Microbial population shift and metabolic characterization of silver diamine fluoride treatment failure on dental caries

Bidisha Paul; Maria A. Sierra; Fangxi Xu; Yasmi O. Crystal; Xin Li; Deepak Saxena; Ryan Richard Ruff

doi:10.1371/journal.pone.0242396

Peer Review History

Original SubmissionNovember 5, 2020
8 Dec 2020 Decision Letter - Ping Xu, Editor PONE-D-20-34812 Microbial population shift and metabolic characterization of silver diamine fluoride treatment failure on dental caries PLOS ONE Dear Dr. Ryan Richard Ruff, 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 respond carefully to the reviewer’s comments point by point, especially the rationale for the small sample size and short-term treatment. Please submit your revised manuscript by Jan 18 2021 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, Ping Xu 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. Please ensure that the Limitations of the study, such as the y short duration between sample collection and SDF application and the sample size, are mentioned in the Abstract. 3. Thank you for stating the following in the Funding Section of your manuscript: "This study was funded in part by an award from the New York University Grant Support Initiative (#RA633, Ru PI) and the National Institute of Dental and Craniofacial Research (#R56DE028933, Ru and Saxena, PIs). The content is solely the responsibility of the authors and does not necessarily re ect the ocial views of the National Institutes of Health, New York University, the New York University College of Dentistry, or the New York University School of Medicine." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Please address the following issues: In the last section of the Introduction, remove metabolites from the text since this was not determined. Throughout the text ensure that species names are italicized and Gram is written with a capital G. If available provide more details of the SDF application among the study subjects. Was it applied by the same clinic/person? was a similar size area treated? Discuss the limitations of the study including the small sample size, problems of 16S sRNA amplification bias. Change sugar throughout the text to carbohydrate. Reviewer #2: This manuscript examines the response of oral caries flora to treatment with silver diamine fluoride (SDF), a therapy used for non-surgical caries management in the US. Multiple in vitro studies have shown that SDF inhibits bacterial cell growth through interaction with bacterial cell membranes and enzymes. A primary goal of this study is to examine the efficacy of SDF in a cohort of 20 children 6-13 years of age, and identify the oral bacteria that contribute to a non-responder phenotype, in which the bacteria are resistant to or survive treatment with SDF and have low treatment efficacy. The primary limitations of the study include the small sample size and study design. Comments: 1. The sample size is small, with 5 children in each of 4 separate treatment groups. The authors do clearly state that this is a pilot study, but do not sufficiently emphasize this limitation when drawing their conclusions in the results and discussion. 2. Is SDF bacteriostatic or bacteriocidal? This is a key, but unaddressed point, in the paper. Why would a bacteriostatic agent be an effective therapeutic? 3. In group 3, the authors sample plaque from surface of a tooth with open caries within 10 minutes after application of SDF. A reasonable rationale for this short time period between SDF application and plaque sampling is needed. An important aspect that the authors do not consider is viability of bacteria on the tooth surface. Taxonomic identification by 166 rRNA amplicon sequencing relies only on the presence of bacterial genomic DNA. It does not access viability of bacteria at that site. 4. The accepted term is 16S rRNA amplicon sequencing. 5. The print (text figure labels) in Figures 1-3 is small and will be very difficult to read when the figures are reduced for publication. 6. It is not clear what Fig 3 (PiCrust) contributes to the paper. None of the identified pathways are statistically significant and no meaningful conclusions can be drawn from this analysis. 7. The identification of Granulicatella, Leptotrrichia, and Tannerella in the non-responders is interesting, with a plausible biological basis of their roles in caries presented in the discussion. ****** 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. https://doi.org/10.1371/journal.pone.0242396.r001
Revision 1
1 Jan 2021 Author Response 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 Thank you for your assistance, we have updated the manuscript to align with the listed style requirements. 2. Please ensure that the Limitations of the study, such as the y short duration between sample collection and SDF application and the sample size, are mentioned in the Abstract. We have added these limitations to the revised Abstract as appropriate. 3. Thank you for stating the following in the Funding Section of your manuscript: "This study was funded in part by an award from the New York University Grant Support Initiative (#RA633, Ru PI) and the National Institute of Dental and Craniofacial Research (#R56DE028933, Ru and Saxena, PIs). The content is solely the responsibility of the authors and does not necessarily re ect the ocial views of the National Institutes of Health, New York University, the New York University College of Dentistry, or the New York University School of Medicine." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. We have added the missing funder to the list on the online submission system and removed any funding language from the manuscript. As well, the cover letter is updated as instructed (I could not find the specific funding statement online, only the place to upload the funder information). 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) We sincerely thank the two reviewers who graciously offered their time to review our paper and provide feedback. We address each comment as listed below and provide our responses. Reviewer #1: Please address the following issues: In the last section of the Introduction, remove metabolites from the text since this was not determined. This has been removed Throughout the text ensure that species names are italicized and Gram is written with a capital G. This has been checked and updated. If available provide more details of the SDF application among the study subjects. Was it applied by the same clinic/person? was a similar size area treated? All SDF treatments and oral examinations were performed by a single clinician (YC). All areas treated were similar; since patients were older than 5 and under continuous care at our clinic, they had small lesions into enamel and dentin, confined to one dental surface only. Only one tooth was treated at that visit and sampled as described in the methods (sampling also the contralateral tooth, and using a similar amount of SDF (never more than 1 drop). We note a previous publication by the member of our research team who provided treatment on the average amount of F and Ag in a single drop: an average (SD) drop measuring 32.5 mL (microliters) would have approximately 1.64 (0.04) to 1.76 (0.05) mg of fluoride, and 8.08 (0.13) to 8.71 (0.38) mg of silver. We have added this to the manuscript. Discuss the limitations of the study including the small sample size, problems of 16S sRNA amplification bias. We have added additional information (there is a similar comment regarding sample size from the other reviewer) to the discussions/limitations, specifically: 16S rRNA gene amplicon sequencing captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic shotgun sequencing. Our use of the PICRUSt package to predict metagenome functional content from 16S rRNA extends our analysis. Additionally, bias may have been introduced due to varying 16S rRNA PCR amplification frequencies. Sequencing errors due to PCR amplification were mitigated by removing mismatches to primers, and denoising and trimming reads with low quality scores during sequencing analyses. Change sugar throughout the text to carbohydrate. These have been changed Reviewer #2: This manuscript examines the response of oral caries flora to treatment with silver diamine fluoride (SDF), a therapy used for non-surgical caries management in the US. Multiple in vitro studies have shown that SDF inhibits bacterial cell growth through interaction with bacterial cell membranes and enzymes. A primary goal of this study is to examine the efficacy of SDF in a cohort of 20 children 6-13 years of age, and identify the oral bacteria that contribute to a non-responder phenotype, in which the bacteria are resistant to or survive treatment with SDF and have low treatment efficacy. The primary limitations of the study include the small sample size and study design. Comments: 1. The sample size is small, with 5 children in each of 4 separate treatment groups. The authors do clearly state that this is a pilot study, but do not sufficiently emphasize this limitation when drawing their conclusions in the results and discussion. We agree with the reviewer regarding the limitation of our study. We have added few lines in the results and discussion of the manuscript to address this issue., specifically in the picrust results and in the concluding paragraph of the discussion. This perspective is both from the microbiologic and epidemiologic. 2. Is SDF bacteriostatic or bacteriocidal? This is a key, but unaddressed point, in the paper. Why would a bacteriostatic agent be an effective therapeutic? SDF is bactericidal, we have noted this in the introduction to the paper and provided a citation which states that in a review of studies on the effect of SDF on cariogenic bacteria, specifically Streptococcus mutans (Zhao et al, 2018; IDJ DOI: 10.1111/idj.12320). Others include Abdullah et al, 2020; https://doi.org/10.1371/journal.pone.0241519 & Burgers et al, 2009; https://doi.org/10.1016/j.archoralbio.2009.03.004. 3. In group 3, the authors sample plaque from surface of a tooth with open caries within 10 minutes after application of SDF. A reasonable rationale for this short time period between SDF application and plaque sampling is needed. An important aspect that the authors do not consider is viability of bacteria on the tooth surface. Taxonomic identification by 166 rRNA amplicon sequencing relies only on the presence of bacterial genomic DNA. It does not access viability of bacteria at that site. We admit that this is a limitation. As this was a pilot study, access to subjects and corresponding samples was limited to a single observation. As a result, we elected to investigate the immediate effects of therapy. Though we are aware that identifying DNA of present bacteria does not elucidate their viability, we wanted to study if there was an immediate measurable effect in order to better plan for future studies. The follow-up, full study to this pilot (currently underway) will collect longitudinal data with variable rates of time following application of SDF. We have included this as part of a larger discussion section on the sample size and timing limitations of the study. 4. The accepted term is 16S rRNA amplicon sequencing. We have made the changes in the manuscript. 5. The print (text figure labels) in Figures 1-3 is small and will be very difficult to read when the figures are reduced for publication. Thank you – we will ensure that if accepted for publication this is reviewed in the proofing/typsetting stage. Additionally, in our revision following with PLOS One style guidelines, our figure labels are now embedded within the manuscript. 6. It is not clear what Fig 3 (PiCrust) contributes to the paper. None of the identified pathways are statistically significant and no meaningful conclusions can be drawn from this analysis. Thank you for your comment. Although the data was not statistically significant due to the small sample size, nevertheless we saw a trend in the plots, specifically higher in non-responders and amongst those that are involved in caries. Additionally, Fig 3 compares the role of bacteria in some of the most important metabolic pathways of bacteria. Future studies aiming to study these pathways with a larger sample size may reveal statistically significant data. As we are actively collecting data as part of a new longitudinal study to expand on this pilot, we feel it is important to note these results. 7. The identification of Granulicatella, Leptotrrichia, and Tannerella in the non -responders is interesting, with a plausible biological basis of their roles in caries presented in the discussion. Thank you for your helpful comments and for your review, it is sincerely appreciated. Attachments Attachment Submitted filename: Response to Reviewers v3.docx https://doi.org/10.1371/journal.pone.0242396.r002
25 Jan 2021 Decision Letter - Ping Xu, Editor Microbial population shift and metabolic characterization of silver diamine fluoride treatment failure on dental caries PONE-D-20-34812R1 Dear Dr. Ryan Richard Ruff, 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, Ping Xu 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 ******** 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: I have no further comments, the authors did address all my listed concerns. Thank you for being responsive to my suggestions. Reviewer #2: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0242396.r003
Formally Accepted
24 Feb 2021 Acceptance Letter - Ping Xu, Editor PONE-D-20-34812R1 Microbial population shift and metabolic characterization of Silver Diamine Fluoride treatment failure on dental caries Dear Dr. Ruff: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Ping Xu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242396.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .