{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T10:02:47Z","timestamp":1779876167081,"version":"3.53.1"},"update-to":[{"DOI":"10.12688\/f1000research.161731.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000}},{"DOI":"10.12688\/f1000research.161731.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000}},{"DOI":"10.12688\/f1000research.161731.3","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000}}],"reference-count":32,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100021726","name":"Direktorat Riset and Pengembangan, Universitas Indonesia","doi-asserted-by":"publisher","award":["No:MKB-318\/UN2.RST\/HKP.05.00\/2024"],"award-info":[{"award-number":["No:MKB-318\/UN2.RST\/HKP.05.00\/2024"]}],"id":[{"id":"10.13039\/501100021726","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    Background Despite diabetes mellitus and periodontal diseases are mutually exclusive, little is known about particular types of bacteria that may have exacerbated the development of diabetics\u2019 periodontal inflammation. The purpose of this study was to descriptively characterize and explore the differences in the salivary microbiomes of individuals with type 2 diabetes (20-40 years old) who had gingivitis or periodontitis to those who did not. Additionally, we evaluated the descriptive relationship between the relative abundance of periodontopathogens and nitrate-reducing bacteria in their salivary microbiome. Methods Saliva was collected from all participants. Genomic DNA was isolated and pooled in equimolar quantities from all individuals within each group to create three pooled libraries: type 2 diabetes (T2DM) patients without periodontal disease (G1), T2DM patients with gingivitis (G2), and T2DM patients with periodontitis (G3). Sequencing was performed using Oxford Nanopore MinION Technology. The relative abundance and bacterial diversity were measured using bioinformatic methods, and all analyses of sequencing data were strictly descriptive and exploratory. Salivary nitrite\/nitrate concentrations were measured on individual, un-pooled samples. Results The salivary microbiota among people with type 2 diabetes and periodontal disease (G2; G3) was observed to have greater bacterial diversity and abundance than that of patients without periodontal disease (G1), according to descriptive alpha-diversity analysis. The G3 group exhibited the largest relative abundance of\n                    <ns3:italic>Porphyromonas gingivalis<\/ns3:italic>\n                    , a key periodontopathogen. Descriptive analysis also suggested that periodontopathic bacteria and nitrate-reducing bacteria have different community structures across the groups. Furthermore, comparison of individual salivary samples showed that nitrite\/nitrate concentration was significantly lower in the G3 group compared to the G1 group (p&lt; 0.05). Results reveal that the inverse interaction between increased\n                    <ns3:italic>P. gingivalis<\/ns3:italic>\n                    and decreased nitrate-reducing bacteria serves as a descriptive hallmark for periodontal disease progression in the T2DM population. Conclusion Results of this exploratory study suggest that the relationship between periodontopathic and denitrifying bacteria in the salivary microbiome varies among those with type 2 diabetes mellitus who also have gingivitis or periodontitis. These distinct microbial features observed may be microbiological characteristics associated with the progression of periodontal disease in this population, warranting further validation as potential indicators for early management.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.161731.4","type":"journal-article","created":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T09:09:05Z","timestamp":1779872945000},"page":"297","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Salivary microbiome and periodontopathogen\/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes"],"prefix":"10.12688","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2512-2579","authenticated-orcid":false,"given":"Endang","family":"Bachtiar","sequence":"first","affiliation":[{"name":"Oral Biology Fac. of Dentistry Depok West Java 16424 University of Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boy M.","family":"Bachtiar","sequence":"additional","affiliation":[{"name":"Oral Biology Fac. of Dentistry Depok West Java 16424 University of Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dicky  L","family":"Tahapary","sequence":"additional","affiliation":[{"name":"Clinical Research Unit RSCM, . Metabolic-Endocrine-Diabetes Division, Dept. Internal Medicine Depok West Java Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2340-6647","authenticated-orcid":false,"given":"Turmidzi","family":"Fath","sequence":"additional","affiliation":[{"name":"Oral Biology Fac. of Dentistry Depok West Java 16424 University of Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Citra Fragrantia","family":"Theodora","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry, Department of Oral Biology and Oral Science Research Center Depok West Java 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0472-4009","authenticated-orcid":false,"given":"Natalina","family":"Haerani","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry,Department of Periodontology Jakarta Jakarta 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Selvi Nafisa","family":"Shahab","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Department Microbiology, Clinical Research Unit RSCM Ciptomangunkusumo Hospital. West Java Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuniarti","family":"Soeroso","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry,Department of Periodontology Jakarta Jakarta 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ardy","family":"Wildan","sequence":"additional","affiliation":[{"name":"Metabolic-Endocrine-Diabetes Division, Dept. Internal Medicine Depok West Java Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fergie Marie Joe Grizella","family":"Runtu","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry,Department of Periodontology Jakarta Jakarta 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatimah Maria","family":"Tadjoedin","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry,Department of Periodontology Jakarta Jakarta 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dewi","family":"Ayuningtyas","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry,Department of Periodontology Jakarta Jakarta 10430 Universitas Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,1,13]]},"reference":[{"key":"ref1","doi-asserted-by":"crossref","first-page":"404-16","DOI":"10.1016\/j.ajpath.2017.10.014","article-title":"Porphyromonas gingivalis Differentially Modulates Apoptosome Apoptotic Peptidase Activating Factor 1 in Epithelial Cells and Fibroblasts.","volume":"188","author":"I Bugueno","year":"2018 Feb","journal-title":"Am. J. Pathol."},{"key":"ref2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-33267-4","article-title":"Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model.","volume":"8","author":"I Bugueno","year":"2018 Oct 8","journal-title":"Sci. Rep."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"593-602","DOI":"10.1038\/nrmicro929","article-title":"Nitrate, bacteria and human health.","volume":"2","author":"J Lundberg","year":"2004 Jul","journal-title":"Nat. Rev. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1186\/1741-7007-8-24","article-title":"Denitrification in human dental plaque.","volume":"8","author":"F Schreiber","year":"2010 Mar 22","journal-title":"BMC Biol."},{"key":"ref5","doi-asserted-by":"crossref","first-page":"536","DOI":"10.12688\/f1000research.51270.2","article-title":"Nitric oxide for the prevention and treatment of viral, bacterial, protozoal and fungal infections.","volume":"10","author":"P Bath","year":"2021","journal-title":"F1000Res."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"38-46","DOI":"10.1016\/j.niox.2021.09.001","article-title":"Inorganic nitrate: A potential prebiotic for oral microbiota dysbiosis associated with type 2 diabetes.","volume":"116","author":"Z Bahadoran","year":"2021 Nov 1","journal-title":"Nitric Oxide."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"738-48","DOI":"10.1038\/nrendo.2011.106","article-title":"Diabetes mellitus and periodontitis: a tale of two common interrelated diseases.","volume":"7","author":"E Lalla","year":"2011 Jun 28","journal-title":"Nat. Rev. Endocrinol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"21-31","DOI":"10.1007\/s00125-011-2342-y","article-title":"Periodontitis and diabetes: a two-way relationship.","volume":"55","author":"P Preshaw","year":"2012 Jan","journal-title":"Diabetologia."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1186\/s12903-020-01180-w","article-title":"Epidemiologic relationship between periodontitis and type 2 diabetes mellitus.","volume":"20","author":"C Wu","year":"2020 Jul 11","journal-title":"BMC Oral Health."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"214-24","DOI":"10.1111\/prd.12318","article-title":"The impact of diabetes on periodontal diseases.","volume":"82","author":"D Graves","year":"2020 Feb","journal-title":"Periodontol. 2000."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"658-65","DOI":"10.1177\/0022034520913818","article-title":"Oral Microbiome Signatures in Diabetes Mellitus and Periodontal Disease.","volume":"99","author":"T Matsha","year":"2020 Jun","journal-title":"J. Dent. Res."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"30-6","DOI":"10.1111\/j.1600-0765.2012.01498.x","article-title":"Subgingival biodiversity in subjects with uncontrolled type-2 diabetes and chronic periodontitis.","volume":"48","author":"R Casarin","year":"2013 Feb","journal-title":"J. Periodontal Res."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"511","DOI":"10.3389\/fimmu.2020.00511","article-title":"The Nexus Between Periodontal Inflammation and Dysbiosis.","volume":"11","author":"T Van Dyke","year":"2020","journal-title":"Front. Immunol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"481-90","DOI":"10.1038\/nrmicro2337","article-title":"Periodontitis: a polymicrobial disruption of host homeostasis.","volume":"8","author":"R Darveau","year":"2010 Jul","journal-title":"Nat. Rev. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"887-97","DOI":"10.1177\/00220345221080982","article-title":"The Importance of Nitrate Reduction for Oral Health.","volume":"101","author":"B Rosier","year":"2022 Jul","journal-title":"J. Dent. Res."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"14-9","DOI":"10.1111\/j.1600-0722.2004.00184.x","article-title":"Evaluation of bacterial nitrate reduction in the human oral cavity.","volume":"113","author":"J Doel","year":"2005 Feb","journal-title":"Eur. J. Oral Sci."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1038\/s41522-022-00289-w","article-title":"Early microbial markers of periodontal and cardiometabolic diseases in ORIGINS.","volume":"8","author":"C Marotz","year":"2022 Apr 20","journal-title":"NPJ Biofilms Microbiomes."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"107-31","DOI":"10.1111\/prd.12393","article-title":"The oral microbiome: Role of key organisms and complex networks in oral health and disease.","volume":"87","author":"L Sedghi","year":"2021 Oct","journal-title":"Periodontol. 2000."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"79","DOI":"10.12688\/f1000research.28216.3","article-title":"A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform.","volume":"10","author":"B Bachtiar","year":"2021","journal-title":"F1000Res."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e0049122","DOI":"10.1128\/msystems.00491-22","article-title":"Using Nanopore Sequencing to Obtain Complete Bacterial Genomes from Saliva Samples.","volume":"7","author":"J Baker","year":"2022 Oct 26","journal-title":"mSystems."},{"key":"ref21","doi-asserted-by":"crossref","first-page":"S44-67","DOI":"10.1111\/jcpe.12939","article-title":"Plaque-induced gingivitis: Case definition and diagnostic considerations.","volume":"45","author":"L Trombelli","year":"2018 Jun","journal-title":"J. Clin. Periodontol."},{"key":"ref22","first-page":"31-5","article-title":"Development of a classification system for periodontal diseases and conditions.","volume":"79","author":"G Armitage","year":"2000 Nov-Dec","journal-title":"Northwest Dent."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"197-205","DOI":"10.1111\/jicd.12143","article-title":"Enterococcus faecalis with capsule polysaccharides type 2 and biofilm-forming capacity in Indonesians requiring endodontic treatment.","volume":"6","author":"E Bachtiar","year":"2015 Aug","journal-title":"J. Investig. Clin. Dent."},{"key":"ref24","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giy169","article-title":"NanoPipe-a web server for nanopore MinION sequencing data analysis.","volume":"8","author":"V Shabardina","year":"2019 Feb 1","journal-title":"Gigascience."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"1744","DOI":"10.12688\/f1000research.20099.1","article-title":"Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study.","volume":"8","author":"E Bachtiar","year":"2019","journal-title":"F1000Res."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"409-19","DOI":"10.1111\/j.2041-1014.2012.00663.x","article-title":"Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology.","volume":"27","author":"G Hajishengallis","year":"2012 Dec","journal-title":"Mol. Oral Microbiol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"1016-25","DOI":"10.1038\/ismej.2012.174","article-title":"The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation.","volume":"7","author":"L Abusleme","year":"2013 May","journal-title":"ISME J."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41368-023-00266-9","article-title":"Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability.","volume":"16","author":"B Rosier","year":"2024 Jan 5","journal-title":"Int. J. Oral Sci."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"182-209","DOI":"10.1111\/prd.12358","article-title":"Did Omics change periodontal therapy?","volume":"85","author":"M Feres","year":"2021 Feb","journal-title":"Periodontol. 2000."},{"key":"ref30","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms11071832","article-title":"Periodontal Pathogens and Their Links to Neuroinflammation and Neurodegeneration.","volume":"11","author":"D Visentin","year":"2023 Jul 18","journal-title":"Microorganisms."},{"key":"ref31","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens10050504","article-title":"Drift of the Subgingival Periodontal Microbiome during Chronic Periodontitis in Type 2 Diabetes Mellitus Patients.","volume":"10","author":"I Balmasova","year":"2021 Apr 22","journal-title":"Pathogens."},{"key":"ref32","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28365782.v4","author":"T Fath","year":"2025","journal-title":"figshare."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/14-297\/v4\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/14-297\/v4\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/14-297\/v4\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T09:09:15Z","timestamp":1779872955000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/14-297\/v4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,13]]},"references-count":32,"URL":"https:\/\/doi.org\/10.12688\/f1000research.161731.4","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/f1000research.161731.3","asserted-by":"subject"},{"id-type":"doi","id":"10.12688\/f1000research.161731.2","asserted-by":"subject"},{"id-type":"doi","id":"10.12688\/f1000research.161731.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.194948.r449917","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.194948.r449913","asserted-by":"subject"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,13]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/14-297\/v4#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.194948.r449917, Gaetano Isola, University of Catania, Catania, 15 Jan 2026, version 4, indexed","URL":"https:\/\/f1000research.com\/articles\/14-297\/v4#referee-response-449917","order":0,"name":"referee-response-449917","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.177804.r406077, Sonia Nath, The University of Adelaide, Adelaide, 16 Sep 2025, version 1, 1 approved with reservations, 1 not approved","URL":"https:\/\/f1000research.com\/articles\/14-297\/v1#referee-response-406077","order":1,"name":"referee-response-406077","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Endang Bachtiar<\/b>; \n<i>Posted: 24 Sep 2025<\/i>; RC#2: Was sample size calculations considered. There are more number of participants in group 3 than 2 and 1.&nbsp;  AR: We appreciate the reviewer's insightful assessment of the sample size and participant distribution. We note that no formal statistical procedure was used to establish the number of participants in each category. Instead, we used a consecutive or sequential sampling strategy for a set amount of time (November 2023 to January 2024). Because fewer patients met the requirements for Group 1 (T2DM without periodontal disease) at the time of sampling, this strategy led to an uneven number of participants throughout the groups. We acknowledge that this is a study constraint because it could have an impact on the statistical power of comparisons between groups. However, finding possible microbial markers of illness development was our main goal, and the notable variations in our findings\u2014especially between Group 1 and the other groups\u2014indicate that the sample size was enough for these particular analyses. _We acknowledge that more research with a bigger, more balanced cohort would be helpful to validate our findings, and we have added a sentence to our Limitations section to specifically express this point.    Reviewer\u2019s#1 comment (RC#1): 1.The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the study. AR: Thank you for this important suggestion. Accordingly, at the end of the introduction section, we state the gap of previous studies, as suggested by the Reviewer#2 (yellow highlighted) and the aim of our study (green highlighted).  2. In the central section, should better clarify inclusions and exclusions criteria of the selected sample. AR: Yes, we agree. Accordingly, we have revised the inclusion and exclusion criteria. Many hanks.&nbsp;  3. In the Discussion section\u2026.better discuss the relationship regarding the by periodontitis in and risk of oxidative stress evolution linked with inflammation in periodontitis patients. AR: Thank you for your suggestion. Accordingly, we have added one paragraph to explain the relationships (highlighted in green).  4. The conclusion should reinforce in light of the discussions. AR: We agree. As a result we have revised the conclusion (highlighted in green). Thank you.  Minor Comments: RC#1 Abstract: Better formulate the abstract section by better describing the aim of the study  Introduction: Please refer to major comments  Discussion Please add a specific sentence that clarifies the results obtained in the first part of the discussion  AR: Thank you for all of the reviewer remarks. Consequently, we have caried out as indicated, the updated version in the respective section. Many thanks. Reviewer\u2019s#2 &nbsp;comment (RC#2): Introduction section Include a brief evidence of the existing literature, are there other studies that have looked at a similar link between diabetes and periodontal disease? Was it conducted on a similar population? What gaps were identified in the previous study?&nbsp; How does this study fills the gaps in the literature.&nbsp;  Author\u2019s response (AR) Thank you for the suggestion. As a result, we updated the introduction, including the additional research and explaining how it fills in the gaps by concentrating on specific bacterial families (highlighted in yellow).&nbsp;  RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included.&nbsp;  AR: We appreciate the reviewer's insightful observation. We recognize the possibility of confounding variables being introduced by include patients with different levels of glycemic control, which may be viewed as &quot;unstable.&quot; Our main goal, therefore, was to examine a representative sample of people with Type 2 Diabetes, a group in which glycemic control and associated issues frequently differ. Therefore, our findings are more clinically relevant because we included a wide variety of diabetic individuals rather than a highly selected sample. This was minimized by excluding participants who were taking drugs (such as hormones) or had other serious metabolic conditions that would have an adverse effect on the study's results. In the Discussion section, we have included a phrase acknowledging this restriction, which may necessitate additional research in subsequent studies.  RC#2: How was alveolar bone loss assessed? Did the authors take radiographs?&nbsp; AR: We appreciate the reviewer's wise observation. We recognize that radiographic evaluation is a regular part of a thorough periodontal examination and is an essential tool for assessing alveolar bone loss. However, the main goal of our study was to find non-invasive salivary microbiological markers that could help differentiate between periodontitis and gingivitis. Our patient groups were adequately stratified using clinical characteristics, namely Clinical Attachment Loss (CAL) and probing depth, in accordance with the recognized classifications of periodontal disease. The use of radiographic imaging was deemed outside the purview of this particular study design because our primary focus was on the microbial composition of saliva as a biomarker, a non-invasive sample. _We also think that a more thorough and multifaceted knowledge of the illness progression would be possible with a future study that combines our microbiological findings with radiographic data. To address this problem, we have included a statement in our Limitations section.  RC#2: Was sample size calculations considered. There are more number of participants in group 3 than 2 and 1.&nbsp;  AR: We appreciate the reviewer's insightful assessment of the sample size and participant distribution. We note that no formal statistical procedure was used to establish the number of participants in each category. Instead, we used a consecutive or sequential sampling strategy for a set amount of time (November 2023 to January 2024). Because fewer patients met the requirements for Group 1 (T2DM without periodontal disease) at the time of sampling, this strategy led to an uneven number of participants throughout the groups. We acknowledge that this is a study constraint because it could have an impact on the statistical power of comparisons between groups. However, finding possible microbial markers of illness development was our main goal, and the notable variations in our findings\u2014especially between Group 1 and the other groups\u2014indicate that the sample size was enough for these particular analyses. _We acknowledge that more research with a bigger, more balanced cohort would be helpful to validate our findings, and we have added a sentence to our Limitations section to specifically express this point.  RC#2: &nbsp;How many dental examiners were there for the periodontal assessment? If there were more than one periodontist, were they calibrated?  AR: We thank the reviewer raising this important query. Yes, we accomplished the calibration process and included it in the methods section (highlighted in yellow).  RC#2: &nbsp;Was sample size calculations considered. There are more number of participants in group 3 than 2 and 1. AR: We appreciate the reviewer's insightful assessment of the sample size and participant distribution. We recognize that no formal statistical procedure was used to establish the number of participants in each category. Rather, we used a sequential sampling method for a particular time frame (November 2023 to January 2024). Because fewer patients met the requirements for Group 1 (T2DM without periodontal disease) at the time of sampling, this strategy led to an uneven number of participants throughout the groups. We acknowledge that this is a study constraint because it could have an impact on the statistical power of comparisons between groups. However, finding possible microbial markers of illness development was our main goal, and the notable variations in our findings\u2014especially between Group 1 and the other groups\u2014indicate that the sample size was enough for these particular analyses. We acknowledge that more research with a bigger, more balanced cohort would be helpful to validate our findings, and we have added a sentence to our Limitations section to specifically express this point (kindly see the limitation section).  RC#2: What instrument was used for periodontal assessment? How many sites probing was done AR: I value your questions. We employed the UNC-15 probe and the explanation of the probing site, provided in the Methods section.  RC#2: Was there any media used to store the saliva samples. Were the samples immediately stored at -80C. Were the saliva samples collected after the probing? Probing can cause bleeding, and this causes inflammation of the gums. Can this have an impact on the saliva samples? How was this mitigated?  AR: We did not use any media to store the saliva, and as can be seen in Methods section, \u201cthe sample was immediately stores in -80oC until DNA extraction\u201d. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this.&nbsp;  RC#2: Any environmental samples collected along with saliva. \u201cAfter rinsing the mouth&quot;- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and&nbsp; we used 0.9% normal saline for about 30 s &nbsp;as mentioned in the methods section.  RC#2: How was the DNA extraction carried out? Were there any controls used for the DNA extraction process? Similarly, during PCR, were any controls used?&nbsp; AR: The reviewer's insightful feedback on the PCR and DNA extraction processes is greatly appreciated. We acknowledge that the application of controls is necessary to guarantee the accuracy of the data. As we explained in the Methods, we followed the manufacturer's instructions for the 16S Barcoding Kit and the MonarchTM Genomic DNA purification kit for performing the PCR and DNA extraction processes. The controls were included during the DNA extraction and PCR amplification processes in accordance with the kit's instructions.&nbsp;  RC#2: Describe the sequencing briefly. What primers were used? Any reference data set used for aligning the sequences. AR: We appreciate your inquiries. Yes, as you can see from the Methods, we have followed the company's instructions for both the alignment and the sequencing process.&nbsp;  RC#2: Which packages in R were used for data processing? Mention the names of the packages.&nbsp; Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used.  Reviewer\u2019s comment (RC#2): For the relative abundance table, did the authors apply any filters. Microbiome data are sparse, and did the authors consider transformation of the data or normalisation before analysis for relative abundance?&nbsp;  Author\u2019s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading &quot;DNA extraction, sequencing, and saliva sample collection.&quot;&nbsp;  RC#2 : Which packages in R were used for data processing? Mention the names of the packages.&nbsp; Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used.&nbsp;  AR: Thank you for these queries, As can be seen in the Methods section, OTUs study was performed using the vegan, ggplot2, and dplyr packages while heatmap was performed using pheatmap and R ColorBrewer packages in R Studio software (version 4.3.2). Moreover, &nbsp;the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels.  Reviewer\u2019s#2 comment and suggestion: -Include a demographic table of the included study participants for each group.&nbsp;  -Did the authors consider beta diversity analysis?&nbsp;  Author\u2019s response. I agree. We included a table on characteristics as advised.&nbsp; We recognize that one common method for comparing microbial communities is beta diversity analysis. However, the main goal of our study was to look at the quantity and variety of microorganisms found solely in saliva and no additional oral samples; beta diversity was not taken into account during the analytical process. Thank you very much.","URL":"https:\/\/f1000research.com\/articles\/14-297\/v4#referee-comment-14653","order":2,"name":"referee-comment-14653","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.192091.r439955, Chandrashekar Janakiram, Armita School of Dentistry, Ernakulam, 10 Jan 2026, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/14-297\/v3#referee-response-439955","order":3,"name":"referee-response-439955","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.194948.r449913, Ansam Mahdi Khalel, University of Kufa, Najaf, 28 Jan 2026, version 4, indexed","URL":"https:\/\/f1000research.com\/articles\/14-297\/v4#referee-response-449913","order":4,"name":"referee-response-449913","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.192091.r429427, Susanna Halim, Universitas Prima Indonesia, Medan, 30 Dec 2025, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/14-297\/v3#referee-response-429427","order":5,"name":"referee-response-429427","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Endang Bachtiar<\/b>; \n<i>Posted: 07 Jan 2026<\/i>; Response to Reviewer\u2019s (5) comments  ABSTRACT Reviewer\u2019s comments\/suggestion (RC\/S): In the abstract, the author only demonstrates the relationship between the number of periopathogen bacteria and the number of nitrate-reducing bacteria... based on ROC (Receiver Operating Characteristic) analysis, only Fusobacterium spp. and Neisseria are strong indicators. The author could provide additional descriptions regarding the interaction between P. gingivalis and nitrate-reducing bacteria. \n<b>Author\u2019s Response (AR)<\/b>: Thank you for your valuable feedback. AR: Thank you for the suggestion. Regarding the ROC analysis, we would like to clarify that following the suggestion from Reviewer 3 in the previous revision stage, we have excluded the ROC analysis from the manuscript. This was decided because our DNA samples were pooled, which limits the statistical validity of individual-based diagnostic performance tests like ROC. However, we have addressed your core suggestion by adding a description in the Abstract regarding the interaction between P. gingivalis and nitrate-reducing bacteria. We now highlight that the shift in their relative abundance (the increase of pathogens alongside the decrease of beneficial nitrate-reducers) serves as a key descriptive indicator of periodontal tissue damage in T2DM patients.  INTRODUCTION RC\/S: The author focuses more on clarifying the periopathogen and nitrate-reducing bacteria, rather than their interaction concerning gingivitis or periodontitis in T2DM patients. Several references should be added to strengthen the research objective. \n<b>Author\u2019s Response (AR):<\/b> We agree. We have revised the Introduction to emphasize the biological interaction between these bacterial groups. Specifically, we have added references explaining how the unique metabolic environment of T2DM patients may disrupt the homeostasis maintained by nitrate-reducing bacteria, thereby facilitating the overgrowth of periodontopathogens. This provides a stronger rationale for our study's focus on their relationship.  METHODS RC\/S: In the exclusion criteria, it is necessary to add that samples should not be from one side of the jaw. In the periodontal assessment, radiography is recommended. In the statistical section, the author does not elaborate on individual differences because of the DNA pooling method. \n<b>Author\u2019s Response (AR)<\/b>: Thank you for these technical insights. 1.&nbsp;&nbsp; &nbsp;Clinical Assessment: We have noted the importance of comprehensive jaw assessment in our protocol description. 2.&nbsp;&nbsp; &nbsp;Radiography: We acknowledge that radiographic data were not obtained. We have added this as a study limitation in the Methods section, clarifying that our diagnosis was based on clinical parameters (CAL and Probing Depth) in accordance with the 2017 World Workshop classification. 3.&nbsp;&nbsp; &nbsp;Statistics: In response to the DNA pooling method, we have removed all inferential statistics (including the ROC curves previously mentioned). The revised manuscript now adopts a strictly descriptive and exploratory approach, which is more appropriate for pooled sequencing data.  RESULTS RC\/S: There are inaccuracies in Figure 2C\/D; Fusobacterium spp. was more numerous than Porphyromonas..... the number of denitrifying bacteria (e.g., Neisseria) should be lower in group G3 compared to G2 and G1. \n<b>Author\u2019s Response (AR): <\/b>We appreciate the reviewer\u2019s careful review of our data. We have re-verified the taxonomic data from our sequencing results. Figures 2C and 2D have been carefully checked to ensure they accurately represent the relative abundances found in our T2DM cohort. We have also added text to clarify these findings, noting that the observed microbial distribution reflects the specific dysbiotic state of the sampled groups.  DISCUSSION RC\/S: The author could add explanations regarding the shift in the proportion of periopathogen bacteria relative to denitrifying bacteria, as well as the effects of T2DM on this relationship. \n<b>Author\u2019s Response (AR):<\/b> We have significantly expanded the Discussion section. We now provide a more detailed analysis of the shift from a nitrate-reducing (beneficial) environment to a periodontopathogen-dominant environment. We specifically discuss how T2DM-induced systemic factors (such as oxidative stress) may contribute to this microbial imbalance, exacerbating periodontal tissue destruction.  CONCLUSION RC\/S: In the conclusion, the author could add the names of the periopathogen and denitrifying bacteria related to the severity of periodontal disease in T2DM patients. \n<b>Author\u2019s Response (AR): <\/b>As suggested, the Conclusion has been revised to be more specific. We now explicitly mention key genera, including Porphyromonas, Fusobacterium, Neisseria, and Rothia, and highlight their shifting interactions as a descriptive hallmark of periodontal disease severity in patients with T2DM.","URL":"https:\/\/f1000research.com\/articles\/14-297\/v4#referee-comment-15218","order":6,"name":"referee-comment-15218","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"The authors declared financial support was received for the research, authorship, and\/or publication of this article. This study was supported by grant provided by Universitas Indonesia\r\n (No: MKB-318\/UN2.RST\/HKP.05.00\/2024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":7,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}