Periodontitis may induce gut microbiota dysbiosis via salivary microbiota

Periodontitis is a prevalent inflammatory disease linked to many systemic conditions. Prior work suggested that gut microbiota may mediate the impact of periodontitis on systemic diseases, but mechanisms remain unclear. Historically, translocation of periodontal microbes or their components into the bloodstream via ulcerated periodontal pockets has been proposed to drive low-grade systemic inflammation and influence distal sites, including the gut microbiota. Given that humans swallow 1–1.5 L of saliva daily, patients with periodontitis may deliver more pathogenic oral microbes to the gut, potentially disrupting intestinal microbial balance. The authors previously showed that periodontitis salivary microbiota can worsen DSS-induced colitis in mice. This study asks whether swallowing of periodontitis-associated salivary microbiota can independently disrupt healthy gut microbiota composition and intestinal immune homeostasis. The approach combined a clinical correlation study of saliva–faeces relationships with an experimental salivary microbiota transplantation into healthy mice at clinically simulated doses, and a persistence assay tracing salivary microbes in the gut.

The introduction references evidence linking periodontitis to systemic diseases and suggests two major mechanistic routes: (1) periodontal pocket–blood circulation leading to systemic low-grade inflammation and downstream effects on the gut microbiota; and (2) saliva-mediated transfer of oral microbes to the gut through swallowing. Prior studies reported extensive microbial transmission along the gastrointestinal tract and highlighted periodontopathogens (e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) in experimental models influencing gut barrier and microbiota. The authors’ prior work indicated that periodontitis salivary microbiota exacerbated colitis. Literature also indicates enrichment of anaerobic periopathogens (Porphyromonadaceae, Tannerella, Treponema) in periodontitis-associated oral communities, and potential roles for Fusobacterium in intestinal inflammation and as early dysbiosis markers. The study positions itself to test the independence of the saliva–gut pathway from blood-mediated routes by using human salivary microbiota gavage into healthy mice without inducing local periodontal inflammation.

Clinical study: 37 systemically healthy participants were recruited at Nanjing Stomatological Hospital: severe periodontitis (SP, n=21) and periodontally healthy (PH, n=16). Indices (debris, plaque, calculus, gingival) were recorded; participants provided unstimulated saliva and fresh fecal samples (stored at −80°C). 16S rRNA sequencing was performed: human samples targeted V4–V5 (primers 515F/907R), sequenced on Illumina PE250; quality control included length (220–500 bp), Q20 threshold, and chimera removal. OTUs at 97% identity (UPARSE/Usearch). Taxonomy by RDP classifier (80% confidence). Alpha diversity (QIIME), beta diversity (UniFrac PCoA, Adonis), LDA effect size (LEfSe), and SourceTracker (Bayesian) assessed oral–gut microbial source contributions. Saliva preservation used 20% glycerol/PBS at −80°C with viability checks by CFU culture. Animal study: Male C57BL/6J mice (6 weeks old) housed SPF were randomized (n=6/group) to receive oral gavage of pooled human salivary microbiota from PH or SP donors daily for 2 weeks (200 µL/mouse; dose scaled to simulate human 1–1.5 L/day based on body weight; saliva processed by sequential centrifugation to remove debris/exfoliated cells and pellet microbiota; resuspended in PBS). After 2 weeks, mice were euthanized; cecal contents collected for 16S rRNA V3–V4 (primers 341F/806R) sequencing and analysis as above. Intestinal barrier and inflammation: Proximal colon was processed for H&E to measure crypt depth (5 well-oriented crypts/section), and immunofluorescence staining for ZO-1 (anti-ZO-1, DAPI) quantified by ImageJ average optical density. qRT-PCR measured mRNA expression (normalized to GAPDH) of tight junctions (ZO-1, Jam3, Cldn2, Cldn3, Cldn15, occludin) and inflammatory mediators (IL-1β, IL-6, IL-10, TNF-α, Csf1, Cxcl1, PAI-1) using the 2^-ΔΔCt method. Persistence assay: To trace survival and distribution of salivary bacteria, pooled salivary microbiota suspensions from PH and SP groups were stained with CFSE (5 µM, 30 min at 37°C; washed) at ~10^7 CFU/mL (OD600 calibration; OD600≈1 ~ 10^9 CFU/mL). Mice were assigned to Stain-PH-S, Stain-SP-S (CFSE-stained), and corresponding unstained controls (Con-PH-S, Con-SP-S) (n=6/group). After a single 200 µL gavage, half the mice were euthanized at 2 h and half at 24 h. Contents from small intestine, cecum, and colon were homogenized, filtered, pelleted, resuspended, plated into 96-well black plates, and mean fluorescence intensity measured (Ex 496 nm/Em 516 nm). Unstained Con-PH-S served as negative control. Statistics: Data expressed as mean or mean±SD. Group comparisons used t-test or chi-square for demographics; Wilcoxon for alpha diversity; t-test or one-way ANOVA for other measures. Significance at P<0.05.

- Clinical cohort (PH n=16; SP n=21): No significant differences in fecal alpha diversity (Chao1, Shannon), but significant beta diversity differences between PH-F and SP-F (PCoA/Adonis P<0.05; e.g., unweighted UniFrac Adonis P=0.004, R2=0.06). LDA indicated SP-F enrichment of Erysipelotrichaceae, Lachnospiracea_incertae_sedis, Blautia; PH-F enrichment of Formosa and Mitsuokella. - Saliva: SP group had higher Shannon diversity (P<0.05). PCoA showed clear separation of PH-S vs SP-S. LDA showed enrichment in SP-S of Porphyromonadaceae, Tannerella, Treponema; PH-S enriched for Streptococcaceae, Veillonellaceae, Pasteurellaceae. - Oral–gut overlap and source tracking: Shared OTUs between saliva and feces were greater in SP than PH. SourceTracker detected saliva-derived bacteria in 18.75% of PH vs 52.38% of SP individuals; saliva-derived bacteria comprised 0.6% of gut microbiota in PH vs 5.88% in SP, indicating increased oral bacterial influx/retention in SP. - Mouse salivary microbiota transplantation (n=6/group, 2 weeks): Cecal alpha diversity similar (Chao1, Shannon), but beta diversity differed (unweighted UniFrac Adonis P=0.009, R2=0.17; weighted UniFrac Adonis P=0.004, R2=0.275). LDA: Akkermansia enriched in C-PH; Porphyromonadaceae and Fusobacterium enriched in C-SP. - Intestinal barrier and inflammation: C-SP mice had significantly reduced colonic crypt depth and decreased ZO-1 protein (IF). mRNA of tight junction genes (Jam3, ZO-1, Cldn2, Cldn3, Cldn15, occludin) were significantly increased (P<0.05), interpreted as compensatory upregulation. Pro-inflammatory mediators IL-1β, IL-6, and Csf1 were upregulated; anti-inflammatory IL-10 was downregulated (all P<0.05). No significant differences in Cxcl1 and PAI-1. - Persistence of salivary bacteria: Microbial suspension concentration was higher for SP than PH (OD-based: SP 0.707×10^9 CFU/mL vs PH 0.158×10^9 CFU/mL). CFSE staining markedly increased fluorescence versus unstained controls (e.g., mean fluorescence intensity: Stain-PH-S 2217.43±405.05; Stain-SP-S 4195.47±236.66; vs Con-PH-S 975.66±1.16; Con-SP-S 752.67±31.19). At 2 h post-gavage, cecal fluorescence was higher in Stain-SP-S vs controls and Stain-PH-S (P<0.05), indicating greater entry of SP saliva-derived bacteria. At 24 h, stained groups retained significantly higher cecal fluorescence than unstained controls, indicating persistence of salivary bacteria for at least 24 h.

The study demonstrates that severe periodontitis associates with altered gut microbiota composition and increased oral–gut microbial overlap, with more saliva-derived bacteria detectable in feces. The salivary microbiota from SP individuals, when transplanted into healthy mice, independently induced gut microbial dysbiosis with enrichment of taxa implicated in both periodontitis and intestinal inflammation (Porphyromonadaceae, Fusobacterium). This was accompanied by impaired intestinal barrier features (reduced crypt depth, decreased ZO-1 protein) and a shift toward low-grade inflammation (increased IL-1β, IL-6, Csf1; decreased IL-10). The paradox of reduced ZO-1 protein but increased tight junction gene transcripts likely reflects a compensatory transcriptional response to barrier challenge. CFSE tracing confirmed that salivary bacteria from both PH and SP donors can reach and persist in the gut for at least 24 h, with higher early cecal signals from SP-derived samples, supporting a saliva–gut route. Collectively, findings support that the saliva–gut pathway is an independent mechanism by which periodontitis can influence intestinal microbiota and mucosal homeostasis, beyond blood-borne routes. The severity of periodontitis may modulate the magnitude of gut microbiota changes, suggesting dose- or composition-dependent effects of oral microbial influx.

Periodontitis may induce gut microbiota dysbiosis through the influx of salivary microbes. Clinically, patients with severe periodontitis exhibit distinct gut and salivary microbiota and greater oral–gut microbial overlap than healthy controls. Experimentally, transplantation of periodontitis-associated salivary microbiota into healthy mice disrupts gut microbial composition, impairs intestinal barrier integrity, and promotes low-grade inflammation. Salivary bacteria can persist in the intestinal tract for at least 24 h, supporting the plausibility of sustained oral–gut microbial influence. Future research should identify specific oral taxa driving gut dysbiosis, delineate mechanisms of ectopic colonization and host responses, assess the role of disease severity, and employ humanized or germ-free models and larger multicenter clinical cohorts to validate these findings and explore therapeutic interventions (e.g., periodontal treatment) for gut microbiota-associated diseases.

- Human saliva was used to inoculate conventional mice; interspecies differences in oral and gut microbiota may limit translatability. Humanized, germ-free, or oral microbiota-associated models and fecal microbiota transplantation frameworks are suggested for future work. - The clinical study was single-center with a modest sample size; although focusing on severe periodontitis reduced intra-group variability, larger multicenter cohorts are needed to generalize findings and examine effects of disease severity. - The exact oral taxa responsible for inducing gut dysbiosis and barrier alterations were not pinpointed; functional and mechanistic studies are warranted. - While the design aimed to isolate the saliva–gut pathway independent of periodontal pocket–blood circulation effects, systemic factors in donors and complex microbial interactions may still contribute; causative pathways in humans remain to be fully validated.