Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes

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Bachtiar" }, { "@type": "Person", "name": "Dicky L Tahapary" }, { "@type": "Person", "name": "Turmidzi Fath" }, { "@type": "Person", "name": "Citra Fragrantia Theodora" }, { "@type": "Person", "name": "Natalina Haerani" }, { "@type": "Person", "name": "Selvi Nafisa Shahab" }, { "@type": "Person", "name": "Yuniarti Soeroso" }, { "@type": "Person", "name": "Ardy Wildan" }, { "@type": "Person", "name": "Fergie Marie Joe Grizella Runtu" }, { "@type": "Person", "name": "Fatimah Maria Tadjoedin" }, { "@type": "Person", "name": "Dewi Ayuningtyas" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " 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’ periodontal inflammation. This study’s aim was to compare 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 relationship between the number of periodontopathogens and the amount of nitrate-reducing bacteria in their salivary microbiome. Methods Saliva was collected, DNA was isolated, the entire 16S ribosomal RNA gene was amplified, and sample libraries were prepared in accordance to the Oxford Nanopore MinION Technology procedure. The relative abundance and bacterial diversity in saliva samples that were pooled according to three groups; T2DM patients without periodontal disease (G1), T2DM patients with gingivitis (G2), and T2DM patients with periodontitis (G3), was measured using bioinformatic methods. Additionally, the relationships between the periodontopathic bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium spp.) and denitrifying community (Haemophilus, Neisseria, Rothia, and Veillonella) were assessed. Results Alpha-diversity analysis revealed, the G1 group had significantly lower bacterial diversity and abundance than groups G2 and G3 (p< 0.0001). However, the microbiota profiles of diabetic patient groups with periodontitis and gingivitis were comparable. Using receiver operating characteristic (ROC) analysis, potential biomarkers for differentiating between gingivitis and periodontitis were discovered. Areas under the curve (AUC) between Fusobacterium spp. and Neisseria were found to be 0.94 (p = 0.43), while the AUC between P. gingivalis and Rothia was not significant (0.84, p = 0.08). Conclusion People with type 2 diabetes mellitus who also have gingivitis or periodontitis exhibit different relationships between periodontopathic and denitrifying bacteria in their salivary microbiome. These features might be essential indicators for early identification and treatment of gingivitis in order to prevent periodontitis. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-297/v1", "name": "Salivary microbiome and periodontopathogen/denitrifying bacteria associated..." } } ] } Home Browse Salivary microbiome and periodontopathogen/denitrifying bacteria associated... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Bachtiar E, Bachtiar BM, Tahapary DL et al. Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.12688/f1000research.161731.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] Endang Bachtiar https://orcid.org/0000-0002-2512-2579 1 , Boy M. Bachtiar 1 , Dicky L Tahapary 2 , [...] Turmidzi Fath https://orcid.org/0000-0003-2340-6647 1 , Citra Fragrantia Theodora 3 , Natalina Haerani https://orcid.org/0000-0003-0472-4009 4 , Selvi Nafisa Shahab 5 , Yuniarti Soeroso 4 , Ardy Wildan 6 , Fergie Marie Joe Grizella Runtu 4 , Fatimah Maria Tadjoedin 4 , Dewi Ayuningtyas 4 Endang Bachtiar https://orcid.org/0000-0002-2512-2579 1 , Boy M. Bachtiar 1 , [...] Dicky L Tahapary 2 , Turmidzi Fath https://orcid.org/0000-0003-2340-6647 1 , Citra Fragrantia Theodora 3 , Natalina Haerani https://orcid.org/0000-0003-0472-4009 4 , Selvi Nafisa Shahab 5 , Yuniarti Soeroso 4 , Ardy Wildan 6 , Fergie Marie Joe Grizella Runtu 4 , Fatimah Maria Tadjoedin 4 , Dewi Ayuningtyas 4 PUBLISHED 14 Mar 2025 Author details Author details 1 Oral Biology Fac. of Dentistry, University of Indonesia, Depok, West Java, 16424, Indonesia 2 Clinical Research Unit RSCM, . Metabolic-Endocrine-Diabetes Division, Dept. Internal Medicine, Universitas Indonesia, Depok, West Java, Indonesia 3 Faculty of Dentistry, Department of Oral Biology and Oral Science Research Center, Universitas Indonesia, Depok, West Java, 10430, Indonesia 4 Faculty of Dentistry,Department of Periodontology, Universitas Indonesia, Jakarta, Jakarta, 10430, Indonesia 5 Faculty of Medicine, Department Microbiology, Clinical Research Unit RSCM, Universitas Indonesia, Ciptomangunkusumo Hospital., West Java, Indonesia 6 Metabolic-Endocrine-Diabetes Division, Dept. Internal Medicine, Universitas Indonesia, Depok, West Java, Indonesia Endang Bachtiar Roles: Formal Analysis, Funding Acquisition, Resources, Supervision, Writing – Review & Editing Boy M. Bachtiar Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation Dicky L Tahapary Roles: Investigation, Methodology Turmidzi Fath Roles: Investigation, Software, Validation, Visualization Citra Fragrantia Theodora Roles: Project Administration, Supervision, Writing – Review & Editing Natalina Haerani Roles: Project Administration Selvi Nafisa Shahab Roles: Formal Analysis Yuniarti Soeroso Roles: Supervision Ardy Wildan Roles: Supervision Fergie Marie Joe Grizella Runtu Roles: Supervision, Validation Fatimah Maria Tadjoedin Roles: Supervision, Validation Dewi Ayuningtyas Roles: Visualization OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Nanopore Analysis gateway. Abstract 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’ periodontal inflammation. This study’s aim was to compare 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 relationship between the number of periodontopathogens and the amount of nitrate-reducing bacteria in their salivary microbiome. Methods Saliva was collected, DNA was isolated, the entire 16S ribosomal RNA gene was amplified, and sample libraries were prepared in accordance to the Oxford Nanopore MinION Technology procedure. The relative abundance and bacterial diversity in saliva samples that were pooled according to three groups; T2DM patients without periodontal disease (G1), T2DM patients with gingivitis (G2), and T2DM patients with periodontitis (G3), was measured using bioinformatic methods. Additionally, the relationships between the periodontopathic bacteria ( Porphyromonas gingivalis , Treponema denticola , Tannerella forsythia , and Fusobacterium spp.) and denitrifying community ( Haemophilus , Neisseria , Rothia , and Veillonella ) were assessed. Results Alpha-diversity analysis revealed, the G1 group had significantly lower bacterial diversity and abundance than groups G2 and G3 (p< 0.0001). However, the microbiota profiles of diabetic patient groups with periodontitis and gingivitis were comparable. Using receiver operating characteristic (ROC) analysis, potential biomarkers for differentiating between gingivitis and periodontitis were discovered. Areas under the curve (AUC) between Fusobacterium spp. and Neisseria were found to be 0.94 (p = 0.43), while the AUC between P. gingivalis and Rothia was not significant (0.84, p = 0.08). Conclusion People with type 2 diabetes mellitus who also have gingivitis or periodontitis exhibit different relationships between periodontopathic and denitrifying bacteria in their salivary microbiome. These features might be essential indicators for early identification and treatment of gingivitis in order to prevent periodontitis. READ ALL READ LESS Keywords Diabetes; gingivitis; periodontitis; salivary microbiome; nanopore sequencing Corresponding Author(s) Endang Bachtiar ( [email protected] ) Close Corresponding author: Endang Bachtiar Competing interests: No competing interests were disclosed. Grant information: 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 (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. Copyright: © 2025 Bachtiar E et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Bachtiar E, Bachtiar BM, Tahapary DL et al. Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.12688/f1000research.161731.1 ) First published: 14 Mar 2025, 14 :297 ( https://doi.org/10.12688/f1000research.161731.1 ) Latest published: 13 Jan 2026, 14 :297 ( https://doi.org/10.12688/f1000research.161731.4 )  There is a newer version of this article available. Suppress this message for one day. Introduction The development of periodontal disease is known to be significantly influenced by the oral microbiota, which may also be an important variable in systemic disorders including diabetes and heart disease. 1 Additionally, recent studies indicate that those with type 2 diabetes (T2DM) are more likely to develop periodontitis and to have more severe forms of disease. 2 – 4 Even though numerous studies have looked at the bidirectional relationship between diabetes mellitus and periodontal disorders, there are still gaps in the quantity and quality of study on the subject. 5 , 6 The most common types of periodontal disease are gingivitis and periodontitis. In many respects, gingivitis is a state of stable inflammation that represents equilibrium (homeostasis). Hence, gingivitis is mostly reversible. 7 However, the prolonged or recurrent occurrence of the condition, particularly in predisposed individuals, may lead to irreversible destruction of hard and soft tissues (periodontitis). Both gingivitis and periodontitis have been recognized as polymicrobial, biofilm-based inflammatory diseases 8 in which a disruption of the ecological balance between the biofilm and the periodontal tissue homeostasis plays a more important role than specific infections, 9 – 11 alongside other environmental, lifestyle and genetic risk factors. In patients with periodontitis, the number of periodontopathogens ( Porphyromonas gingivalis , Treponema denticola , and Tannerella forsythia ) and Fusobacterium spp. is increasing, while the number of normal flora bacteria is decreasing. 12 – 14 Healthy microbial populations include all genera; Rothia, Neisseria, Actinomyces, Veillonella, Kingella , Propionibacterium , Prevotella, Granulicatella, and Haemophilus. They are all recognized to be nitrate-reducing bacteria (NRB), 15 , 16 and have antimetabolic disease activities. 17 These NRB have attracted much attention currently given their crucial function in the human nitrogen cycle’s nitrate (NO3-) - nitrite (NO2-) - nitric oxide (NO) pathway. 18 , 19 Additionally, blood pressure regulation and insulin resistance are positively impacted by oral nitrate-reducing bacteria. 20 Therefore, the purpose of this study was to evaluate both the composition of the oral microbiota in diabetic individuals with and without periodontal disease, along with the relationship between the periodontopathic and the NRB, with a particular emphasis on Neisseria , Veillonella , Haemophilus , and Rothia. Saliva was chosen as the sample type to accurately capture the oral microbiome’s composition, since it is commonly used to explain the microbial shifts that occur in periodontal sites as they progress from health to disease. 21 – 24 Furthermore, the Oxford Nanopore Technology (ONT) long-read sequencing approach was performed to identify taxa based on whole 16S rRNA sequences, which allows the species-level identification of the representative microbes, including periodontopathogen 25 and oral bacteria associated with nitrate-reducing. 26 Methods Participants and patient characteristic Participants in this cross-sectional study were Indonesian adult patients recruited from the Dr. Cipto Mangunkusumo Hospital in Jakarta between November 2023 and January 2024. Saliva was collected from 171 participants who met the inclusion and exclusion criteria. The first criteria was 20-40 years old with type 2 diabetes (T2DM, non-insulin-dependent diabetes) diagnosed by the internist of the Division of Endocrinology, based on the conditions of blood glucose level 2 hours after oral glucose load ≥ 200 mg/dL, HbA1c ≥ 6.5%, or plasma glucose was ≥ 200 mg/dl with classic hyperglycemic crisis. In addition, all participants fulfilled the following conditions: 1) did not use antibiotics or nonsteroidal anti-inflammatory drugs and did not smoke within three months prior to saliva collection; 2) had at least 20 teeth at the time of sample collection; 3) had no overt symptoms of oral mucosal or root caries; 4) had not undergone periodontal surgery or therapy in the previous six months; 5) did not consume any food for at least one hour prior to sample collection. Patients with following conditions were excluded from the study: 1) diagnosed with a metabolic disorder other than diabetes such as hyperthyroidism or cancer that could have influenced the development of the periodontitis; 2) used certain medication such as hormones within six months prior to the sample collection; 3) were pregnant or breastfeeding. Participants were further divided into three groups: diabetic patients without evidence of periodontal diseases (Group 1, n = 28); diabetic patients with gingivitis (Group 2, n = 54); diabetic patients with periodontitis (Group 3, n = 89). Age (20-40 years old) and sex (35–50% male) were balanced across the three groups. The periodontal evaluations were carried out by competent periodontists. The diagnosis of gingivitis was made by bleeding on probing (BOP) score, 27 while chronic periodontitis was diagnosed based on the standard classification of the American academic of periodontology. 28 Participants diagnosed with chronic periodontitis were identified as having at least 30% of sites with alveolar bone resorption, as well as more than 4 sites with probing depth (PD) ≥ 4 mm and clinical attachment loss (CAL) ≥ 2 mm. In accordance with the criteria of the institutional ethics committee, All participants provided written informed consent before they participated part in the study, and the Dr. Cipto Mangunkusumo Hospital’s Ethics Committee approved the study’s protocols (Ethics Reference Number: KET-1203/UN2.F1/ETIK/PPM.00.02/2023). Saliva sample collection, DNA extraction, and sequencing Participants’ saliva samples (3-5 mL) were collected according to procedure reported elsewhere 29 one hour after they refrained from eating, drinking, or brushing their teeth. After rinsing their mouths, the participants immediately spit 3–5 mL of unstimulated whole saliva into a 25 mL conical tube. Samples were kept at -80°C until DNA extraction. DNA was extracted using the Monarch ® , ™ Genomic DNA purification kit, NEB #T3010S/L (New England Biolabs, Bruningstrasse Frankfurt am Main, Germany) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbag, CA, USA). Following PCR amplification, PCR products from each sample were purified and quantified using the Qubit. 30 The genomic DNA of each study group’s samples was ligated in equimolar quantities to create a pool of three group libraries for nanopore sequencing. Each barcoding kit contained 50 ng of starting DNA. Nanopore amplicon library was prepared using the 16S Barcoding Kit 24 V14 (SQK-RAB204, Oxford Nanopore Technologies, UK) following the manufacturer’s instruction. The kit contains primer 27F and 1492R for amplification of the full-length 16S rRNA gene. Each barcoding kit contained 50 ng of starting DNA. Sequencing was conducted using the MinION (Oxford Nanopore Technologies, UK) with a MinION flow cell (R10.4.1) for 8 hours. 25 Subsequently, the basecalling were generated using MinKNOW (Oxford Nanopore Technologies, UK). For microbiota profiling analysis, we followed the EPI2ME for wf-metagenomic workflow for real time analysis. The analysis results were further generated in the form of a report in the EPI2ME for wf-metagenomic. 25 Measuring the nitrite and nitrate content in saliva Nitrate and nitrite levels in unstimulated saliva samples were quantified using the Griess Reagent System, Promega #TB229, Madison, WI 53711-5399 USA). 20 The procedure involved mixing 50 μL of the supernatant with 50 μL of Griess reagents, allowing the mixture remain at room temperature for 10 minutes in the dark, and then using a spectrophotometer (AccuReader. M965/M965+, Nangang, Taipei, Taiwan), to quantify the mixture at 450 nm. Bioinformatic and statistical analysis For microbiota profiling analysis (OTU, alpha diversity, and rarefaction curve), the EPI2ME for wf-metagenomic was used. To endure data quality, only high-quality reads (“pass”, >5 cumulative reads) were included in the analysis. 31 The operational taxonomic units (OTUs) in each group and the rarefaction curve were developed using EPI2ME, and analysed using RStudio 4.3.3. The software was also used to assess alpha diversity (representing the taxonomic abundance profiles) between groups, which was examined using the Simpson and Shannon indices (representing species evenness and richness). We used the Kruskal-Wallis test to evaluate the groups’ differences in alpha diversity, while statistical difference of the comparing between two groups (G1/G2 and G3) was determine by Student’s t-tests and that among all groups (G2, G3, and G3) by one-way ANOVA test followed by Barlet’s test. The interaction between periodontopathic bacteria and NRB as a predictor of the development of periodontal damage in diabetic subjects was also evaluated for sensitivity and specificity using the receiver operating characteristic (ROC) approach. All statistical analyses were conducted using GraphPad Prism software version 10 for Mac, which is licensed and requires an annual subscription (GraphPad Software, San Diego, CA, USA). A significance level of p< 0.05 was employed. R Studio is an alternative to GraphPad for statistical analysis. Results Read analysis, the rarefaction curve and alpha diversity of each group’s microbiota As shown in Figures 1A,B,C , the majority of the 16S rRNA gene’s V1-V9 hypervariable regions were covered by the 287,215 sequence reads that were produced. Each read in this study was a unidirectional base calling sequence (i.e. it was either forward or backward). A shift in the microbial composition between the groups was shown by rarefaction curves and alpha diversity indices (Shannon and Simpson). There are significant variations in the overall bacterial diversity between G1, G2, and G3. According to the Shannon index, the gingivitis (G2 group) showed a higher relative abundance of bacteria (p< 0.05) than either the periodontitis (G3 group) or oral health (G1 group). However, the Simpson index revealed that the species richness of groups G2 and G3 was comparable. Figure 1. The salivary microbiome's rarefaction curve and alpha diversity among different T2DM patient groups. The total number of sequences is displayed on the horizontal axis, while the number of operational taxonomic units (OTUs) at a 97% intersequence similarity level is shown on the vertical axis of the rarefaction curve (A). The diversity indices of Shannon (B) and Simpson (C) show how alpha diversity varies among the three groups in terms of evenness and richness. Species diversity and evenness seem to be larger in gingivitis group (G2) and periodontitis group (G3) compared to oral health group (G1). Asterisk (*) indicates a statistically significant difference (p-value < 0.05). Bacterial variability comparison between T2DM patient groups Operational taxonomic unit (OTU) clustering was performed and a total of 1123 OTUs were obtained with 97% identity of coverage for each group. As shown by the histogram in Figures 2A , the phylum-level compositions of the G1 and G2 groups are comparable. However, Bacillota ( Firmicutes ) consistently represent the dominant phyla in all groups, although their relative abundance in the G2 and G3 groups appears to be slightly lower than in the G1 group. Next the most prevalent phyla were Pseudomonadota ( Proteobacteria ), although their numbers were lower in the G3 group than in the G1 and G2 groups. Bacteroidota ( Bacteroides ) came next, with almost equal numbers in each group, and the prevalence other phyla was less than 5%. Figure 2. Saliva microbiota taxonomic composition in T2DM patients with and without periodontal diseases. Relative abundance of mayor of salivary bacterial taxa, which relative abundance >5%, are presented at the phylum (A) and genus (B) level. “Others” refers to the remaining phyla. A comparison of the relative abundance of periodontopathic bacterial species (C) and genus of NO 3 - -reducing bacteria (D) between three groups of patient with T2DM. G1 through G3 represent the participant group. Although the phylum-level microbiome is dominated by Firmicutes , the relative abundance of certain genera varies significantly amongst the groups. Streptococcus was the most prevalent taxa in each group. Haemophilus was most prevalent in group G1 (14%), followed by groups G2 and G3 (3% and 1.5%, respectively). Neisseria (22%) and Gemella (24%) were two other prominent genera that were shown to be abundant in the G2 and G3 groups, respectively. Although the three groups’ relative abundances of Porphyromonas seem to vary, group G3 still has higher numbers of these genera than the other two groups ( Figure 2B ). Comparative abundance of salivary periodontopathogens and nitrate-reducing bacteria based on the pooled samples’ 16S rRNA gene sequencing Periodontopathic and nitrate-reducing bacteria in each of the patient groups were the focus of an expanded evaluation of changes in the salivary microbiome. Figure 2C shows that T. forsythia was more prevalent in the saliva of diabetic individuals without periodontal diseases (G1), whereas Fusobacterium spp. were the bacteria that were found in higher proportion in the saliva of all groups under study (G1, G2, and G3) (p<0.005). Although P. gingivalis was present in all groups, the G3 group exhibited a greater amount of this species than the other groups. In terms of the nitrate-reducing bacteria ( Haemophilus , Rothia , Veillonella , and Neisseria ), the G1 group had a considerably higher level of Haemophilus than the G2/G3 group (p< 0.05). Rothia proportions were consistently lower in all groups than Veillonella , whose numbers increased gradually from the G1-G2 and G3 groups. The proportion of Neisseria was significantly lower in the G1 group than in the G2 and G3 groups, where the bacterial counts were comparable ( Figure 2D ). Furthermore, the correlation between specific NRB taxa and periodontopathic bacteria found in the salivary microbiomes of the participants was evaluated using a heat map ( Figure 3 ). It was shown that each group of diabetic patients had their own unique salivary bacterial profiles. The results also demonstrated that there was a significant positive correlation between the abundance and presence of all periodontopathic bacteria and Haemophilus in the group G1, but negative correlations with Actinomyces and Veillonella. In the G2 and G3 groups of periodontal disorders, Neisseria and each periodontopathogen showed a positive association. However, Fusobacteria was the only periodontopathogen species in those groups with a strong and moderate interaction. In contrast, Rothia was found to be negatively correlated with each periodontopathogen. Given the distinct patterns of relationships, we considered to performing phylogenetic analysis for analyzing the genera Neisseria and Rothia. We noticed that, of the two NRB species, the gingivitis (G2) group had more variation than the periodontitis (G3) group ( Figure 4 ). To further contextualize the impacts of the periodontopathogen/NRP relationship, we evaluated their association using a ROC curve, and the findings are illustrated in Figure 5A,B . The correlations that existed between Fusobacterium and Neisseria and between P. gingivalis and Rothia had respective area under the curves (AUCs) of 0.93 (p-value = 0.04) and 0.8 (p-value = 0.08). Figure 3. Phylogenetic variations among nitrate-reducing bacteria between T2DM patients with periodontitis (G3) and gingivitis (G2). Overall phylogenetic diversity of nitrate-reducing bacteria was considerably lower in T2DM individuals with periodontitis than in those with gingivitis. Patients with periodontitis (A and B) or gingivitis (C and D), but not both, have specific Neisseria species (Blue box). The distribution of Rothia species (Red box) also differed among the groupings. Figure 4. The heatmap displays the quantity of periodontopathic and nitrate-reducing bacteria in the groups under study. The study groups and the targeted bacteria were represented by rows and columns, respectively. The relative proportion of the bacterial assignment within each group is represented by the colors in the heatmap. A shift in color toward dark red denotes a higher abundance. Significant correlations after p-value adjustment are market by *p = 0.05, and **p = 0.01. Figure 5. A ROC curve illustrates the relationship between periodontopathic and denitrification bacteria. The interaction between Fusobacteria spp. and Neisseria is a useful indicator for differentiating between gingivitis and periodontitis, as evidenced by the high AUC (Area Under the Curve) of 0.94 and p-value of 0.04 (A). However, while still a reasonable diagnostic marker (AUC 0.8, p = 0.08), the association between P. gingivalis and Rothia is not as strong for a prediction (B). Nitrate-Nitrite measurement in saliva Participants with T2DM accompany with gingivitis or periodontitis (G2 or G3 groups) had significantly lower salivary nitrite concentrations than those without periodontal disease (G1 group) ( Figure 6 ). Figure 6. Salivary nitrite concentration in unstimulated saliva from T2DM subject groups. All participants with gingivitis (G2), periodontitis (G3), and no periodontal disease (G1) had their salivary nitrite levels measured using the Griess reaction merthod. Bars represent mean + SD. An asterisk (*) indicates a statistically significant difference (p-value < 0.05) and ns = not significant. Discussion This study’s main finding is that diabetes mellitus and periodontal diseases together have significantly greater effects on alterations in the composition of salivary microbiota than do diabetes mellitus alone. This suggests that the most important factors modifying the composition of salivary microorganisms are periodontal diseases (gingivitis and periodontitis). Thus, we evaluated which oral bacteria increase the risk of periodontal diseases and how specifically diabetes affects them from the perspective of the oral microbiome. Since both polymicrobial synergy and dysbiosis have a major influence on periodontal disorders, 32 , 33 we intended to better understand any potential relationships between the relative abundance of periodontopathogens and nitrate-reducing bacteria, in diabetics with and without periodontal diseases. First, we found that the 16S rRNA-based MinION technology’s sequencing depth allowed for the identification of 97% of the bacterial population, allowing the ability to identify bacterial cells in pooled saliva samples. Subsequently, these studies’ findings showed that T2DM patients with periodontal diseases had significantly greater alpha diversity in their saliva than those without the disease. The result suggests that the salivary microbiota’s composition significantly shifted from symbiosis to dysbiosis as our subjects’ periodontal health deteriorated. The findings may also imply that the diversity of individual microbial patterns among our diabetes group members may have led to the difference in alpha diversity seen in this study. Additionally, using the Shannon and Simpson indices, we found that T2DM participants with gingivitis (G2 group) and periodontitis (G3 group) had higher species variety than those without periodontal diseases (G1 group). However, people with T2DM and gingivitis may have more low-abundance bacterial species in their saliva, which could explain the higher Shannon index. Although they have little effect on the Simpson’s index, these rare species add to the total diversity measured by the Shannon index. This implies that, despite the fact that gingivitis and periodontitis displayed different clinical symptoms, species abundance rather than species diversity is the primary factor influencing the differences in salivary microbiome between the two groups. Further research is necessary to fully understand the implications of these findings and their potential therapeutic relevance. However, it should be remembered that these diversity shifts may not always be associated with changes in the relative abundances of the microbiome; they may instead be explained by certain ecological conditions that influence the patterns of microbial succession. 34 These results allowed us to distinguish between the two distinct “microbiota states” associated with the G2 and G3 groups of gingivitis and periodontitis, respectively. At the phylum level, Bacillota ( Firmicutes ) are consistently the most prevalent bacteria across all groups, but their relative abundance in the G2 and G3 groups appears to be slightly higher than in the G1 group (those without periodontal disease). Shannon’s diversity index additionally indicated that the changes were most noticeable at the genus level, with Streptococcus being the most prevalent bacteria found in the current study. The results were similar to those published by Shaalan et al. (2022) 35 and Omori et al. (2022). 36 Since the phylum Bacillota , which includes numerous genera, is involved in both periodontal health and diseases, 37 , 38 higher proportions of patients with type 2 diabetes who have periodontal disease may indicate a dysbiosis, in which bacteria from this phylum predominate and exacerbate the inflammatory process of periodontal disease. Additionally, we discovered that the gingivitis (G1 group) and periodontitis G2 group) have a greater proportion of Pseudomonadota. This phyla is the second largest group in the oral microbiome, 39 and oral pathobionts may belong to this phylum. 40 By comparing to Bacteroidota , Pseudomonadota was considerably more common in patients with gingivitis (p< 0.0001) but less prevalent in patients with periodontitis. A similar finding was also reported in China, where younger patients with T2DM had a significant abundance of Proteobacteria (synonym Pseuodomonadota ). 41 Even though our adult diabetic participants may be more susceptible to early inflammation of periodontal disease due to Pseudomonadota -related bacteria, the relative abundance of Actinomycetota did not differ significantly across all groups assessed, which is consistent with previous reports. 42 One possible explanation for the bacterial shifting mentioned above is the decrease in number or extinction of bacterial species related to nitrate reducers. Therefore, we aimed to determine if the observed compositionality of microbiota in each group could be explained by different prevalence rates of nitrate reducer bacteria. We noticed, that several genera, including Porphyromonas , Fusobacterium , Haemophylus , Neisseri a, Rothia , and Veillonela , were found in this study. All of them have the ability to regulate nitrate-nitrite-nitric oxide (NO). 16 , 43 – 45 Among these bacteria, Porphyromonas and Fusobacterium are periodontopathic bacteria, 46 while the remaining bacteria are linked to oral health. 47 , 48 This study found that among T2DM patients with periodontal diseases (G2 and G3 groups), Neisseria and periodontopathic bacteria ( P. gingivalis and Fusobacteria spp.) had a significant positive correlation (p< 0.005), as compared with those without periodontal diseases (G1 group). Furthermore, the existence of Rothia species in both groups raises the possibility that the main differences or functional role of the species may change depending on the severity of periodontal disease and type 2 diabetes. On the other hand, this highlights Neisseria ’s significance as a key element of the oral cavity’s core microbiota, 49 and this studies demonstrated how the nitrate reducer bacteria and periodontopathogen collaborate to boost periodontal inflammation in individuals with type 2 diabetes. 50 Indeed, finding that people with type 2 diabetes who also have periodontal disease have a low amount of Rothia in their saliva indicates that the bacteria could benefit people with periodontal health. 45 , 51 , 52 Therefore, in our diabetic patients, Neisseria spp. and Rothia spp. could be the main oral nitrate-reducing bacteria 51 in responsible for periodontal health and inflammation. Consequently, to simplify the interpretation and presentation of our results, we disregarded the other nitrate-reducing bacteria that did not demonstrate a significant association. As a result, we discovered that T2DM patients with periodontitis had much less phylogenetic diversity of these bacteria than those with gingivitis. We noticed, Neisseria flavescens , N. iguanae , N. macacae , N. oralis and N. zoodegmatis were only found in the pooled metagenomic salivary samples of the gingivitis patient (G2 group), while N. weaveri was detected only in the periodontitis patients (G3). The zoonotic pathogens Neisseria zoodegmatis and N. weaveri have been reported to cause soft tissue infections, 53 , 54 even though their presence in the human salivary microbiome has not been reported. For Rothia , R. aeria was exclusively present in the G2 group, while R. mucilaginosa and R. dentocariosa were discovered in both patient groups (G2 and G3). Indeed, we speculated that the way that bacteria interact with periodontopathogen in T2DM patients may be indicative that gingivitis develops into periodontitis. In this sense, the differences between Neisseria or Rothia association with the specific bacteria that are commonly associated with periodontal disease can be used to predict the progress of periodontal disease in patients with type 2 diabetes. First, we focused on the red complex bacteria ( P. gingivalis , T. denticola , and T. forsythia ), and Fusobacterium spp. which are the most threatening or potential pathogens that contribute to periodontal disease in adults. 55 We noted, that the red complex bacteria were found in the saliva of each participant group, supporting previous findings that periodontal pathogens were present in individuals with both periodontal disease and periodontal health,. 56 , 57 Accordingly, group G3 (diabetic individuals with periodontitis) had the highest abundance of periodontopathogens, especially P. gingivalis. This finding suggests a more severe dysbiosis in this population, where P. gingivalis plays a crucial role. 9 The result is in line with a study that examined the subgingival microbiota of individuals with periodontal disease. 34 However, it should be mentioned that variations in the host’s specific response to the opportunistic infection may have important variable in the disease’s severity. 58 Furthermore, it’s also noteworthy that our diabetes patients have a significantly higher density of bridging periodontopathic bacteria ( Fusobacterium spp.), which have been shown to restrict and prepare the environment for the colonization of pathogenic red complex species that lead to periodontitis. 59 , 60 The high concentration of Fusobacterium DNA found in this study suggests that more periodontopathic bacteria colonized the oral cavity of patients with T2DM. In addition, Fusobacterium spp. are associated to bleeding on probing, 61 a symptom that is more common in our T2DM patients who do not have any periodontal disease (not shown). Subsequently, our finding which include the dendrogram analysis, indicate a higher diversity of Neisseria and Rothia species in gingivitis (G2 group) than in periodontitis (G3 group). Our result suggests that specific species within these genera may be more influential in the initiation and early progression of gingival inflammation, while their roles might shift or diminish in chronic periodontitis. 62 Neisseria may contribute synergistically with periodontopathic bacteria like Fusobacterium to establish the inflammation, whereas Rothia appears to have a unique, potentially protective, association with the inflammatory process. 62 Moreover, it has been reported that the abundance of Neisseria in saliva is associated with periodontal health 63 and the abundance of Rothia decreased in periodontitis. 13 Both bacteria are essential for reducing oral nitrate. 51 , 52 Another study found that while the overall diversity of the oral microbiome declined in mice with diabetes mellitus, Proteobacteria along with Firmicutes numbers increased. 64 The information support our findings that elevated Proteobacteria abundance, with Neisseria being the most prevalent, seemed to be triggered by inflammation of the periodontal tissue in people with type 2 diabetes. However, we recommend more studies to validate these findings. Since Actinomycetota ’s Rothia are known NO - 3 reducers, 65 the lower salivary nitric oxide content indicated by the Griess reaction could be the reason why our T2DM patients with periodontal disease (G2 and G3 groups) had a smaller number of these bacteria than those without periodontal disease (G1 group). Nonetheless, the nitrate concentration of the G2 and G3 groups were comparable. This suggests that the advancement of periodontal disease (gingivitis and periodontitis) may impair the activity of nitrate reducer in either periodontal inflammation conditions. 12 Interestingly, groups G2 and G3 displayed a decrease in salivary nitrate concentration despite a higher number of Neisseria. Hence, this study indicate that there is a complex interaction between different bacterial species and their metabolic activity in the oral microbiome of our T2DM subjects, which is not covered in our study. Indeed, additional study is required to ascertain whether the increased Neisseria proportion in the microbiome of T2DM subjects with gingivitis or periodontitis causes or results from periodontal inflammation. It should be mentioned that the significant variation in bacterial counts between the gingivitis and periodontitis groups in our T2DM participants is not necessarily indicative of a biomarker’s diagnostic value. 66 Hence, the capacity to distinguish between cases of gingivitis and periodontitis in individuals with type 2 diabetes was enhanced by the use of the ROC curve, which showed a cut-off point that established a sensitivity and specificity relationship. According to the ROC analysis, the association between Fusobacteria and Neisseria was a more reliable predictor of gingivitis than the association between P. gingivalis and Rothia for periodontitis prediction. A stronger AUC (0.93 vs. 0.8) and a lower p-value (0.04 vs. 0.08) indicate a more robust and statistically significant association. Literature shows, that periodontopathic bacteria ( P. gingivalis , T. denticola , T. forsythia , and Fusobacterium spp.) are responsible for the development and progress of periodontal disorders. 67 The study’s findings clearly showed that people with type 2 diabetes accompanied with gingivitis or periodontitis have reduced nitrate reduction efficiency, with the exception of Neisseria , which affects the ability of microorganisms to reduce nitrate in saliva. 12 Therefore, the shifting of oral microbial equilibrium, particularly the balance of nitrate-reducing bacteria activities, may be the cause of periodontal inflammation in our diabetic subjects. Furthermore, a previous study 68 demonstrated that the activity of bacteria that reduce nitrate may help minimize the risk of systemic diseases like hypertension and insulin resistance. Our research showed that these bacteria induce gingivitis prior to generate periodontitis. Nevertheless, more study is required to validate this finding. Limitation First, the cross-sectional approach to the study renders it difficult to determine causal relationships. Second, the pooled metagenomes are not representative enough to reveal any significant differences between the groups being studied. 69 Nonetheless, the findings confirm and reinforce the results of the 16S rRNA gene sequencing analysis. Lastly, we did not include smoking habit, which may be a confounder in the study results. However, data are emerging that the oral microbiota, which is associated with periodontal disease, may be strongly correlated with the incidence of type 2 diabetes, even when confounders are excluded. Conclusion The results of the study revealed that the microbial communities in saliva change significantly between the conditions of oral health and periodontal disease, with the progression of periodontal inflammation in diabetics being an important contributor in these variations. Overall, the findings demonstrate that individuals with type 2 diabetes mellitus who also have gingivitis or periodontitis may have unique relationships between periodontopathic and nitrate-reducing bacteria in their salivary microbiome. These characteristic could be crucial microbiological indicators for the early detection and management of gingivitis in order to prevent periodontitis. Author contributions BB: Data curation, Funding acquisition, Writing-review & editing. DT: Validation, Visualization, Review & editing. CT: Resources, Supervision, Review &editing. NH:, Resources, Validation. CT: Project administration, Validation.YS: Data curation, Validation. SS: Validation, Visualization, Review & editing. AW: Data curation, Validation. FR: Resources and Supervision FT: Data curation, Validation, Visualization. DA: Resources and Supervision. EB: Conceptualization, Writing-Original draft. Data availability statement Figshare: Raw data salivary microbiome using 16s barcoding kit 24 V14, ONT (Oxford Nanopore Technology), https://doi.org/10.6084/m9.figshare.28365782.v4 70 This project contains the following underlying data: • FASTQ files in folder of barcode 18, 19, 20 • Subject data in.xlsx format • Figures in PNG and JPG format Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). 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Internal Medicine, Universitas Indonesia, Depok, West Java, Indonesia 3 Faculty of Dentistry, Department of Oral Biology and Oral Science Research Center, Universitas Indonesia, Depok, West Java, 10430, Indonesia 4 Faculty of Dentistry,Department of Periodontology, Universitas Indonesia, Jakarta, Jakarta, 10430, Indonesia 5 Faculty of Medicine, Department Microbiology, Clinical Research Unit RSCM, Universitas Indonesia, Ciptomangunkusumo Hospital., West Java, Indonesia 6 Metabolic-Endocrine-Diabetes Division, Dept. Internal Medicine, Universitas Indonesia, Depok, West Java, Indonesia Endang Bachtiar Roles: Formal Analysis, Funding Acquisition, Resources, Supervision, Writing – Review & Editing Boy M. Bachtiar Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation Dicky L Tahapary Roles: Investigation, Methodology Turmidzi Fath Roles: Investigation, Software, Validation, Visualization Citra Fragrantia Theodora Roles: Project Administration, Supervision, Writing – Review & Editing Natalina Haerani Roles: Project Administration Selvi Nafisa Shahab Roles: Formal Analysis Yuniarti Soeroso Roles: Supervision Ardy Wildan Roles: Supervision Fergie Marie Joe Grizella Runtu Roles: Supervision, Validation Fatimah Maria Tadjoedin Roles: Supervision, Validation Dewi Ayuningtyas Roles: Visualization Competing interests No competing interests were disclosed. Grant information 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 (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. Article Versions (4) version 4 Revised Published: 13 Jan 2026, 14:297 https://doi.org/10.12688/f1000research.161731.4 version 3 Revised Published: 09 Dec 2025, 14:297 https://doi.org/10.12688/f1000research.161731.3 version 2 Revised Published: 06 Oct 2025, 14:297 https://doi.org/10.12688/f1000research.161731.2 version 1 Published: 14 Mar 2025, 14:297 https://doi.org/10.12688/f1000research.161731.1 Copyright © 2025 Bachtiar E et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Bachtiar E, Bachtiar BM, Tahapary DL et al. Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.12688/f1000research.161731.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 14 Mar 2025 Views 0 Cite How to cite this report: Nath S. Reviewer Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406077 ) The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406077 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Sep 2025 Sonia Nath , The University of Adelaide, Adelaide, Australia Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.177804.r406077 Here, the authors report the changes in the salivary microbiome among type 2 diabetes patients, with gingivitis or periodontitis. Introduction: Include a brief evidence of the existing literature, are there other studies that have looked at a similar ... Continue reading READ ALL Here, the authors report the changes in the salivary microbiome among type 2 diabetes patients, with gingivitis or periodontitis. Introduction: 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? How does this study fills the gaps in the literature. Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. How was alveolar bone loss assessed? Did the authors take radiographs? Was sample size calculations considered. There are more number of participants in group 3 than 2 and 1. How many dental examiners were there for the periodontal assessment? If there were more than one periodontist, were they calibrated? Was the score of their consistency. What instrument was used for periodontal assessment? How many site probing was done. 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? Any environmental samples collected along with saliva. "After rinsing the mouth"- what was used to rinse the mouth. How was the DNA extraction carried out? Were there any controls used for the DNA extraction process? Similarly, during PCR, were any controls used? Describe the sequencing briefly. What primers were used? Any reference data set used for aligning the sequences. Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Results: Include a demographic table of the included study participants for each group. Did the authors consider beta diversity analysis? For the realtive 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? Figure 2B shows only 10 genera. What about the others? Were there any statistical tests performed to compare the differences in the phylum and genera? In Figure 3, there is no legend for the blue colour. What does the * in the blue indicate? Figure 3 is hard to follow. What is A and B. Did the authors consider differential abundance analysis, Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Periodontist and microbiolgy researcher I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Nath S. Reviewer Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406077 ) The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406077 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 26 Sep 2025 Endang Bachtiar , Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia 26 Sep 2025 Author Response RC#2: 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 ... Continue reading RC#2: 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 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—especially between Group 1 and the other groups—indicate 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’s#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. 3. In the Discussion section….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’s#2 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? How does this study fills the gaps in the literature. Author’s 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). RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. 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 "unstable." 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? 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. 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—especially between Group 1 and the other groups—indicate 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: 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: 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—especially between Group 1 and the other groups—indicate 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, “the sample was immediately stores in -80oC until DNA extraction”. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this. RC#2: Any environmental samples collected along with saliva. “After rinsing the mouth"- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and we used 0.9% normal saline for about 30 s 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? 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. 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. RC#2: Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Reviewer’s 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? Author’s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading "DNA extraction, sequencing, and saliva sample collection." RC#2 : Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. 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, the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels. Reviewer’s#2 comment and suggestion: -Include a demographic table of the included study participants for each group. -Did the authors consider beta diversity analysis? Author’s response. I agree. We included a table on characteristics as advised. 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. RC#2: 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 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—especially between Group 1 and the other groups—indicate 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’s#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. 3. In the Discussion section….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’s#2 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? How does this study fills the gaps in the literature. Author’s 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). RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. 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 "unstable." 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? 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. 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—especially between Group 1 and the other groups—indicate 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: 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: 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—especially between Group 1 and the other groups—indicate 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, “the sample was immediately stores in -80oC until DNA extraction”. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this. RC#2: Any environmental samples collected along with saliva. “After rinsing the mouth"- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and we used 0.9% normal saline for about 30 s 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? 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. 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. RC#2: Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Reviewer’s 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? Author’s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading "DNA extraction, sequencing, and saliva sample collection." RC#2 : Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. 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, the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels. Reviewer’s#2 comment and suggestion: -Include a demographic table of the included study participants for each group. -Did the authors consider beta diversity analysis? Author’s response. I agree. We included a table on characteristics as advised. 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. Competing Interests: No Competing Interests Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 26 Sep 2025 Endang Bachtiar , Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia 26 Sep 2025 Author Response RC#2: 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 ... Continue reading RC#2: 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 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—especially between Group 1 and the other groups—indicate 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’s#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. 3. In the Discussion section….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’s#2 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? How does this study fills the gaps in the literature. Author’s 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). RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. 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 "unstable." 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? 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. 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—especially between Group 1 and the other groups—indicate 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: 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: 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—especially between Group 1 and the other groups—indicate 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, “the sample was immediately stores in -80oC until DNA extraction”. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this. RC#2: Any environmental samples collected along with saliva. “After rinsing the mouth"- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and we used 0.9% normal saline for about 30 s 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? 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. 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. RC#2: Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Reviewer’s 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? Author’s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading "DNA extraction, sequencing, and saliva sample collection." RC#2 : Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. 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, the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels. Reviewer’s#2 comment and suggestion: -Include a demographic table of the included study participants for each group. -Did the authors consider beta diversity analysis? Author’s response. I agree. We included a table on characteristics as advised. 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. RC#2: 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 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—especially between Group 1 and the other groups—indicate 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’s#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. 3. In the Discussion section….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’s#2 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? How does this study fills the gaps in the literature. Author’s 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). RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. 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 "unstable." 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? 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. 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—especially between Group 1 and the other groups—indicate 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: 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: 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—especially between Group 1 and the other groups—indicate 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, “the sample was immediately stores in -80oC until DNA extraction”. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this. RC#2: Any environmental samples collected along with saliva. “After rinsing the mouth"- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and we used 0.9% normal saline for about 30 s 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? 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. 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. RC#2: Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Reviewer’s 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? Author’s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading "DNA extraction, sequencing, and saliva sample collection." RC#2 : Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. 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, the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels. Reviewer’s#2 comment and suggestion: -Include a demographic table of the included study participants for each group. -Did the authors consider beta diversity analysis? Author’s response. I agree. We included a table on characteristics as advised. 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. Competing Interests: No Competing Interests Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Isola G. Reviewer Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406078 ) The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406078 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 03 Sep 2025 Gaetano Isola , University of Catania, Catania, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.177804.r406078 In the manuscript entitled: “Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes" the author aimed to compare the salivary microbiomes of individuals with type 2 diabetes (20–40 years old) who had gingivitis ... Continue reading READ ALL In the manuscript entitled: “Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes" the author aimed to compare the salivary microbiomes of individuals with type 2 diabetes (20–40 years old) who had gingivitis or periodontitis to those who did not. Additionally, were evaluated the relationship between the number of periodontopathogens and the amount of nitrate-reducing bacteria in their salivary microbiome. The author found that people with type 2 diabetes mellitus who also have gingivitis or periodontitis exhibit different relationships between periodontopathic and denitrifying bacteria in their salivary microbiome. These features might be essential indicators for early identification and treatment of gingivitis in order to prevent periodontitis. Major comments: In general, the idea and innovation of this study regards the analysis of the link between mediators, and periodontitis is interesting and novel because the role these aspects in medicine are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design. The study was well conducted by the authors; However, there are some concerns to revise that are described below. The introduction section resumes the existing knowledge regarding the important factor linked with the relationship between periodontitis and related mediators associated with systemic disease risk. However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the relationship and impact of chlorexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration: 1) Ref 1 2) Ref 2 The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the study. In the central section, should better clarify inclusions and exclusions criteria of the selected sample. Please better state the results obtained in the abstract. The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship regarding the by periodontitis in and risk of oxidative stress evolution linked with inflammation in periodontitis patients. The conclusion should reinforce in light of the discussions. In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for indexing in this journal and requests a revision before a futher re-evaluation of the manuscript. Minor Comments: 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 Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly References 1. Polizzi A, Alibrandi A, Lo Giudice A, Distefano A, et al.: Impact of periodontal microRNAs associated with alveolar bone remodeling during orthodontic tooth movement: a randomized clinical trial. Journal of Translational Medicine . 2024; 22 (1). Publisher Full Text 2. Isola G, Polizzi A, Santagati M, Alibrandi A, et al.: Effect of Nonsurgical Mechanical Debridement With or Without Chlorhexidine Formulations in the Treatment of Peri‐Implant Mucositis. A Randomized Placebo‐Controlled Clinical Trial. Clinical Oral Implants Research . 2025; 36 (5): 566-577 Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Periodontology, Oral medicine I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Isola G. Reviewer Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406078 ) The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406078 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 26 Sep 2025 Endang Bachtiar , Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia 26 Sep 2025 Author Response Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : ... Continue reading Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : Thank you for these important suggestions. Thus, in the revised version, we have formulated both the study’s aim and the result (highlighted in green). RC#1 : ……”introduce and discuss the relationship and impact of chlorhexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration” AR : We thank the reviewer's insightful recommendation. We also agree that including current research on microRNA and chlorhexidine will improve our manuscript by setting our investigation in a more comprehensive scientific framework. Although the microbial associations are the main focus of our work, we have included a few phrases in the Introduction (green highlighted) to recognize their involvement in the intricate interactions between oral and systemic health. RC#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: We agree. this suggestion was also provided the Reviewer#1. Accordingly, we have revised it as suggested. Thank you. Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : Thank you for these important suggestions. Thus, in the revised version, we have formulated both the study’s aim and the result (highlighted in green). RC#1 : ……”introduce and discuss the relationship and impact of chlorhexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration” AR : We thank the reviewer's insightful recommendation. We also agree that including current research on microRNA and chlorhexidine will improve our manuscript by setting our investigation in a more comprehensive scientific framework. Although the microbial associations are the main focus of our work, we have included a few phrases in the Introduction (green highlighted) to recognize their involvement in the intricate interactions between oral and systemic health. RC#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: We agree. this suggestion was also provided the Reviewer#1. Accordingly, we have revised it as suggested. Thank you. Competing Interests: No Competing Interests Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 26 Sep 2025 Endang Bachtiar , Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia 26 Sep 2025 Author Response Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : ... Continue reading Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : Thank you for these important suggestions. Thus, in the revised version, we have formulated both the study’s aim and the result (highlighted in green). RC#1 : ……”introduce and discuss the relationship and impact of chlorhexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration” AR : We thank the reviewer's insightful recommendation. We also agree that including current research on microRNA and chlorhexidine will improve our manuscript by setting our investigation in a more comprehensive scientific framework. Although the microbial associations are the main focus of our work, we have included a few phrases in the Introduction (green highlighted) to recognize their involvement in the intricate interactions between oral and systemic health. RC#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: We agree. this suggestion was also provided the Reviewer#1. Accordingly, we have revised it as suggested. Thank you. Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : Thank you for these important suggestions. Thus, in the revised version, we have formulated both the study’s aim and the result (highlighted in green). RC#1 : ……”introduce and discuss the relationship and impact of chlorhexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration” AR : We thank the reviewer's insightful recommendation. We also agree that including current research on microRNA and chlorhexidine will improve our manuscript by setting our investigation in a more comprehensive scientific framework. Although the microbial associations are the main focus of our work, we have included a few phrases in the Introduction (green highlighted) to recognize their involvement in the intricate interactions between oral and systemic health. RC#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: We agree. this suggestion was also provided the Reviewer#1. Accordingly, we have revised it as suggested. Thank you. Competing Interests: No Competing Interests Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 14 Mar 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 5 Version 4 (revision) 13 Jan 26 read read Version 3 (revision) 09 Dec 25 read read read read Version 2 (revision) 06 Oct 25 read read read Version 1 14 Mar 25 read read Gaetano Isola , University of Catania, Catania, Italy Sonia Nath , The University of Adelaide, Adelaide, Australia Chandrashekar Janakiram , Armita School of Dentistry, Ernakulam, India Ansam Mahdi Khalel , University of Kufa, Najaf, Iraq Susanna Halim , Universitas Prima Indonesia, Medan, Indonesia Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 Jan 2026 | for Version 4 Ansam Mahdi Khalel , University of Kufa, Najaf, Iraq 0 Views copyright © 2026 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have no further comments to make. Competing Interests No competing interests were disclosed. Reviewer Expertise immunology and microbiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Khalel AM. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.194948.r449913) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v4#referee-response-449913 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 15 Jan 2026 | for Version 4 Gaetano Isola , University of Catania, Catania, Italy 0 Views copyright © 2026 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Unfortunately, my previous comments were not still addressed and I am not able to review any advancement compared of the previous version: I suggest that the authors should address my previous comments fully. Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontology, Oral medicine I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Isola G. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.194948.r449917) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v4#referee-response-449917 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Janakiram C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 10 Jan 2026 | for Version 3 Chandrashekar Janakiram , Armita School of Dentistry, Ernakulam, India 0 Views copyright © 2026 Janakiram C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Thank you for your detailed rebuttal to the reviewers’ comments. The responses are appropriate and adequately address the concerns raised, resulting in a clear improvement in the scientific rigour of the manuscript. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Janakiram C. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.192091.r439955) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v3#referee-response-439955 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Halim S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 Dec 2025 | for Version 3 Susanna Halim , Universitas Prima Indonesia, Medan, Indonesia 0 Views copyright © 2026 Halim S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions ABSTRACT It is known that the main cause of periodontitis is Porphyromonas gingivalis, which serves as the primary pathogen causing inflammation and damage to the supporting tissues of the teeth, and is highly dominant in chronic periodontitis. Meanwhile, Fusobacterium nucleatum is a periopathogen that aids in colonizing plaque and triggering inflammation. In the abstract, the author only demonstrates the relationship between the number of periopathogen bacteria and the number of nitrate-reducing bacteria in saliva microbes, with research results indicating that the greatest abundance of periopathogen bacteria is Porphyromonas. Based on ROC (Receiver Operating Characteristic) analysis, only Fusobacterium spp. and Neisseria are strong indicators for distinguishing between gingivitis and periodontitis. In the abstract, the author could provide additional descriptions regarding the interaction between Porphyromonas gingivalis and nitrate-reducing bacteria in relation to the damage to the supporting tissues of the teeth (gingivitis or periodontitis), thus serving as an indicator. INTRODUCTION In the introduction, the author focuses more on clarifying the periopathogen bacteria and nitrate-reducing bacteria, thereby not concentrating on the interaction between periopathogen bacteria and denitrifying bacteria concerning gingivitis or periodontitis in patients with type 2 diabetes mellitus (T2DM). There is a reciprocal relationship between periodontal disease and type 2 diabetes mellitus. The author needs to add several references in the introduction to strengthen the research objective regarding the relationship between periopathogen bacteria and denitrifying bacteria as factors exacerbating periodontal disease in T2DM patients. METHODS In the exclusion criteria, it is necessary to add that samples should not be from one side of the jaw, as chewing on one side can exacerbate the condition of periodontal disease, thus affecting the number of periopathogen and denitrifying bacteria. In the periodontal assessment, it is recommended to perform radiography as a clinical evaluation to support the probing results with the UNC-15 periodontal probe. In the statistical section, the author does not elaborate on the differences among the three groups based on variations between individuals within a single group because the author combines all DNA samples from each group into a single library for sequencing, making statistical tests like ANOVA inappropriate since each group only has one data point on the microbiome profile. RESULTS In the research results, there are inaccuracies, particularly in figure 2C, where Fusobacterium spp. is found in higher proportions compared to other periopathogen bacterial groups, with the highest number in group G2 and the lowest in group G3, whereas it should be lowest in group G1 (without periodontal disease). Porphyromonas gingivalis was found in the highest proportion in group G3 compared to groups G2 and G1. However, Fusobacterium spp. was more numerous than Porphyromonas, whereas Porphyromonas should be more prevalent than Fusobacterium as the main pathogenic bacteria causing inflammation and damage to the supporting tissues of the teeth. Figure 2D shows that the proportion of Neisseria, as nitrate-reducing bacteria, is significantly lower in group G1 (without periodontal disease) compared to G2 (gingivitis) and G3 (periodontitis), while the abstract and introduction state that there is a relationship between periopathogen bacteria and nitrate-reducing bacteria; that is, an increase in periopathogen bacteria will lead to a decrease in denitrifying bacteria. Thus, the number of denitrifying bacteria should be lower in group G3 compared to G2 and G1. The author could discuss this matter in the discussion section to explain why this occurs. DISCUSSION In the discussion section, the author could add explanations regarding the shift in the proportion of periopathogen bacteria relative to denitrifying bacteria, as well as the effects and reciprocal relationship of non-insulin-dependent T2DM on the severity of periodontal disease and changes in the proportions of periopathogen and denitrifying bacteria. CONCLUSION In the conclusion, the author could add the names of the periopathogen and denitrifying bacteria related to the severity of periodontal disease (gingivitis or periodontitis) in T2DM patients, as well as the interaction between periopathogen bacteria and denitrification in gingivitis and periodontitis concerning T2DM. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise biomedical, oral biology, public health, implantology, I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 14 Jan 2026 Endang Bachtiar, Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia Response to Reviewer’s (5) comments ABSTRACT Reviewer’s 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. Author’s Response (AR) : 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. Author’s Response (AR): 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. Author’s Response (AR) : Thank you for these technical insights. 1. Clinical Assessment: We have noted the importance of comprehensive jaw assessment in our protocol description. 2. 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. 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. Author’s Response (AR): We appreciate the reviewer’s 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. Author’s Response (AR): 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. Author’s Response (AR): 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. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Halim S. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.192091.r429427) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v3#referee-response-429427 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 Dec 2025 | for Version 3 Ansam Mahdi Khalel , University of Kufa, Najaf, Iraq 0 Views copyright © 2026 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions No other comment, I approved the article. Competing Interests No competing interests were disclosed. Reviewer Expertise immunology and microbiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Khalel AM. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.192091.r439956) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v3#referee-response-439956 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 27 Dec 2025 | for Version 3 Gaetano Isola , University of Catania, Catania, Italy 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors still not fully addressed the previous comments of the first round of revision Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontology, Oral medicine I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Isola G. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.192091.r439958) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v3#referee-response-439958 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 20 Nov 2025 | for Version 2 Ansam Mahdi Khalel , University of Kufa, Najaf, Iraq 0 Views copyright © 2025 Khalel A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Based on a thorough review of the manuscript, here are comments for each section, focusing on key areas for improvement. Abstract The abstract identifies a potential biomarker (the Fusobacterium-Neisseria relationship) for distinguishing gingivitis from periodontitis, which is a strong finding. However, it currently states that the G3 (periodontitis) group had the "largest abundance of Porphyromonas gingivalis ", but fails to contextualize this by comparing it to the other groups or mentioning other key bacterial shifts, which slightly weakens the summary of the overall microbiome changes observed. Introduction The introduction effectively establishes the bidirectional link between diabetes and periodontal disease. However, the rationale for focusing specifically on the interplay between periodontopathogens and denitrifying bacteria could be strengthened. While denitrifying bacteria are introduced, the text should more explicitly state the central hypothesis: that a disruption in this specific functional group's relationship with pathogens is a key driver of disease progression from gingivitis to periodontitis in the unique metabolic environment of diabetic patients. Materials and Methods The methodology of pooling all DNA samples from each of the three groups into a single library for sequencing is a significant limitation that is not adequately addressed. This approach prevents any statistical analysis of inter-individual variation within groups, making it impossible to determine if the observed differences are truly representative of the groups or are skewed by a few outlier individuals. Consequently, the statistical tests mentioned (like Student's t-tests and ANOVA) seem inappropriate as there is only one data point (n=1) for each group's microbiome profile. Results The presentation of the heatmap in Figure 4 is critically flawed and misleading. The figure legend claims that "A shift in color toward dark red denotes a higher abundance," but the color key provided clearly indicates the scale represents p-values of statistical significance, not bacterial abundance. This misrepresents the data, as a cell could be dark red (highly significant) but represent a very low abundance. The results section should be revised to present this data accurately, for instance by using a heatmap where color intensity reflects relative abundance and using asterisks to denote statistical significance, rather than conflating the two. Discussion The discussion effectively interprets the finding that the Fusobacterium-Neisseria interaction may be a strong predictor for the transition from gingivitis to periodontitis. To enhance this section, the authors should propose a more detailed biological mechanism for this specific interaction. For example, they could discuss how Fusobacterium 's role as a bridging organism might facilitate the colonization of certain Neisseria species that, in the inflammatory diabetic environment, switch from a commensal to a pro-inflammatory role, thereby driving the destructive phase of periodontitis. Conclusion The conclusion concisely summarizes that unique relationships between denitrifying and periodontopathic bacteria exist in diabetic patients with periodontal disease. To be more impactful, it should directly state the study's most novel finding as the primary takeaway: that the specific salivary interaction between Fusobacterium and Neisseria emerges as a promising, non-invasive biomarker for distinguishing between gingivitis and the more severe, irreversible state of periodontitis in individuals with type 2 diabetes. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise immunology and microbiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 09 Dec 2025 Endang Bachtiar, Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia We sincerely appreciate the reviewer's enthusiasm regarding the biomarker potential of this descriptive observation. However, due to the methodological constraint of pooling the genomic DNA samples, the calculated Receiver Operating Characteristic (ROC) curve and the Area Under the Curve (AUC) value were statistically invalid. 1. Abstract/Conclusion: Action Taken: We have removed all ROC analysis from the manuscript. We have revised the Conclusion to state that this specific microbial feature is a "promising, descriptive characteristic that warrants further validation" for distinguishing disease states, tempering the claim to match the study's revised descriptive scope. 2. Heatmap: Action Taken: We have revised the Heatmap ( Figure 4 ) and its caption. The figure now uses color intensity solely to reflect Relative Abundance (the descriptive data). All asterisks and p-values have been completely removed from the figure, and the caption explicitly states that the data is strictly descriptive and exploratory . 3 . Introduction: Action Taken: The final paragraph of the Introduction has been revised to more explicitly state that the central aim is to descriptively characterize the disruption of the oral nitrate-nitrite-NO pathway in the context of T2DM. 3 . Introduction: Action Taken: We have incorporated a discussion proposing a hypothetical mechanism based on descriptive findings, while emphasizing the need for future functional studies to validate this observation. 4. Results: Action Taken: We have completely restructured the Results section to use strictly descriptive language for all microbial findings (Rarefaction Curve (Figure 1A), Diversity (Figure 1B, 1C), Phylogenetic Tree ( Figure 3 ), and Heatmap ( Figure 4 )), while reserving inferential terms only for the clinical and nitrite/nitrate data ( Figure 5 ). View more View less Competing Interests Non-Financial Competing Interests reply Respond Report a concern Khalel AM. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.188726.r426666) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v2#referee-response-426666 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Janakiram C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Nov 2025 | for Version 2 Chandrashekar Janakiram , Armita School of Dentistry, Ernakulam, India 0 Views copyright © 2025 Janakiram C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This cross-sectional study compares pooled salivary microbiomes (ONT MinION whole-16S sequencing) from type-2 diabetes (T2DM) patients grouped by periodontal status (no disease G1, gingivitis G2, periodontitis G3) to explore relationships between classic periodontopathogens and nitrate-reducing/denitrifying bacteria. The authors report higher alpha diversity in diseased groups, greater abundance of P. gingivalis in G3, altered nitrate-reducer profiles (notably Neisseria , Rothia ) and an ROC signal (Fusobacterium–Neisseria AUC ≈ 0.93) distinguishing gingivitis from periodontitis. Data and FASTQ files are made available. Major concerns Pooled libraries per group — The methods state that genomic DNA “of each study group’s samples was ligated in equimolar quantities to create a pool of three group libraries for nanopore sequencing” (i.e., one pooled library per group). If true, no individual-level sequence data exist for sequencing analyses, so all downstream diversity statistics (OTU counts, alpha indices, tests comparing groups, correlation heatmaps, ROC analyses) are not valid as inferential tests across subjects. Group-level pooling destroys inter-individual variance and prevents estimation of within-group dispersion, making p-values meaningless. The manuscript must explicitly state whether samples were pooled and justify this choice — but pooling precludes almost every statistical comparison reported. Clarify and, if pooling occurred, rework the paper to present only descriptive, exploratory group-level summaries (no inferential statistics), or (ideally) reprocess/sequences individual samples. Mismatch between analyses reported and sequencing design / sample unit Related to (1): the authors report OTU counts (n=1123), alpha diversity tests (Kruskal-Wallis, t-tests), heatmaps of correlations, differential abundance claims and ROC analysis with p-values. These require per-sample counts. The methods must: (a) clearly describe library preparation and whether sequencing reads came from individual barcodes or pooled group barcodes; (b) if barcodes represent individuals, provide per-sample read counts and QC thresholds; (c) if truly pooled, remove inferential testing and reframe conclusions as descriptive. Bioinformatic preprocessing and compositional data treatment insufficiently described Important details are missing or ambiguous: the pipeline steps (Dorado basecaller v7.6.8, length filter 200–1500 bp, Kraken2 with ncbi_16s_18s_28s_ITS), thresholds for read inclusion (>5 cumulative reads), normalization / transformation used for relative abundance comparisons, and whether differential abundance tools that account for compositionality (e.g., ANCOM, DESeq2 with appropriate normalization, ALDEx2, Songbird) were used. Microbiome relative-abundance data are compositional and sparse; simple comparisons of raw relative abundances can be misleading. The authors must (a) list exact commands/versions and R packages used; (b) describe normalization, filtering, rarefaction decisions; (c) justify the choice of Kraken2 reference and discuss species-level assignment accuracy with ONT long 16S reads. Statistical methods: inappropriate tests and missing beta diversity Authors used Shannon/Simpson for alpha diversity (fine) but then mix Kruskal-Wallis, t-tests and one-way ANOVA in ways that are unclear and possibly inappropriate given data distribution. Beta-diversity analysis (ordination: PCoA/NMDS on Bray-Curtis/UniFrac) is standard to show community shifts between groups and is missing. Differential abundance testing with multiple-test correction is also absent. explicit beta diversity analysis and clearer delineation of which software did which tests. Study design: selection, grouping and sample size imbalance The three groups are uneven (G1 N=28, G2 N=54, G3 N=89) and authors acknowledge no formal sample-size calculation. The manuscript must discuss potential bias from imbalance, how confounders (age, sex, HbA1c, medication, smoking) were handled, and whether statistical models adjusted for them. Clinical periodontal phenotyping lacks radiographic confirmation and full description Diagnosis relied on clinical CAL/PD and BOP but the manuscript states radiographs were not used. Radiographic bone loss is a standard component of periodontitis staging and its absence weakens case definition; authors must justify omission and discuss how misclassification might affect findings. Also report examiner calibration statistics (kappa or ICC) for PD/CAL measurements Biological interpretation overreaches given cross-sectional design Authors imply mechanistic or predictive roles (e.g., “these features may be crucial markers for early detection”) and use ROC analyses to suggest biomarkers. Given cross-sectional data (and the pooling issue), such claims are premature. Tone down causal/predictive language and limit claims to associations. If ROC analyses are retained, justify them technically (need per-sample data & independent validation). Figures and legends need rework Figure legends misplaced earlier (authors note swapping of Fig 3/4 legends). Heatmap (Fig.4) lacks clear legend for colors, significance marks, and clustering annotations. ROC plots must show sample counts, confidence intervals, and cutoffs. Ensure axis labels, units and p-value reporting are consistent. Nitrite/nitrate measurements — methods and link to sequencing Griess assay is reported, but authors should specify sample handling (timing of collection relative to probing, storage), limits of detection, and whether nitrite levels correlate at the individual level with microbial taxa (again, requires per-sample data). Address whether saliva was collected before probing (probing can cause bleeding and alter analytes). Taxonomic claims at species level require caution Long-read 16S can improve resolution, but Kraken2 on 16S requires careful interpretation; species assignments (e.g., N. zoodegmatis , N. weaveri ) that are normally animal-associated need cautious wording and consideration of misclassification / database contamination. Provide assignment confidence metrics (percent identity, read counts supporting species). Reproducibility / code and data Good that FASTQ and subject data are on Figshare; also provide the full analysis scripts (Nextflow / EPI2ME parameters, R scripts) so reviewers can reproduce analyses. Minor Fix typos, duplicated sentences and figure-legend order (authors note they fixed some previously). Define abbreviations at first use (NRB, OTU, ONT, Q-score). Clarify whether reads are unidirectional (authors say forward or backward) and how that affects assembly/classification. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Epidemilogist and ORal Health and Genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 09 Dec 2025 Endang Bachtiar, Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia We fully agree with this critical assessment. Action Taken: 1. Methods Section: We have explicitly stated that DNA samples were pooled in equimolar quantities and that saliva for nitrite/nitrate measurement was NOT pooled . 2. Analytical Framework: We have revised the entire manuscript to adopt a strictly descriptive and exploratory scope for all sequencing results. 3. ROC Removal: We have removed all ROC analysis from the Abstract, Results, Figures, and Discussion, and the Methods section now explicitly states this omission and the reason. Action Taken: We have removed all p-values, asterisks, and inferential language (e.g., 'significantly different') associated with alpha diversity, OTU counts, and taxonomic comparisons throughout the entire manuscript, including all figure captions and legends for Figures 1, 2, 3, and 4. Action Taken: We have included Table 1: Baseline Demographic and Clinical Characteristics (on individual-level data). The Methods now explicitly state that comparison across groups was performed using ANOVA for Age and Kruskal-Wallis for non-parametric clinical indices (PD, CAL, % BOP) to ensure statistical validity. Action Taken: This clarification is now explicit in the Methods. We confirm that appropriate inferential statistics (ANOVA/Kruskal-Wallis) were correctly applied to this individual data set and are reported with p-values in the Results ( Figure 5 ). View more View less Competing Interests Non-Financial Competing Interest reply Respond Report a concern Janakiram C. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.188726.r429433) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v2#referee-response-429433 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 07 Oct 2025 | for Version 2 Gaetano Isola , University of Catania, Catania, Italy 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Unfortunately, the original bias have not been solved. In this version there are some lacks in study design, methodology and results. Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontology, Oral medicine I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Isola G. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.188726.r420541) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v2#referee-response-420541 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Nath S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Sep 2025 | for Version 1 Sonia Nath , The University of Adelaide, Adelaide, Australia 0 Views copyright © 2025 Nath S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Here, the authors report the changes in the salivary microbiome among type 2 diabetes patients, with gingivitis or periodontitis. Introduction: 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? How does this study fills the gaps in the literature. Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. How was alveolar bone loss assessed? Did the authors take radiographs? Was sample size calculations considered. There are more number of participants in group 3 than 2 and 1. How many dental examiners were there for the periodontal assessment? If there were more than one periodontist, were they calibrated? Was the score of their consistency. What instrument was used for periodontal assessment? How many site probing was done. 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? Any environmental samples collected along with saliva. "After rinsing the mouth"- what was used to rinse the mouth. How was the DNA extraction carried out? Were there any controls used for the DNA extraction process? Similarly, during PCR, were any controls used? Describe the sequencing briefly. What primers were used? Any reference data set used for aligning the sequences. Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Results: Include a demographic table of the included study participants for each group. Did the authors consider beta diversity analysis? For the realtive 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? Figure 2B shows only 10 genera. What about the others? Were there any statistical tests performed to compare the differences in the phylum and genera? In Figure 3, there is no legend for the blue colour. What does the * in the blue indicate? Figure 3 is hard to follow. What is A and B. Did the authors consider differential abundance analysis, Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontist and microbiolgy researcher I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 26 Sep 2025 Endang Bachtiar, Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia RC#2: 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 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—especially between Group 1 and the other groups—indicate 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’s#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. 3. In the Discussion section….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’s#2 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? How does this study fills the gaps in the literature. Author’s 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). RC#2: Methods: In the inclusion criteria, T2DM patients who were under medication, but still had an unstable condition, were included. 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 "unstable." 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? 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. 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—especially between Group 1 and the other groups—indicate 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: 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: 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—especially between Group 1 and the other groups—indicate 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, “the sample was immediately stores in -80oC until DNA extraction”. Thank you. Saliva samples were collected as well before the probing was carried out. In the Method section, we describe this. RC#2: Any environmental samples collected along with saliva. “After rinsing the mouth"- what was used to rinse the mouth. AR: NO, we only collected saliva as oral sample, and we used 0.9% normal saline for about 30 s 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? 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. 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. RC#2: Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. Reviewer’s 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? Author’s response (AR): I appreciate your queries. Indeed, we trimmed and filtered them, and we included them in the procedure part under the subheading "DNA extraction, sequencing, and saliva sample collection." RC#2 : Which packages in R were used for data processing? Mention the names of the packages. Describe clearly for which analysis R Studio was used, and for which analysis GraphPad was used. 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, the Receiver Operating Characteristic (ROC) curves and one-way ANOVA were conducted using GraphPad for the study of salivary nitrite levels. Reviewer’s#2 comment and suggestion: -Include a demographic table of the included study participants for each group. -Did the authors consider beta diversity analysis? Author’s response. I agree. We included a table on characteristics as advised. 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. View more View less Competing Interests No Competing Interests reply Respond Report a concern Nath S. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406077) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406077 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 03 Sep 2025 | for Version 1 Gaetano Isola , University of Catania, Catania, Italy 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the manuscript entitled: “Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes" the author aimed to compare the salivary microbiomes of individuals with type 2 diabetes (20–40 years old) who had gingivitis or periodontitis to those who did not. Additionally, were evaluated the relationship between the number of periodontopathogens and the amount of nitrate-reducing bacteria in their salivary microbiome. The author found that people with type 2 diabetes mellitus who also have gingivitis or periodontitis exhibit different relationships between periodontopathic and denitrifying bacteria in their salivary microbiome. These features might be essential indicators for early identification and treatment of gingivitis in order to prevent periodontitis. Major comments: In general, the idea and innovation of this study regards the analysis of the link between mediators, and periodontitis is interesting and novel because the role these aspects in medicine are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design. The study was well conducted by the authors; However, there are some concerns to revise that are described below. The introduction section resumes the existing knowledge regarding the important factor linked with the relationship between periodontitis and related mediators associated with systemic disease risk. However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the relationship and impact of chlorexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration: 1) Ref 1 2) Ref 2 The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the study. In the central section, should better clarify inclusions and exclusions criteria of the selected sample. Please better state the results obtained in the abstract. The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship regarding the by periodontitis in and risk of oxidative stress evolution linked with inflammation in periodontitis patients. The conclusion should reinforce in light of the discussions. In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for indexing in this journal and requests a revision before a futher re-evaluation of the manuscript. Minor Comments: 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 Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly References 1. Polizzi A, Alibrandi A, Lo Giudice A, Distefano A, et al.: Impact of periodontal microRNAs associated with alveolar bone remodeling during orthodontic tooth movement: a randomized clinical trial. Journal of Translational Medicine . 2024; 22 (1). Publisher Full Text 2. Isola G, Polizzi A, Santagati M, Alibrandi A, et al.: Effect of Nonsurgical Mechanical Debridement With or Without Chlorhexidine Formulations in the Treatment of Peri‐Implant Mucositis. A Randomized Placebo‐Controlled Clinical Trial. Clinical Oral Implants Research . 2025; 36 (5): 566-577 Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontology, Oral medicine I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 26 Sep 2025 Endang Bachtiar, Oral Biology Fac. of Dentistry, University of Indonesia, Depok, 16424, Indonesia Major comment Abstract: Abstract: - Better formulate the abstract section by better describing the aim of the study. - Please better state the results obtained in the abstract. AR : Thank you for these important suggestions. Thus, in the revised version, we have formulated both the study’s aim and the result (highlighted in green). RC#1 : ……”introduce and discuss the relationship and impact of chlorhexidine and micro RNA on periodontal tissues and mediators that could impact diabetes amelioration” AR : We thank the reviewer's insightful recommendation. We also agree that including current research on microRNA and chlorhexidine will improve our manuscript by setting our investigation in a more comprehensive scientific framework. Although the microbial associations are the main focus of our work, we have included a few phrases in the Introduction (green highlighted) to recognize their involvement in the intricate interactions between oral and systemic health. RC#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: We agree. this suggestion was also provided the Reviewer#1. Accordingly, we have revised it as suggested. Thank you. View more View less Competing Interests No Competing Interests reply Respond Report a concern Isola G. Peer Review Report For: Salivary microbiome and periodontopathogen/denitrifying bacteria associated with gingivitis and periodontitis in people with type 2-diabetes [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :297 ( https://doi.org/10.5256/f1000research.177804.r406078) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-297/v1#referee-response-406078 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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