Salivary large extracellular vesicle associated bacterial DNA reflects periodontal disease status and non-surgical periodontal therapy response

Abstract

Periodontitis is associated with microbial dysbiosis and an imbalanced host response. We investigated whether salivary large extracellular vesicles (EVs) associated bacterial DNA can reflect periodontal status and monitor changes after non-surgical periodontal therapy (NSPT). In our longitudinal cohort (18 healthy and 18 Stage Ⅲ/Ⅳ periodontitis), saliva was collected at baseline for all participants and at 3 and 6 months after NSPT for the periodontitis group. Clinical parameters, including plaque index (PI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL), were recorded. Salivary large EVs were isolated, characterized and analyzed by RT-qPCR for bacterial genomic DNA from 11 oral pathogens. At baseline, the periodontitis group showed higher PI, BOP, and mean PPD than the healthy group (P<0.0001). After NSPT, PI, BOP, mean PPD, mean CAL, and percentage of deep pockets were lower at 3 and 6 months versus baseline, with no difference between follow-ups. Large EVs-associated DNA forPorphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Prevotella intermedia, Eikenella corrodens, and Streptococcus mutans was higher in periodontitis than in health (P<0.01). Following NSPT, large EV-DNA levels for P. gingivalis, F. nucleatum, and A. actinomycetemcomitans declined at 3 months (P<0.05 to <0.0001), and P. gingivalis, F. nucleatum, and P. intermedia were reduced at 6 months relative to baseline (P<0.05 to <0.001). These findings indicate that bacterial DNA in salivary large EVs, particularly from P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, can differentiate health from periodontitis and be useful for monitoring the response to NSPT. This suggests that bacterial gDNA in salivary large EVs may be useful as a non-invasive biomarker for monitoring periodontal activity and treatment outcomes, though validation in larger multicenter cohorts with longer follow-up is warranted to enable clinical translation.

Keywords

Saliva / Large extracellular vesicles / Bacterial DNA / Periodontal Disease / Non-Surgical Periodontal Therapy

References

[1] Peres M A, Macpherson L M D, Weyant R J, et al. Oral diseases: a global public health challenge[J/OL]. Lancet, 2019, 394(10194): 249-260. https://pubmed.ncbi.nlm.nih.gov/31327369/. DOI: 10.1016/S0140-6736(19)31146-8.

[2] Hajishengallis G, Chavakis T, Lambris J D . Current understanding of periodontal disease pathogenesis and targets for host-modulation therapy[J/OL]. Periodontol 2000, 2020, 84(1): 14-34. https://pubmed.ncbi.nlm.nih.gov/32844416/. DOI: 10.1111/prd.12331.

[3] Saleh M H A, Dias D R, Kumar P . The economic and societal impact of periodontal and peri-implant diseases[J/OL]. Periodontol 2000, 2025, 98(1): 100-118. https://pubmed.ncbi.nlm.nih.gov/38693603/. DOI: 10.1111/prd.12568.

[4] Curtis M A, Diaz P I, Van Dyke T E . The role of the microbiota in periodontal disease[J/OL]. Periodontol 2000, 2020, 83(1): 14-25. https://pubmed.ncbi.nlm.nih.gov/32385883/. DOI: 10.1111/prd.12296.

[5] Nanci A, Bosshardt D D . Structure of periodontal tissues in health and disease[J/OL]. Periodontol 2000, 2006, 40: 11-28. https://pubmed.ncbi.nlm.nih.gov/16398683/. DOI: 10.1111/j.1600-0757.2005.00141.x.

[6] Wang H, Sun J, Ma G, et al. The role and mechanism of bacterial outer membrane vesicles in the development of periodontitis[J/OL]. Frontiers in Microbiology, 2025, 16: 1654137. https://pubmed.ncbi.nlm.nih.gov/40969433/. DOI: 10.3389/fmicb.2025.1654137.

[7] Wang J, Li X, Zhang Y, et al. Microbial- and host immune cell-derived extracellular vesicles in the pathogenesis and therapy of periodontitis: a narrative review[J/OL]. Journal of Periodontal Research, 2024, 59(6): 1115-1129. https://pubmed.ncbi.nlm.nih.gov/38758729/. DOI: 10.1111/jre.13283.

[8] Hajishengallis G, Darveau R P, Curtis M A . The keystone-pathogen hypothesis[J/OL]. Nature Reviews Microbiology, 2012, 10(10): 717-725. https://pubmed.ncbi.nlm.nih.gov/22941505/. DOI: 10.1038/nrmicro2873.

[9] Sanz M, Herrera D, Kebschull M, et al. Treatment of stage I-Ⅲ periodontitis: The EFP S3 level clinical practice guideline[J/OL]. Journal of Clinical Periodontology, 2020, 47(Suppl 22): 4-60. https://pubmed.ncbi.nlm.nih.gov/32383274/. DOI: 10.1111/jcpe.13290.

[10] Tomasi C, Abrahamsson K H, Apatzidou D . Subgingival instrumentation[J/OL]. Periodontol 2000, 2023; 00: 1-13. https://pubmed.ncbi.nlm.nih.gov/37162295/. DOI: 10.1111/prd.12485.

[11] Bostanci N, Grant M M, Kebschull M . Biological definition of periodontal diseases: a historical review of host-response diagnostics and their implications for disease classification[J/OL]. Periodontol 2000, 2025; 00: 1-13. https://pubmed.ncbi.nlm.nih.gov/40936359/. DOI: 10.1111/prd.70005.

[12] Umapathy V R, Natarajan P M, Swamikannu B . Review insights on salivary proteomics biomarkers in oral cancer detection and diagnosis[J/OL]. Molecules, 2023, 28(13): 5283. https://pubmed.ncbi.nlm.nih.gov/37446943/. DOI: 10.3390/molecules28135283.

[13] Gilbert Klaczko C, Alkhars N, Zeng Y, et al. The oral microbiome and cross-kingdom interactions during pregnancy[J/OL]. Journal of Dental Research, 2023, 102(10): 1122-1130. https://pubmed.ncbi.nlm.nih.gov/37431832/. DOI: 10.1177/00220345231176459.

[14] Corana M, Baima G, Iaderosa G, et al. Salivary proteomics for detecting novel biomarkers of periodontitis: a systematic review[J/OL]. Journal of Periodontal Research, 2025, 60(7): 633-655. https://pubmed.ncbi.nlm.nih.gov/39620241/. DOI: 10.1111/jre.13357.

[15] Han P, Bartold P M, Ivanovski S . The emerging role of small extracellular vesicles in saliva and gingival crevicular fluid as diagnostics for periodontitis[J/OL]. Journal of Periodontal Research, 2022, 57(1): 219-231. https://pubmed.ncbi.nlm.nih.gov/34773636/. DOI: 10.1111/jre.12950.

[16] Cai R, Wang L, Zhang W, et al. The role of extracellular vesicles in periodontitis: pathogenesis, diagnosis, and therapy[J/OL]. Frontiers in Immunology, 2023, 14: 1151322. https://pubmed.ncbi.nlm.nih.gov/37114060/. DOI: 10.3389/fimmu.2023.1151322.

[17] Welsh J A, Goberdhan D C I, O'Driscoll L, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches[J/OL]. Journal of Extracellular Vesicles, 2024, 13(2): e12404. https://pubmed.ncbi.nlm.nih.gov/38326288/. DOI: 10.1002/jev2.12404.

[18] Yu L, Zhu G, Zhang Z, et al. Apoptotic bodies: bioactive treasure left behind by the dying cells with robust diagnostic and therapeutic application potentials[J/OL]. Journal of Nanobiotechnology, 2023, 21(1): 218. https://pubmed.ncbi.nlm.nih.gov/37434199/. DOI: 10.1186/s12951-023-01969-1.

[19] Han P, Lai A, Salomon C, et al. Detection of salivary small extracellular vesicles associated inflammatory cytokines gene methylation in gingivitis[J/OL]. International Journal of Molecular Sciences, 2020, 21(15): 5273. https://pubmed.ncbi.nlm.nih.gov/32722322/. DOI: 10.3390/ijms21155273.

[20] Park J, Kim N E, Yoon H, et al. Fecal microbiota and gut microbe-derived extracellular vesicles in colorectal cancer[J/OL]. Frontiers in Oncology, 2021, 11: 650026. https://pubmed.ncbi.nlm.nih.gov/34595105/. DOI: 10.3389/fonc.2021.650026.

[21] Han P, Bartold P M, Salomon C, et al. Salivary outer membrane vesicles and DNA methylation of small extracellular vesicles as biomarkers for periodontal status: a pilot study[J/OL]. International Journal of Molecular Sciences, 2021, 22(5): 2423. https://pubmed.ncbi.nlm.nih.gov/33670900/. DOI: 10.3390/ijms22052423.

[22] Mizgier M L, Nardocci G, Ramírez V, et al. Proteomic insights into gingival crevicular extracellular vesicles in periodontitis and gestational diabetes: an exploratory study[J/OL]. Journal of Clinical Periodontology, 2025, 52(2): 225-236. https://pubmed.ncbi.nlm.nih.gov/39532703/. DOI: 10.1111/jcpe.14083.

[23] Ye Q, Xu H, Liu S, et al. Apoptotic extracellular vesicles alleviate Pg-LPS-induced inflammatory responses of macrophages via AMPK/SIRT1/NF-κB pathway and inhibit osteoclast formation[J/OL]. Journal of Periodontology, 2022, 93(11): 1738-1751. https://pubmed.ncbi.nlm.nih.gov/35499816/. DOI: 10.1002/JPER.21-0657.

[24] Han P, Jiao K, Moran C S, et al. TNF-α and OSX mRNA of salivary small extracellular vesicles in periodontitis: a pilot study[J/OL]. Tissue Engineering Part C: Methods, 2023, 29(7): 298-306. https://pubmed.ncbi.nlm.nih.gov/37358387/. DOI: 10.1089/ten.tec.2023.0051.

[25] Liaw A, Liu C, Bartold P M, et al. Salivary histone deacetylase in periodontal disease: a cross-sectional pilot study[J/OL]. Journal of Periodontal Research, 2023, 58(2): 433-443. https://pubmed.ncbi.nlm.nih.gov/36717759/. DOI: 10.1111/jre.13104.

[26] Liu C, Seneviratne C J, Palma C, et al. Immunoaffinity-enriched salivary small extracellular vesicles in periodontitis[J/OL]. Extracellular Vesicles and Circulating Nucleic Acids, 2023, 4(4): 698-712. https://pubmed.ncbi.nlm.nih.gov/39697803/. DOI: 10.20517/evcna.2023.48.

[27] Sulugodu Ramachandra S, Woodford V, Han P, et al. Systemic azithromycin as an adjunct to non-surgical subgingival instrumentation in the treatment of stage Ⅲ/Ⅳ, grade C periodontitis: 12-month clinical, microbiological and cytokine results of a randomised controlled trial[J/OL]. Journal of Clinical Periodontology, 2025, 52(5): 666-680. https://pubmed.ncbi.nlm.nih.gov/40123306/. DOI: 10.1111/jcpe.14150.

[28] Vandenbroucke J P, von Elm E, Altman D G, et al. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration[J/OL]. International Journal of Surgery, 2014, 12(12): 1500-1524. https://pubmed.ncbi.nlm.nih.gov/25046751/. DOI: 10.1016/j.ijsu.2014.07.014.

[29] Papapanou P N, Sanz M, Buduneli N, et al. Periodontitis: consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions[J/OL]. Journal of Clinical Periodontology, 2018, 45(Suppl 20): S162-S170. https://pubmed.ncbi.nlm.nih.gov/29926490/. DOI: 10.1111/jcpe.12946.

[30] Santavanond J P, Rutter S F, Atkin-Smith G K, et al. Apoptotic bodies: mechanism of formation, isolation and functional relevance[J/OL]. Subcellular Biochemistry, 2021, 97: 61-88. https://pubmed.ncbi.nlm.nih.gov/33779914/. DOI: 10.1007/978-3-030-67171-6_4.

[31] Kawano T, Okamura K, Shinchi H, et al. Differentiation of large extracellular vesicles in oral fluid: combined protocol of small force centrifugation and sedimentation pattern analysis[J/OL]. Journal of Extracellular Biology, 2024, 3(2): e143. https://pubmed.ncbi.nlm.nih.gov/38939901/. DOI: 10.1002/jex2.143.

[32] Miceli R T, Chen T Y, Nose Y, et al. Extracellular vesicles, RNA sequencing, and bioinformatic analyses: challenges, solutions, and recommendations[J/OL]. Journal of Extracellular Vesicles, 2024, 13(12): e70005. https://pubmed.ncbi.nlm.nih.gov/39625409/. DOI: 10.1002/jev2.70005.

[33] Tsering T, Li M, Chen Y, et al. EV-ADD, a database for EV-associated DNA in human liquid biopsy samples[J/OL]. Journal of Extracellular Vesicles, 2022, 11(10): e12270. https://pubmed.ncbi.nlm.nih.gov/36271888/. DOI: 10.1002/jev2.12270.

[34] Miron R J, Estrin N E, Sculean A, et al. Understanding exosomes: part 3-therapeutic and diagnostic potential in dentistry[J/OL]. Periodontol 2000, 2024, 94(1): 415-482. https://pubmed.ncbi.nlm.nih.gov/38546137/. DOI: 10.1111/prd.12557.

[35] Han P, Bartold P M, Salomon C, et al. Salivary small extracellular vesicles associated miRNAs in periodontal status: A pilot study[J/OL]. International Journal of Molecular Sciences, 2020, 21(8): 2809. https://pubmed.ncbi.nlm.nih.gov/32316600/. DOI: 10.3390/ijms21082809.

[36] Yamaguchi A, Tsuruya Y, Igarashi K, et al. Changes in the components of salivary exosomes due to initial periodontal therapy[J/OL]. Journal of Periodontal and Implant Science, 2023, 53(5): 347-361. https://pubmed.ncbi.nlm.nih.gov/36919005/. DOI: 10.5051/jpis.2203700185.

[37] Huang X, Hu X, Zhao M, et al. Analysis of salivary exosomal proteins in young adults with severe periodontitis[J/OL]. Oral Diseases, 2020, 26(1): 173-181. https://pubmed.ncbi.nlm.nih.gov/31630466/. DOI: 10.1111/odi.13217.

[38] Reseco L, Molina-Crespo A, Atienza M, et al. Characterization of extracellular vesicles from human saliva: effects of age and isolation techniques[J/OL]. Cells, 2024, 13(1): 95. https://pubmed.ncbi.nlm.nih.gov/38201299/. DOI: 10.3390/cells13010095.

[39] Guo W, Ye P, Yu H, et al. CD24 activates the NLRP3 inflammasome through c-Src kinase activity in a model of the lining epithelium of inflamed periodontal tissues[J/OL]. Immunity, Inflammation and Disease, 2014, 2(4): 239-253. https://pubmed.ncbi.nlm.nih.gov/25866631/. DOI: 10.1002/iid3.42.

[40] Zanoni I, Ostuni R, Marek L R, et al. CD14 controls the LPS-induced endocytosis of Toll-like receptor 4[J/OL]. Cell, 2011, 147(4): 868-880. https://pubmed.ncbi.nlm.nih.gov/22078883/. DOI: 10.1016/j.cell.2011.09.051.

[41] Chen Y, Wang H, Ni Q, et al. B-cell-derived TGF-β1 inhibits osteogenesis and contributes to bone loss in periodontitis[J/OL]. Journal of Dental Research, 2023, 102(7): 767-776. https://pubmed.ncbi.nlm.nih.gov/37082865/. DOI: 10.1177/00220345231161005.

[42] Corbella S, Alberti A, Donos N, et al. Efficacy of different protocols of non-surgical periodontal therapy in patients with type 2 diabetes: a systematic review and meta-analysis[J/OL]. Journal of Periodontal Research, 2025, 60(5): 417-437. https://pubmed.ncbi.nlm.nih.gov/39343708/. DOI: 10.1111/jre.13327.

[43] Citterio F, Gualini G, Chang M, et al. Pocket closure and residual pockets after non-surgical periodontal therapy: a systematic review and meta-analysis[J/OL]. Journal of Clinical Periodontology, 2022, 49(1): 2-14. https://pubmed.ncbi.nlm.nih.gov/34517433/. DOI: 10.1111/jcpe.13547.

[44] Chang J, Meng H W, Lalla E, et al. The impact of smoking on non-surgical periodontal therapy: a systematic review and meta-analysis[J/OL]. Journal of Clinical Periodontology, 2021, 48(1): 60-75. https://pubmed.ncbi.nlm.nih.gov/33022758/. DOI: 10.1111/jcpe.13384.

[45] Silbereisen A, Lira-Júnior R, Afacan B, et al. TREM-1 pathway biomarkers for classification of periodontal diseases and monitoring of treatment response in grade B and C periodontitis[J/OL]. Journal of Clinical Periodontology, 2025, 52(9): 1288-1297. https://pubmed.ncbi.nlm.nih.gov/40528650/. DOI: 10.1111/jcpe.14195.

[46] Teles F, Martin L, Patel M, et al. Gingival crevicular fluid biomarkers during periodontitis progression and after periodontal treatment[J/OL]. Journal of Clinical Periodontology, 2025, 52(1): 40-55. https://pubmed.ncbi.nlm.nih.gov/39278722/. DOI: 10.1111/jcpe.14061.

[47] Dilber E, Hagenfeld D, Ehmke B, et al. A systematic review on bacterial community changes after periodontal therapy with and without systemic antibiotics: an analysis with a wider lens[J/OL]. Journal of Periodontal Research, 2020, 55(6): 785-800. https://pubmed.ncbi.nlm.nih.gov/32990996/. DOI: 10.1111/jre.12803.

[48] Krajewski A, Perussolo J, Ercal P, et al. The effect of non-surgical periodontal therapy on subgingival microbiota: a systematic review and meta-analysis[J/OL]. Journal of Periodontal Research, 2025, 60(10): 963-993. https://pubmed.ncbi.nlm.nih.gov/40347039/. DOI: 10.1111/jre.13409.

[49] Darveau R P, Hajishengallis G, Curtis M A . Porphyromonas gingivalis as a potential community activist for disease[J/OL]. Journal of Dental Research, 2012, 91(9): 816-820. https://pubmed.ncbi.nlm.nih.gov/22772362/. DOI: 10.1177/0022034512453589.

[50] Socransky S S, Haffajee A D, Cugini M A, et al. Microbial complexes in subgingival plaque[J/OL]. Journal of Clinical Periodontology, 1998, 25(2): 134-144. https://pubmed.ncbi.nlm.nih.gov/9495612/. DOI: 10.1111/j.1600-051x.1998.tb02419.x.

[51] Belibasakis G N, Maula T, Bao K, et al. Virulence and pathogenicity properties of Aggregatibacter actinomycetemcomitans[J/OL]. Pathogens, 2019, 8(4): 222. https://pubmed.ncbi.nlm.nih.gov/31698835/. DOI: 10.3390/pathogens8040222.

[52] Kalfas S, Pour Z K, Claesson R, et al. Leukotoxin A production and release by JP2 and non-JP2 genotype Aggregatibacter actinomycetemcomitans in relation to culture conditions[J/OL]. Pathogens, 2024, 13(7): 569. https://pubmed.ncbi.nlm.nih.gov/39057796/. DOI: 10.3390/pathogens13070569.

[53] Shenker B J, Korostoff J, Walker L P, et al. Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces cellugyrin (synaptogyrin 2)-dependent cellular senescence in oral keratinocytes[J/OL]. Pathogens, 2024, 13(2): 155. https://pubmed.ncbi.nlm.nih.gov/38392893/. DOI: 10.3390/pathogens13020155.

[54] Kolenbrander P E, Palmer R J Jr, Rickard A H, et al. Bacterial interactions and successions during plaque development[J/OL]. Periodontol 2000, 2006, 42: 47-79. https://pubmed.ncbi.nlm.nih.gov/16930306/. DOI: 10.1111/j.1600-0757.2006.00187.x.

[55] Xu J, Yu L, Ye S, et al. Oral microbiota-host interaction: the chief culprit of alveolar bone resorption[J/OL]. Frontiers in Immunology, 2024, 15: 1254516. https://pubmed.ncbi.nlm.nih.gov/38455060/. DOI: 10.3389/fimmu.2024.1254516.

[56] Hong M, Li Z, Liu H, et al. Fusobacterium nucleatum aggravates rheumatoid arthritis through FadA-containing outer membrane vesicles[J/OL]. Cell Host & Microbe, 2023, 31(5): 798-810.e7. https://pubmed.ncbi.nlm.nih.gov/37054714/. DOI: 10.1016/j.chom.2023.03.018.

[57] Johnston W, Rosier B T, Artacho A, et al. Mechanical biofilm disruption causes microbial and immunological shifts in periodontitis patients[J/OL]. Scientific Reports, 2021, 11(1): 9796. https://pubmed.ncbi.nlm.nih.gov/33963212/. DOI: 10.1038/s41598-021-89002-z.

[58] Nakagawa K, Watanabe K, Mizutani K, et al. Genetic analysis of impaired healing responses after periodontal therapy in type 2 diabetes: clinical and in vivo studies [J/OL]. Journal of Periodontal Research, 2024, 59(4): 712-727. https://pubmed.ncbi.nlm.nih.gov/38501307/. DOI: 10.1111/jre.13249.

[59] Duran-Pinedo A E, Solbiati J, Frias-Lopez J, et al. Subgingival host-microbiome metatranscriptomic changes following scaling and root planing in grade Ⅱ/Ⅲ periodontitis[J/OL]. Journal of Clinical Periodontology, 2023, 50(3): 316-330. https://pubmed.ncbi.nlm.nih.gov/36281629/. DOI: 10.1111/jcpe.13742.

[60] Kim J, Shin H, Park J . RNA in salivary extracellular vesicles as a possible tool for systemic disease diagnosis[J/OL]. Journal of Dental Research, 2017, 96(8): 938-944. https://pubmed.ncbi.nlm.nih.gov/28410004/. DOI: 10.1177/0022034517702100.

[61] Bi R, Li Y, Yang Q, et al. Porphyromonas gingivalis induces an inflammatory response via the cGAS-STING signaling pathway in a periodontitis mouse model[J/OL]. Frontiers in Microbiology, 2023, 14: 1183415. https://pubmed.ncbi.nlm.nih.gov/37405166/. DOI: 10.3389/fmicb.2023.1183415.

[62] Crump K E, Sahingur S E . Microbial nucleic acid sensing in oral and systemic diseases[J/OL]. Journal of Dental Research, 2016, 95(1): 17-25. https://pubmed.ncbi.nlm.nih.gov/26438211/. DOI: 10.1177/0022034515609062.