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Ann Lab Med
Ann Lab Med
Annals of Laboratory Medicine
2234-3806
2234-3814
Korean Society for Laboratory Medicine

10.3343/alm.2024.0043
alm-44-6-600
Letter to the Editor
Clinical Microbiology
Fretibacterium Species to Fusobacterium periodonticum Ratio as a Potential Biomarker of Periodontitis Based on Salivary Microbiome Profiling
https://orcid.org/0009-0003-6790-8451
Oh Eun Ji D.D.S. 1*
https://orcid.org/0009-0003-6790-8451
Jang Hyun Hee M.S. 2*
https://orcid.org/0009-0003-6790-8451
Park Sangwon D.D.S., Ph.D. 1
https://orcid.org/0009-0003-6790-8451
Lim Hyun-Pil D.D.S., Ph.D. 1
https://orcid.org/0009-0003-6790-8451
Yun Kwi-Dug D.D.S., Ph.D. 1
https://orcid.org/0009-0003-6790-8451
Jang Woohyung D.D.S., Ph.D. 1
https://orcid.org/0009-0003-6790-8451
Kim Ok-Su D.D.S., Ph.D. 3
https://orcid.org/0009-0003-6790-8451
Park Chan D.D.S., Ph.D. 1
https://orcid.org/0009-0003-6790-8451
Won Eun Jeong M.D., Ph.D. 4
1 Department of Prosthodontics, Chonnam National University, Gwangju, Korea
2 Biomedical Sciences Graduate Program (BMSGP), Chonnam National University, Hwasun, Korea
3 Department of Periodontology, Chonnam National University, Gwangju, Korea
4 Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Corresponding author: Eun Jeong Won, M.D., Ph.D. Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea E-mail: ejwon@amc.seoul.kr
Co-corresponding author: Chan Park, D.D.S., Ph.D. Department of Prosthodontics, Chonnam National University, 33 Yongbong-ro, Buk-gu, Gwangju 61186, Korea E-mail: upgradepc@hanmail.net
* These authors contributed equally to this study as co-first authors.

1 11 2024
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© Korean Society for Laboratory Medicine
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
National Research Foundation of Korea http://dx.doi.org/10.13039/501100003725 NRF-2022R1C1C1002741 Ministry of Education, Science and Technology http://dx.doi.org/10.13039/501100004085 NRF-2022R1C1C1002741 Chonnam National University Hwasun Hospital http://dx.doi.org/10.13039/501100019780 HCRI22005
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pmcDear Editor,

Periodontitis is an inflammatory disease with a high global prevalence and is characterized by microbially associated host-mediated inflammation that results in lost periodontal attachment [1]. Several cultivable periodontal pathogens are used as markers of chronic periodontitis [2], but a consensus has not been reached regarding the definitive periodontal pathogens. In this study, we assessed the role of the salivary microbiome as a potential biomarker of periodontitis based on comparisons with a periodontitis-resistant control group in the Korean population. We included 13 patients with stage IV periodontitis [3] and 19 periodontitis-free control patients recruited at Chonnam National University Dental Hospital, Gwangju, Korea, from May 2022 to December 2022. We excluded patients taking medications that can affect periodontitis progression within 3 months of the start of this investigation. No statistically significant differences were observed in terms of factors known to influence the oral microbiome between the two groups, such as age, smoking habits, or medication usage (Table 1). This study was carried out in accordance with all the relevant institutional guidelines. Ethical approval was obtained from the Chonnam National University Hospital (CNUDH-2023-015), and written informed consent was obtained from all participants.

Each participant provided an unstimulated saliva sample using the OMNIgene ORAL OM-505 collection device (DNAGenotek, Ontario, Canada), according to a previously reported protocol [4]. Genomic DNA was extracted using a GeneAll Exgene Blood SV Mini Kit from GeneAll (Seoul, Korea) according to the manufacturer’s instructions. The V3–V4 regions of bacterial 16S rRNA genes were amplified and sequenced by Macrogen (Seoul, Korea) using the MiSeq platform. Sequences were trimmed, merged, and then clustered into operational taxonomic units using CLC Genomics Workbench v. 10.1.1 and CLC Microbial Genomics Module v. 2.5 (Qiagen, Hilden, Germany), as previously reported [5]. The Mann–Whitney test and the χ2 test were used to test for phenotypic differences in the microbiomes using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). P<0.05 was considered to reflect a statistically significant difference.

The overall diversity of the salivary microbiome in patients with periodontitis was lower than that of the controls, and the overall composition of the salivary microbiome seemed to differ between both groups. However, this difference was not statistically significant (data not shown). When we compared the bacterial compositions of the salivary microbiomes of both groups at the species level, the periodontitis group showed a predominance of Selenomonas and Rothia species. In contrast, the control group showed a predominance of Prevotella species (Fig. 1A). Linear discriminant analysis revealed several taxa that correlated highly with the periodontitis or control group (Fig. 1B). A volcano plot confirmed that Fretibacterium spp. were associated with patients in the periodontitis group (Fig. 1C). Furthermore, we compared the ratio of Fretibacterium species to Fusobacterium periodonticum (Freti/FPO ratio) between both groups. The periodontitis group showed a significantly higher Freti/FPO ratio than the control group (median, Freti/FPO ratio, 20.16 vs. 0.28, periodontitis group vs. control group, P=0.0054; Fig. 1D). Controversy remains as to whether dysbiosis is related to disease severity of periodontitis [6, 7].

Our findings suggest that dysbiosis is commonly observed in patients with periodontitis, but the degree of dysbiosis could not be quantified absolutely. However, the periodontitis group harbored different salivary microbial community compositions, consistent with previous findings [7]. Among several taxa specifically associated with the periodontitis group in this study, we focused on Fretibacterium species. This focus was supported by previous findings showing significant positive correlations between the levels of Fretibacterium sp. HOT 360 and periodontal parameters [8] and peri-implantitis (F. fastidiosum) [9]. We suggest the detection and quantification of Fretibacterium species as a saliva-based diagnostic bacterial biomarker for periodontitis screening. When considering potential protective markers, we observed several commensal taxa, including Haemophilus species and F. periodonticum [10]. Previously, those taxa were inversely associated with S. mutans, the key pathogen causing dental caries [11]. Hence, we speculate that the abundances of Fretibacterium species and F. periodonticum might be inversely correlated. Finally, we observed the potential of a high Freti/FPO ratio as a biomarker of periodontitis.

Our study has some limitations. The V3–V4 region of the 16S rRNA gene is limited in its ability to resolve fine-level taxonomic differences. As an observational study, causality was not tested. Larger studies of other populations, incorporating shotgun metagenomic sequencing, quantification of absolute microbial loads, and site-specific measures of oral bacterial communities, are warranted. The broader implications of this study may include the potential identification of salivary microbial biomarkers, particularly Fretibacterium species, and the Freti/FPO ratio, for periodontitis screening, offering insights into diagnostic strategies and paving the way for further research on oral microbiome-related interventions.

ACKNOWLEDGEMENTS

We truly appreciate Sung Hoon Kim for assistance in sample collection and preparation.

Fig. 1 Salivary microbiome analysis for patients in the periodontitis and control groups. (A) The periodontitis group showed a predominance of Selenomonas and Rothia species, whereas the control group showed a predominance of Prevotella species. (B) Linear discriminant analysis (LDA) scores for differentially abundant taxa in the salivary microbiome among patients in the periodontitis group (purple) and the control group (blue). The length denotes the effect size for a given taxon. Among the taxa shown, we observed P=0.05 via Kruskal–Wallis testing and an LDA score of ≥4.0. (C) Volcano plot showing several bacterial taxa specifically associated with patients in the periodontitis or control group. (D) Ratio of Fretibacterium species to Fusobacterium periodonticum (Freti/FPO ratio) between the periodontitis and control groups. The periodontitis group showed a significantly higher Freti/FPO ratio than the control group did (median, Freti/FPO ratio, 20.16 vs. 0.28, periodontitis group vs. control group, P=0.0054).

Abbreviations: Ambig, ambiguous; Uncultu, unculturable.

Table 1 Clinical characteristics of participants in the study cohort

Characteristics	Periodontitis group (N=13)	Periodontitis-free control group (N=19)	
Sex (female/male), N	7/6	9/10	
Age, yrs, mean±SD	64.3±8.2	30.9±4.4	
Smoking habit, N of cases			
Non-smoker/ex-smoker/current smoker	12/1/0	17/2/0	
N of missing teeth, mean±SD	16.5±8.2	0	
N of periodontal pockets >4 mm, mean±SD	4.8±2.4	0	
N of periodontal pockets >6 mm, mean±SD	3.1±1.6	0	
Periodontitis classification, N		
Stage I/II/III/IV	0/0/0/13	0/0/0/0	
Underlying disease, N (%)		
Any cancer or tumor type	3 (23.1)	0 (0.0)	
Hypertension	3 (23.1)	0 (0.0)	
Any cardiovascular disease	2 (15.4)	0 (0.0)	
Diabetes mellitus	1 (7.7)	0 (0.0)	

AUTHOR CONTRIBUTIONS

Conceptualization: Won EJ, Park C. Methodology: Jang HH, Oh EJ. Investigation: Jang HH, Oh EJ. Visualization: Jang HH, Won EJ. Funding acquisition: Won EJ, Park C. Project administration: Won EJ, Park C. Supervision: Park S, Lim HP, Yun KD, Jang W, Kim OS. Writing – original draft: Jang HH, Oh EJ, Park C, Won EJ. Writing – review & editing: Jang HH, Oh EJ, Park S, Lim HP, Yun KD, Jang W, Kim OS, Park C, Won WJ.

CONFLICTS OF INTEREST

None declared.

RESEARCH FUNDING

This study was supported by grants from the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Education, Science, and Technology (grant number NRF-2022 R1C1C1002741) and by the Chonnam National University Hwasun Hospital Institute for Biomedical Research (grant number HCRI22005).
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References

1 Kassebaum NJ Bernab E Dahiya M Bhandari B Murray CJL Marcenes W 2014 Global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression J Dent Res 93 1045 53 10.1177/0022034514552491 25261053
2 Riep B Edesi-Neuss L Claessen F Skarabis H Ehmke B Flemmig TF 2009 Are putative periodontal pathogens reliable diagnostic markers? J Clin Microbiol 47 1705 11 10.1128/JCM.01387-08 19386852
3 Tonetti MS Greenwell H Kornman KS 2018 Staging and grading of periodontitis: framework and proposal of a new classification and case definition J Periodontol 89 S1 S159 72 10.1002/JPER.18-0006 29926952
4 Vogtmann E Chen J Kibriya MG Amir A Shi J Chen Y 2019 Comparison of oral collection methods for studies of microbiota Cancer Epidemiol Biomarkers Prev 28 137 43 10.1158/1055-9965.EPI-18-0312 30262598
5 Kim MJ Lee YJ Kim TJ Won EJ 2021 Gut microbiome profiles in colonizations with the enteric protozoa Blastocystis in Korean populations Microorganisms 10 34 10.3390/microorganisms10010034 24d66d738444448eab43407af3112513 35056483
6 Lu C Zhao Q Deng J Chen K Jiang X Ma F 2022 Salivary microbiome profile of diabetes and periodontitis in a Chinese population Front Cell Infect Microbiol 12 933833 10.3389/fcimb.2022.933833 7d36d69fb45d4cea909e87b16e50cc7a 35979090
7 Lu H He L Xu J Song W Feng X Zhao Y 2020 Well-maintained patients with a history of periodontitis still harbor a more dysbiotic microbiome than health J Periodontol 91 1584 94 10.1002/JPER.19-0498 32490546
8 Khemwong T Kobayashi H Ikeda Y Matsuura T Sudo T Kano C 2019 Fretibacterium sp. human oral taxon 360 is a novel biomarker for periodontitis screening in the Japanese population PLoS One 14 e0218266 10.1371/journal.pone.0218266 fa275b977b4844cf99e8db033efb575a 31216300
9 Belibasakis GN Mir-Mari J Sahrmann P Sanz-Martin I Schmidlin PR Jung RE 2016 Clinical association of Spirochaetes and Synergistetes with peri-implantitis Clin Oral Implants Res 27 656 61 10.1111/clr.12690 26354174
10 Kim M Yun SY Lee Y Lee H Yong D Lee K 2022 Clinical differences in patients infected with Fusobacterium and antimicrobial susceptibility of Fusobacterium isolates recovered at a tertiary-care hospital in Korea Ann Lab Med 42 2 188 95 10.3343/alm.2022.42.2.188 34635612
11 Blostein F Bhaumik D Davis E Salzman E Shedden K Duhaime M 2022 Evaluating the ecological hypothesis: early life salivary microbiome assembly predicts dental caries in a longitudinal case-control study Microbiome 10 240 10.1186/s40168-022-01442-5 d6e2ae20f4cf43dfafce4805f2d4f828 36567334
