What the salivary microbiome reveals about cardiovascular and neurological diseases

The human oral microbiome is a sophisticated community of microbes, including viruses, bacteria, bacteriophages, and fungi. More than 700 species of oral bacteria have been identified so far, with around 250 of these species living in the average person’s mouth.¹

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Though these microbes have a wide range of ecological roles, they share several common traits. For example, their ability to thrive in a wet, warm environment and evade the antimicrobial mechanisms of saliva.

Closer inspection reveals that the mouth contains multiple niches, including the gingival sulcus, teeth, cheeks, throat, tongue, and tonsils. Microbes living within millimeters of one another can, therefore, possess very different characteristics.

Sampled via saliva, the oral microbiome represents a collection of these heterogeneous niches.

Recent findings in microbiology have revealed candidate phyla radiation (CPR)—a new branch of previously unidentified bacteria estimated to encompass 15 % of microbial diversity.²

CPR bacteria are ultramicroscopic, with reduced genomes that can only exist in a symbiotic relationship on the surface of their bacterial host.

Their inability to live independently makes it difficult to culture these bacteria. Recent estimates suggest that one group, the Saccharibacteria phylum (previously known as TM7), is pervasive within the human oral microbiome, accounting for up to 20 % of the population.

Saccharibacteria exhibit extremely dynamic interactions with their host, along with the ability to kill or parasitize other microbes to acquire molecules they cannot produce themselves. They likely influence the oral microbiome’s ecological balance by altering its overall structure, hierarchy, and functionality.^3,4

Humans are so reliant on the oral microbiome that some digestive functions are outsourced to microbes. For example, humans do not possess the enzyme needed to convert dietary nitrate to nitrite, but microbes in the mouth can transform nitrate from fruits and vegetables into nitrite and, subsequently, nitric oxide,⁵ which is an important regulator of blood pressure.

Multiple studies have also shown that the use of chlorhexidine-containing mouthwash can negatively impact blood pressure by triggering a shift in the oral microbiome.^6,7

Beyond its role in the initiation of digestion, the oral microbiome appears to be key to maintaining both oral and systemic health. This article examines how the composition of the salivary microbiome, as well as expressed genetic material, can serve as an indicator of overall health and various diseases.

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The salivary microbiome in systemic disease

Collecting saliva samples is easy, making the oral microbiome one of the best-studied microbiomes. This has led to the discovery of links between the oral microbiome and a diverse array of diseases, including inflammatory bowel disease, cardiovascular disease, Alzheimer’s disease, rheumatoid arthritis, diabetes, preterm birth, pancreatic cancer, and colon cancer.

Researchers are consistently observing changes in the oral microbiome, with the hope that these patterns can be used to predict disease and track the effectiveness of treatments.⁸

Bacteria can have systemic effects across the body through a series of direct and indirect routes. One direct route involves swallowing bacteria present in saliva. Humans swallow approximately 1.5 liters of saliva daily, containing approximately 10⁸–10¹⁰ microbes.

The number of live microbes reaching the gut is limited by various mechanisms, including the low acidity of the stomach and the presence of bile acids in the duodenum. However, evidence suggests that continual transfer from the oral cavity to the gut does occur.

Oral bacteria can also travel throughout the body via the microvasculature of the gums and tight junctions.⁹ In addition to direct bacterial transfer into the vascular system, bacterial products such as lipopolysaccharide (LPS) are also transported throughout the body.

For example, LPS from P. gingivalis has been found to bind to Toll-like Receptor 4 (TLR4) on host cell membranes, activating the NF-κB signaling pathway and resulting in the production of inflammatory cytokines like Tumor Necrosis Factor α (TNF-α) and Interleukin 6 (IL-6), as well as chemokines like Monocyte Chemoattractant Protein 1 (MCP-1).¹⁰

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Oral microbiome and cardiovascular health

It is estimated that cardiovascular diseases (CDs) are the primary cause of death, claiming the lives of 17.9 million individuals annually. The ability to use the oral microbiome as an early warning signal or a method for monitoring treatment could have a significant positive impact.

A wide range of comprehensive studies has revealed a link between periodontal disease (PD) and cardiovascular disease. One study of more than 1,600 patients found that the risk of a first myocardial infarction was considerably higher in patients with PD.

The large number of patients in this study required that results be rigorously controlled for a range of potentially confounding factors, including smoking and medication.¹¹

The results of these associative studies are supported by more mechanistic research. For example, another study analyzed hospitalized patients for cardiac biomarkers, with metagenomic and metatranscriptomic analysis performed on subgingival plaque and coronary thrombi.

Cardiac trauma, measured by Troponin-I, which was found to increase alongside multiple markers of periodontal disease, including probing pocket depth, inflamed surface area, and clinical attachment loss.

Levels of P. gingivalis (measured using 16S copy numbers) were also found to increase with myocardial injury and coronary artery disease burden. The upregulation of virulence genes was confirmed via gene expression analysis and correlated with increased myocardial injury and disease burden.

The upregulated genes are involved in pathogenic phenotypes. For example, bacterial adhesion is necessary for intracellular and transepithelial invasion (finA of P. gingivalis), for mounting pro-inflammatory responses via TLR (bioF-3 of P. gingivalis), and for degradation of intercellular adhesins, enabling bacterial invasion beyond the epithelial/endothelial barrier and host-defense evasion (prtH and prtP genes of T. forsythia and T. denticola).

Physical evidence of bacterial translocation was also observed, with a correlation noted between bacterial loads detected in coronary thrombi and corresponding subgingival plaque for P. gingivalis and T. forsythia from the same patient.¹²

Mimotope Variation Analyses (MVA)—a new immunoprofiling technique—was used to explore the impact of the oral microbiome on the immune system in the context of CD. This novel technique combines phage display of 12-mer peptide antigens (mimotopes) with NGS to generate quantitative serologic profiles.

MVA was recently used to profile 96 individuals with and without CD, classifying them according to their periodontal health. Peptide antigens captured by the phage display library were sequenced to identify peptide epitopes associated with the antibody immune response in periodontal disease and CAD.

Mapping these 8,088 epitope sequences onto proteomes revealed immunoreactivity to the seven most common periodontal pathogens, highlighting a strong antibody response to P. gingivalis.

It was also possible to identify several dominant shared core epitopes, including a strong response to epitope A with the core pattern P.T.PR, previously mapped to Epstein-Barr Virus (EBV VP26). Subjects in the periodontitis group exhibited high immunoreactivity to this C-terminal epitope.

Multivariable models were also constructed using antibody responses to these epitopes, enabling the stratification of subjects with periodontitis from healthy controls. This was based on their antibody responses to EBV—particularly VP26 and sequence-mimicking bacterial antigens.¹³

Oral microbiome and Alzheimer's disease

There is a growing body of evidence suggesting that periodontal pathogens, and the inflammatory response prompted by their presence, play a role in the development of neurodegenerative diseases such as Alzheimer’s disease (AD).

Mechanisms behind this link were revealed in a recent mouse study, where mice were gavaged with oral microbes from either healthy subjects or subjects with periodontal disease (PD).

Initially, 16S metagenomic sequencing confirmed that the salivary microbiome differed significantly between subjects with PD and healthy individuals. Approximately 90 % of species were identified via ASVs, which were found to be unique in the PD group. Known pathogens such as Treponema, Porphyromonas, and Fusobacterium were enriched.

The study also showed that mice gavaged with PD saliva displayed decreased cognitive function and increased anxiety levels. Levels of Aβ oligomers and pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, were also significantly more abundant in the cortex of mice gavaged with PD saliva compared to those gavaged with healthy saliva.

The study further examined the effects of PD gavage on the gut microbiome and intestinal health. It was noted that levels of fecal lipocalin-2 (LCN2), which promotes intestinal inflammation, were higher in PD-gavaged mice.

RNA sequencing revealed that treatment with periodontitis-related salivary microbiota led to notable changes in immune response-related gene expression in the intestine, along with the upregulation of cytokine genes (including TNF-α and IL-B) in colon tissue.

There was also evidence that the oral microbiome adversely affected intestinal integrity. This was seen in the increased content of fecal albumin and reduced expression of tight junction-related proteins ZO-1 and occludin, which play key roles in maintaining the intestinal barrier.

Staining further revealed that the PD group exhibited damage to the intestinal mucus layer, with a decreased number of goblet cells per crypt. ELISA analysis showed that plasma concentrations of TNF-α and IL-1β (indicators of systemic inflammation) were considerably higher in mice treated with periodontitis-related salivary microbiota than in healthy controls.¹⁴

Together, these results demonstrate a link between PD-associated oral microbes and increased levels of AD- and inflammation-associated biomarkers, which are connected to the gut through elevated intestinal inflammation markers and reduced tight junction integrity.

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The future of saliva as a tool for diagnostic and treatment decisions

Saliva is an abundant biological fluid that is well suited to non-invasive collection. Insights gained from saliva analysis can support the diagnosis of a range of diseases by complementing clinical and histopathological findings.

As sequencing methods continue to improve, new metagenomic and metatranscriptomic data will help reveal microbial trends associated with a growing number of diseases.

As we gain a deeper understanding of the mechanisms behind these diseases, the composition of the oral microbiome could be used for early diagnosis and to monitor treatment progress.

New techniques are also emerging to help rebalance the dysbiotic oral microbiome. These include targeted antimicrobial peptides, prebiotics and postbiotics, compounds that break down EPS, and modulators of the host immune response.

Norgen's saliva microbiome products

Norgen offers a comprehensive saliva workflow, from preservation devices to NGS sequencing services. The company’s range of saliva collection and preservation devices can inactivate microbes and maintain nucleic acid integrity at room temperature.

A wide range of extraction kits is available, including kits for both saliva DNA and saliva RNA in multiple formats. Kits are also available to accommodate high-throughput options, including magnetic beads and 96-well plates.

References and further reading

1. Caselli, E., et al. (2020). Defining the oral microbiome by whole-genome sequencing and resistome analysis: the complexity of the healthy picture. BMC Microbiology, 20(1). https://doi.org/10.1186/s12866-020-01801-y.
2. Brown, C.T., et al. (2015). Unusual biology across a group comprising more than 15% of domain Bacteria. Nature, (online) 523(7559), pp.208–211. https://doi.org/10.1038/nature14486.
3. Tian, J., et al. (2022). Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment. Proceedings of the National Academy of Sciences, 119(2). https://doi.org/10.1073/pnas.2114909119.
4. Bor, B., et al. (2019). Saccharibacteria (TM7) in the Human Oral Microbiome. Journal of Dental Research, 98(5), pp.500–509. https://doi.org/10.1177/0022034519831671.
5. Rosier, B.T., et al. (2020). Nitrate as a potential prebiotic for the oral microbiome. Scientific Reports, (online) 10(1), p.12895. https://doi.org/10.1038/s41598-020-69931-x.
6. Kapil, V., et al. (2013). Physiological role for nitrate-reducing oral bacteria in blood pressure control. Free Radical Biology and Medicine, (online) 55, pp.93–100. https://doi.org/10.1016/j.freeradbiomed.2012.11.013.
7. Cutler, C., et al. (2019). Post-exercise hypotension and skeletal muscle oxygenation is regulated by nitrate-reducing activity of oral bacteria. Free Radical Biology and Medicine, (online) 143, pp.252–259. https://doi.org/10.1016/j.freeradbiomed.2019.07.035.
8. Peng, X., et al. (2022). Oral microbiota in human systematic diseases. International Journal of Oral Science, (online) 14(1), pp.1–11. https://doi.org/10.1038/s41368-022-00163-7.
9. Ljubomir et al. (2023). Breaking the Gingival Barrier in Periodontitis. 24(5), pp.4544–4544. https://doi.org/10.3390/ijms24054544.
10. Nativel, B., et al. (2017). Porphyromonas gingivalis lipopolysaccharides act exclusively through TLR4 with a resilience between mouse and human. Scientific Reports, (online) 7(1). https://doi.org/10.1038/s41598-017-16190-y.
11. Rydén, L., et al. (2016). Periodontitis Increases the Risk of a First Myocardial Infarction: A Report From the PAROKRANK Study. Circulation, 133(6), p.CIRCULATIONAHA.115.020324. https://doi.org/10.1161/circulationaha.115.020324.
12. Joshi, C., A. et al. (2022). Myocardial infarction risk is increased by periodontal pathobionts: a cross-sectional study. Scientific reports, 12(1). https://doi.org/10.1038/s41598-022-19154-z.
13. Mariliis Jaago, et al. (2022). Antibody response to oral biofilm is a biomarker for acute coronary syndrome in periodontal disease. Communications Biology, (online) 5(1). https://doi.org/10.1038/s42003-022-03122-4.
14. Lu, J., et al. (2022). Periodontitis-related salivary microbiota aggravates Alzheimer’s disease via gut-brain axis crosstalk. Gut Microbes, 14(1). https://doi.org/10.1080/19490976.2022.2126272.

Acknowledgments

Produced from materials originally authored by Norgen Biotek Corporation.

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