Treponema denticola as a prognostic biomarker for periodontitis in dogs

Daehyun Kwon; Kisuk Bae; HyeonJo Kim; Sang-Hyun Kim; Dongbin Lee; Jae-Hoon Lee

doi:10.1371/journal.pone.0262859

Peer Review History

Original SubmissionAugust 13, 2021
25 Nov 2021 Decision Letter - Catherine A. Brissette, Editor PONE-D-21-24139Treponema denticola as a prognostic biomarker for periodontitis in dogsPLOS ONE Dear Dr. Lee, 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 address the comments from all three reviewers, paying particular attention to R1 and R2's concerns about the data supporting the conclusions. Please submit your revised manuscript by Jan 09 2022 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. 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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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: Partly Reviewer #2: Partly Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? 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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 Reviewer #3: 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 screened prognosis marker bacteria for dog periodontitis from dental plaque using quantification real-time PCR. The investigation of the dog periodontitis is a little and more investigation is required. This manuscript focused on the human periodontal pathogen in dog periodontitis. The viewpoint is interesting. Author had better to revise several points to improve manuscript. 1. Specificity of the primer/probe set of quantification real-time PCR was not clear. The composition of the dental plaque bacteria in dog has not completely investigated. It is possible that the primers/probes cross reacted to closely related species to human periodontal pathogen. For example, the sequence of P. gingivalis and P. gluae shows very high similarity. In the present condition, the specificity of the detection of the species by the authors was not clear. Authors should describe the specificity of the primers used in this study. 2. Author showed the number of each organism in this paper. In the present condition, the percentage of the total microorganisms is unclear. It is helpful to understand the total number of the bacteria in the sample. 3. In the prevalence of the pathogen in the present study is significantly difference especially in P. gingivalis and A. actinomycetemcomitans. Authors mentioned about the condition of the disease such as PD3, PD4 for the comparison. The microflora changed according to the age and type of dog. Authors had better to include the condition of the dog for comparison of the prevalence. Minor comments 1. Page 4, line 9. … alveolar bone, and gingiva. The description of periodontitis need the reference. 2. Page 4, line 16-17. However, many studies …disease in dogs [7]. Author only 1 report for the “many studies”. Reviewer #2: This manuscript is straightforward and presents data demonstrating a strong association of Treponema denticola to be associated with canine periodontitis. A strength of this manuscript compared to previous literature is the larger sample size and collection of clinical samples from multiple sites around the tooth. Association analysis seems appropriate but the authors need to make sure to not overstate the relevance of this data. 1. The manuscript is fairly well written, but there are still quite a few minor grammatical edits required. Also the authors should refer to " this current study" in places throughout the text of the discussion, as it is not always clear that they are referring to their own data presented in this manuscript. 2. Please clarify method for sample collection and DNA isolation. Were 6 individual paper points used as shown inFigure 1? Were these 6 paper points then pooled together to give 1 sample which was then treated with 180ul lysozyme as indicated the methods? 3. Most figures are depicting fold change while Figure 4 indicates absolute numbers of bacteria. indicate in the methods how these values were calculated ( compared to standard curve of each bacteria known numbers?) If bacterial DNA was directly used ( or bacteria cultured) this information needs to be included. 4. table 4 refers to "each bacterial species" in the title, but only association for Aa and Pg is shown 5. THE methods do refer to the number of total dogs studied yet There is no mention of the number of animals that were divided into each classification group ( ie. PD1, Pd2 etc). How many animals were in the PD and P1 group ( reversible) or the PD2. Pd3 and Pd4 group ( irreversible). Were these classifications of the clinical sample collected made on each tooth collected or the overall radiographic appearance of the animal? Reviewer #3: The manuscript presents an original and interesting point of view of the study of microbiota in canine periodontitis. My overall impression is that the section results is too long, and some parts should be deleted, considering that same data are presented also in the tables and figures. Specific comments: Table 1 could be moved to supplementary material. Table 2, title ( and also headline in the results): “donated” is not appropriate, please substitute. Figures: it is not necessary to comment the results. So delete sentences: Figure 2, legenda: , starting from Fm, Pn, etc Fig. 3: please delete last sentence, starting fromTreponema denticola Fig 4: delete 3 sentences, starting from The mean number Fig 5: delete starting from mean numbers ****** 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 Reviewer #3: 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.0262859.r001
Revision 1
5 Jan 2022 Author Response Catherine A. Brissette, Ph.D. Academic Editor, PLOS ONE Dear Dr. Catherine A. Brissette We thank the editor and referees of the “PLOS ONE” for taking their time to review our report. We have made some corrections and clarifications in the manuscript after going over the review’s comments. The changed are summarized below: 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 include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Answer: It was approved by the Institutional Animal Care and Use Committees (IACUC) of Gyeongsang National University, an ethics committee related to animal experiments, and it was described in the animals section of materials and methods. 3. 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. Review Comments to the Author Reviewer #1: This manuscript screened prognosis marker bacteria for dog periodontitis from dental plaque using quantification real-time PCR. The investigation of the dog periodontitis is a little and more investigation is required. This manuscript focused on the human periodontal pathogen in dog periodontitis. The viewpoint is interesting. Author had better to revise several points to improve manuscript. 1. Specificity of the primer/probe set of quantification real-time PCR was not clear. The composition of the dental plaque bacteria in dog has not completely investigated. It is possible that the primers/probes cross reacted to closely related species to human periodontal pathogen. For example, the sequence of P. gingivalis and P. gluae shows very high similarity. In the present condition, the specificity of the detection of the species by the authors was not clear. Authors should describe the specificity of the primers used in this study. Answer & Correction: We appreciate this point of specific comment asking us to provide the qPCR specificity/sensitivity data with the primer/probe set designed for this study. Accordingly, we provided additional data regarding the specificity/sensitivity test results in the Supporting Information section. In the Supporting Table S1 and S2, Porphyromoas gulae, the putative periodontopathic bacteria in dogs, was newly included to show the differential detection ability of the qPCR with the primer/probe set designed for specific detection of P. gingivalis (the human periodontopathic bacteria) in the subgingival plaque samples of dogs (Please see the highlighted portion of the tables). In the revised manuscript, page 9, we added sentence “The qPCR performed with the primer-probe sets used in this study are as shown in the Table 1, and the data for assessment of specificity and sensitivity of the primer-probe sets detecting 11 bacteria from the subgingival plaques of dogs can be found in Supporting Information section.” 2. Author showed the number of each organism in this paper. In the present condition, the percentage of the total microorganisms is unclear. It is helpful to understand the � Answer : In the present study, the total number of samples is 336, and the detection rate for each bacteria is expressed by dividing the number of samples detected for each bacteria. For example, in Figure 2, 5/336=1.49% of samples in which Aa: Aggregatibacter actinomycetemcomitans was detected among 336 samples were indicated. 3. In the prevalence of the pathogen in the present study is significantly difference especially in P. gingivalis and A. actinomycetemcomitans. Authors mentioned about the condition of the disease such as PD3, PD4 for the comparison. The microflora changed according to the age and type of dog. Authors had better to include the condition of the dog for comparison of the prevalence. Answer: As mentioned in Table 2, the age, sex and weight of dogs under the reversible and Irreversible groups (PD3, PD4) were compared, but it was stated that there was no statistical difference. (12 pages) Minor comments 1. Page 4, line 9. … alveolar bone, and gingiva. The description of periodontitis need the reference. Answer & Correction: References in this content correspond to Reference No. 7 in the current manuscript. Accordingly, references were added to the manuscript and references 6 and 7 were changed. 2. Page 4, line 16-17. However, many studies …disease in dogs [7]. Author only 1 report for the “many studies”. According to reviewer 1's comment, the sentence has been modified as follows. � …..Yamasaki et al evaluated putative pathogens related to periodontal disease in dogs Reviewer #2: This manuscript is straightforward and presents data demonstrating a strong association of Treponema denticola to be associated with canine periodontitis. A strength of this manuscript compared to previous literature is the larger sample size and collection of clinical samples from multiple sites around the tooth. Association analysis seems appropriate but the authors need to make sure to not overstate the relevance of this data. 1. The manuscript is fairly well written, but there are still quite a few minor grammatical edits required. Also the authors should refer to " this current study" in places throughout the text of the discussion, as it is not always clear that they are referring to their own data presented in this manuscript. Answer and Corrections: Since the word "this study" may be confused with the current manuscript, our study was revised as "the current study" or "the present study" in the manuscript. 2. Please clarify method for sample collection and DNA isolation. Were 6 individual paper points used as shown in Figure 1? Were these 6 paper points then pooled together to give 1 sample which was then treated with 180ul lysozyme as indicated the methods? Answer and correction: Yes, comments from Reviewer 2, the following sentence has been added to the sample collection section. The total number of samples is 336, and the detection rate for each bacteria is expressed by dividing the number of samples detected for each bacteria. Page 4: Six paper points obtained subgingival plaque from six points around a tooth, and all six paper points were placed in a sterilized sample container, then a unique barcode number was assigned to the sample container and analyzed as one sample 3. Most figures are depicting fold change while Figure 4 indicates absolute numbers of bacteria. indicate in the methods how these values were calculated ( compared to standard curve of each bacteria known numbers?) If bacterial DNA was directly used ( or bacteria cultured) this information needs to be included. Answer: As mentioned in Table 2, the age, sex and weight of dogs under the reversible and Irreversible groups (PD3, PD4) were compared, but it was stated that there was no statistical difference. (12 pages) 4. table 4 refers to "each bacterial species" in the title, but only association for Aa and Pg is shown Answers and corrections: Comments from Reviewer 2 Therefore, the contents of Table 4 have been corrected to the following sentences. Table 4. Associations between the number of each bacterial species and the reversible and irreversible group and each PD group �Table 4. Relatively very low reliability of Aa and Pg in the Associations between the number of each bacterial species and the reversible and irreversible group and each PD group Answers and corrections: In accordance with the amendments to Table 4, the relevant sentences in the document have been corrected to the following sentences. Statistical methods were used to analyze associations between periodontal conditions and the number of bacteria using various comparisons (Table 4). � Statistical methods were used to analyze associations between periodontal conditions and the number of bacteria using various comparisons (Table 4) 5. THE methods do refer to the number of total dogs studied yet There is no mention of the number of animals that were divided into each classification group ( ie. PD1, Pd2 etc). How many animals were in the PD and P1 group ( reversible) or the PD2. Pd3 and Pd4 group ( irreversible). Were these classifications of the clinical sample collected made on each tooth collected or the overall radiographic appearance of the animal? � Answers and correction : The following was added to the results section. Page 13: …... Of the total 336 samples, 190 samples (PD0, 70; PD1 120 samples) were classified into the reversible group, and 146 samples (PD2, 77; PD3, 34; PD4, 35 samples) were classified as the irreversible group. Reviewer #3: The manuscript presents an original and interesting point of view of the study of microbiota in canine periodontitis. My overall impression is that the section results is too long, and some parts should be deleted, considering that same data are presented also in the tables and figures. Specific comments: Table 1 could be moved to supplementary material. Answer: If “Table 1” is not an issue for publication, we would like to leave it in the main manuscript. Table 2, title ( and also headline in the results): “donated” is not appropriate, please substitute. Answers and corrections: As the reviewer's advice, the following corrections were made: “Table 2. Characteristic profiles of dogs that provided the subgingival plaque sample” Figures: it is not necessary to comment the results. So delete sentences: Figure 2, legenda: , starting from Fn, Pn, etc Answers and corrections: The part mentioned by the reviewer has been deleted. Fig. 3: please delete last sentence, starting from Treponema denticola Answers and corrections: The part mentioned by the reviewer was deleted and merged into a part of the related manuscript. Fig 4: delete 3 sentences, starting from The mean number Answers and corrections: The part mentioned by the reviewer has been deleted. Fig 5: delete starting from mean numbers Answers and corrections: The part mentioned by the reviewer has been deleted. Attachments Attachment Submitted filename: Response to Reviewer_R1_F.docx https://doi.org/10.1371/journal.pone.0262859.r002
7 Jan 2022 Decision Letter - Catherine A. Brissette, Editor Treponema denticola as a prognostic biomarker for periodontitis in dogs PONE-D-21-24139R1 Dear Dr. Lee, 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, Catherine A. Brissette, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0262859.r003
Formally Accepted
12 Jan 2022 Acceptance Letter - Catherine A. Brissette, Editor PONE-D-21-24139R1 Treponema denticola as a prognostic biomarker for periodontitis in dogs Dear Dr. Lee: 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 Dr. Catherine A. Brissette Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0262859.r004

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