect the profile within a tubule. The situation is perhaps analogous to the transition between supragingival and subgingival plaque - each harbor distinct communities that contain both gram-positive and gram-negative bacteria.

2. Another issue the small sample size.

Points 1 and 2 are important caveats that need to be addressed and/or clearly stated at least in the abstract.

The distinction between minor and major revision hinges on how the authors wish to interpret and present their findings. For example, a major revision would be the addition of histology for gram-negative bacteria.

Reviewer #2: 1. Include a rationale for the use of P. gingivalis DNA as the standard for bacterial quantitation. It seems an unusual choice for a caries study. How does its rDNA gene load compare with that of the oral streptococci and other species that were identified in the study

2. Also include the rationale for collecting samples for microbiome or for imaging a two different sites and explain how clinical diagnosis was calibrated. Imminent exfoliation is given as a reason for extraction in both control and ECC patients.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Minor point

L-278 "A single fungal species was almost always dominant within an individual carious tooth; co-colonizing fungi of distinct species was seldom observed." 
For these teeth, were multiple ASVs of the same species observed? Did certain ASVs dominate? It maybe of interest to consider intra-specific variation. Considerable variation in biochemical characteristics among strains of a species may exist.

Reviewer #2: As S. mutans and C. albicans can be found intermixed and co-adhered to one another in the surface biofilm, none of the explanations offered for distinct dentinal tubule localization are entirely satisfactory. Some additional discussion would improve the paper.

**********

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

Figure 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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Dear Dr. Beall,

We are pleased to inform you that your manuscript 'Fungi and Bacteria Occupy Distinct Spatial Niches within Carious Dentin' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Alex Andrianopoulos

Section Editor

PLOS Pathogens

Alex Andrianopoulos

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):