Title:Pyrosequencing of supra- and subgingival biofilms from inflamed peri-implant and periodontal sites
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1 Author's response to reviews Title:Pyrosequencing of supra- and subgingival biofilms from inflamed peri-implant and periodontal sites Authors: Joerg Eberhard Simone Schaumann Ingmar Staufenbiel Ralph Scherer Markus Schilhabel Andreas Winkel Sascha N Stumpp (stumpp.nico@mh-hannover.de) Meike Stiesch (stiesch.meike@mh-hannover.de) Version:2Date:24 October 2014 Author's response to reviews: see over
2 MHH, Zahnärztliche Prothetik, OE 7710, Hannover Zentrum Zahn-, Mund- und Kieferheilkunde Klinik für Zahnärztliche Prothetik und Biomedizinische Werkstoffkunde Editor-in-Chief BMC Oral Health Professor Magdalena Morawska Prof. Dr. Jörg Eberhard, MMEd Telefon: Fax: Carl-Neuberg-Straße Hannover Telefon: Ihr Zeichen Ihre Nachricht vom Unser Zeichen Dear Editor-in-Chief, Dear Magdalena Morawska, Please find attached our revised manuscript entitled Pyrosequencing of supra- and subgingival biofilms from inflamed peri-implant and periodontal sites that we hope now is suitable for publication in the Journal BMC Oral Health. We are grateful to the reviewers comments and are the opinion that the manuscript has substantially improved now. Please find below our answers to the reviewers comments. With regards Jörg Eberhard
3 Reviewer's report Title:Pyrosequencing of supra- and subgingival biofilms in peri-implantitis and periodontitis lesions Version:1Date:25 September 2014 Reviewer: purnima kumar Reviewer's report: There are two major problems with this manuscript: 1. The definitions of disease are extremely loose. For example, the authors have used probe depths and bleeding on probing to select cases for sampling. These two criteria define a number of disease states and cannot definitively establish what the disease state was. Answer: The definition of the cases has been corrected. The manuscript has been renamed and the text in the Materials and Methods section has been adapted (p.:4; l.:2-11) so that there are no longer any definition problems. We have current X-rays that show evident radiographic bone loss. This information has been added to the text (p.:4; l.:7, 12-13). 2. Greengenes is capable of resolution at the genus level, not species. The authors have used this level for making comparisons. There are several species within each genus that are health compatible and disease associated. The genus Streptococcus is a prime example. At the genus level, S. mutans and S. mitis will fall into the same clade, but one causes disease and the other is beneficial. Answer: Because of the complexity of the expected microbiomes, the evaluation was performed at the genus level. The number of cases also suggests that analysis at the genus level is more reasonable than at the species level. Several current publications (Bizzarro S. (2013), Hu Y.J. (2013)) also deal with these problems at the genus level. The justification for this approach has been extended in the text (p.:12; l.:4), with additional literature. Others:Please spell check the document. Quality of written English: Needs some language corrections before being published Answer: The manuscript has been edited by a native English speaker with a background in the biological sciences. Reviewer's report Title: Pyrosequencing of supra- and subgingival biofilms in peri-implantitis and periodontitis lesions
4 Version:1Date:18 September 2014 Reviewer: Ana Paula Colombo Reviewer's report: Dear authors, This study aimed to compare the microbial composition of the supra and sub-gingival biofilm of teeth with periodontitis and implants with peri-implantitis. The authors concluded that the composition of the microbiota of both clinical diseases is similar although they are different diseases. The purpose of the study is interesting but there are several problems with writing and methodology that needs to be addressed. 1- The whole manuscript needs to be revised for typing errors and misspelling of bacteria names. Answer: The manuscript has been edited by a native English speaker with a background in the biological sciences. The spelling of the bacteria has been checked and corrected throughout the manuscript : Background: the sentence on lanes needs to be rephrased because Kumar did look at periodontitis and failing implants, although was the subgingival plaque. Answer: The sentence has been corrected, as recommended by the reviewer (p.:3; l.:11+12). 2.2: In fact, was really necessary to evaluate the supragingival plaque of both clinical diseases? What is the rationale? Answer: The aim of the study was to examine and compare the different microbiomes on implants and teeth, in order to be able to evaluate the significance of this as an etiological factor. For this reason, the authors considered that supragingival plaque should be included in the investigation : Lane 22: for the first time should be removed. This is very relative since a paper may come out tomorrow presenting similar methodology. Answer: The reviewer's recommendation has been followed and for the first time has been removed (p.:3; l.:21). 3.2: Remove the sentence on lanes 24-25, this is not the reason why you use the deep sequencing technique. Answer: This recommendation has been followed. 3.3: This whole paragraph should be improved, it is hard to follow. The sentence was invented should also be changed. Answer: This recommendation has been followed. The paragraph has been deleted (p.:3; l.:24ff)
5 4- Methods: smoking is a major risk factor for periodontitis and peri-implantitis, so no smoking within the last 6 months does not make sense. Smoking has a long and cumulative effect on the periodontal tissues. I believe that smoking should be excluded. Former smokers should have stopped for at least 5 years. Answer: The reviewer is right. Smokers have been excluded. The misleading sentence in Materials and Methods has been reformulated (p.:4; l.:10). 5- For a patient to have periodontitis or peri-implantitis, he/she had to have at least 1 site with PPD>4mm? So I could have a patient with several sites with periodontitis or periimplantitis and others with just one diseased site? This is important because measures the extension of disease. Diseased sites are reservoirs for periodontal pathogens that may cross-disseminate to other sites in the mouth. Answer: The missing data has been added in the Materials and Methods section and in Table 1 (p.:4; l.:2). 6- Clinical examination: for the plaque index API add a reference. Answer: The source has been added. 7- Sample collection: how did you collect supragingival plaque with paper points????? All samples in my opinion should have been taken with curettes. Answer: We used Paper Points Iso 30, which were stable enough to collect plaque. Thus, we could use the same technique for supra- and subgingival or supra- and submucosal. 8- DNA isolation, lane 15: sulcus fluid or subgingival plaque??? Answer: As recommended by the reviewer, text changed to for sampling (p.:5; l.:14). 9- How were the concentrations of DNA determined? Answer: The procedure has been added to the manuscript (p.:6+7; l.:25, 1-3). 10-My main criticism is related to the number of patients. Due to the high inter-individual variability of the periodontal microbiota, make conclusion and assumptions with 7 patients is problematic.
6 Answer: As the microbiome was expected to be complex, the evaluation was performed at the level of the genus. The number of cases is an additional reason to perform the analysis at the genus level rather than at the species level. Several current publications (Bizzarro S. (2013), Hu Y.J. (2013)) also study the problem at the level of the genus. The justification in the text has been extended (p.:12; l.:4) and additional literature has been cited : Results: table 1, there is something wrong with the plaque index % of implants? Answer: The numerical error has been corrected. 11.2: The description of the micro data should be improved, it is very confusing. Answer: The sentence structure has been changed to make this section clearer (p.:9; l.:9, 14, 18, 21). 12-What was the reason to do the Gram analysis? How was that done? The subtitle is not self-explanatory, the authors should have mentioned this in the methods. Answer: The Gram classification was intended to be an additional phenotypic classification. At this level, the data could be compared with other publications, e.g. Kumar et. al. (2012). Adjusted in the Materials and Methode section (p.:8; l.:1-2). 13-All figure legends should be improved, including details of the statistical analysis. Answer: All figure legends have been improved. The name of the bacteria in fig 1 should be clearer. What does c stand for? Answer: The quality of the figure has been improved, as well as the legend. 14-Discussion is too long and could be shortened. Answer: The reviewer's recommendation has been followed by deleting sections in the discussion. 15-Other major criticism here was the difficult in providing identification at the species level. This may be one of the reasons the authors found very similar microbiotas between the 2 groups or areas. Answer: One result of the study is indeed that the microbiome is very similar at the level of the genus.
7 16-Why do you think the classical periodontal pathogens were rarely detected and in low numbers? Answer: This sentence was removed when the discussion was abbreviated. 17-On lane 7, page 13: several studies (including Haffajee s studies) have shown that diversity is actually associated with health. Loss of diversity may lead to disease. Discuss that. Answer: Our study did not include healthy and diseased implants, so we are unable to contribute to this discussion. We have deleated the respective sentence in the discussion. 18-The whole discussion about Gram-positive and negative species does not seem relevant. Answer: This section was substantially shortened. The authors are the opinion that the discussion about Gram-positive and Gram-negative staining is relevant for the reader (see comment no. 12). 19-The authors should be careful with the conclusions saying the there are no major differences between the microbiota of periodontitis and peri-implantitis based on the analysis of 7 patients. Answer: The conclusion section has been rephrased according to the reviewer s suggestions (p.:14; l.:15-18). 20-All references need to be reviewed, the order, the names of the journals and some of them are incomplete. Answer: The references have been checked and corrected. Quality of written English: Needs some language corrections before being published Answer: The manuscript has been checked by a native English speaker with a background in the biological sciences. Reviewer's report Title: Pyrosequencing of supra- and subgingival biofilms in peri-implantitis and
8 periodontitis lesions Version:1Date:24 September 2014 Reviewer:Anna Louropoulou Reviewer's report: In the section M&M, line 12-13; the diagnosis of peri-implantitis and periodontitis is based on BOPand PD. The diagnosis of peri-implantitis and periodontitis is based also on the presence of radiographic bone loss. If radiographic data are not provided the use of terms periimplantitis or periodontitis can be misleading. There is a chance that some of the cases included are gingivitis or mucositis cases. The authors should provide radiographic data if they want to continue using these terms otherwise, if radiographic data are not available, they should use other terminology. Answer: We have followed the reviewer's recommendation, see Commentary 1 for Reviewer 1. Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Acceptable Statistical review: Yes, but I do not feel adequately qualified to assess the statistics. Declaration of competing interests: I declare that I have no competing interests
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