Association Between Oral Microbiota and Gastrointestinal/Extra-Gastrointestinal Diseases
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Abstract
Dental clinicians and researchers have recently recommended oral microbial examinations to more accurately diagnose and treat oral diseases, including periodontitis and dental caries. Theoretical and experimental evidence suggests that oral microbiota may also be associated with non-oral diseases, such as gastrointestinal and extra-gastrointestinal diseases. This review highlights studies demonstrating microbial alterations in the oral cavity associated with malignant tumors including gastric, colorectal, esophageal, and lung cancers, implying that these alterations may serve as early indicators for non-invasive diagnosis and risk assessment of cancer development. Furthermore, we addressed the implications of oral microbial co-occurrence with malignant tumors, such as Streptococcus anginosus, Fusobacterium nucleatum, and Veillonella parvula, which are recognized as tumor-enriched oral pathogens involved in the development and progression of cancers in the stomach, colon, and lungs, respectively. Notably, we explored the immune and inflammatory mechanisms underlying reciprocal interactions between oral microbiota and tumors, underscoring that targeting these mechanistic pathways can contribute to preventing cancer development.
INTRODUCTION
The oral cavity, the second-largest microbial habitat in the human body, is colonized by diverse microorganisms that inhabit the buccal mucosa, dental pellicle, tongue, gingiva, and saliv [1]. The microbiota in the oral cavity of newborns is predominantly composed of maternal vaginal or skin microbes, which are immediately replaced by oral microbiota that are common in all infants and adults [2]. The microbial distribution in the oral cavity is easily influenced by various factors, such as food consumption [3], smoking [4], nationality [5], and underlying diseases [6]. Importantly, the chronic dominance of pathogenic bacteria in the oral cavity, referred to as oral dysbiosis, has been widely recognized to contribute to the progression of various oral diseases, including periodontitis [7-16], dental caries [17], and oral cancer [18]. Furthermore, recent studies highlighted the association between oral microbiota and systemic, non-oral diseases mainly due to the microbial translocation to other organs, either by passing through the esophagus via swallowing [19] or entering the bloodstream via leaky gums [6]. This review aimed to explore recent research investigating the potential roles of the oral microbiota in both gastrointestinal and extra-gastrointestinal diseases.
ORAL MICROBIOTA AND GASTROINTESTINAL DISEASES
Oral microbial alterations associated with gastrointestinal diseases
Advances in metagenome sequencing technologies have enhanced our understanding of the strong association between oral microbial alterations and gastrointestinal diseases, including gastric and colorectal cancers (Table 1). Sun et al. [20] analyzed saliva or plaque samples from 37 gastric cancer patients and 13 healthy controls and reported considerable differential abundances in oral microbial compositions between the two groups. Specifically, compared with healthy controls, gastric cancer patients exhibited that relative abundance of Veillonella, Prevotella, Aggregatibacter, and Megasphaera increased, whereas Leptotrichia, Rothia, Capnocytophaga, Campylobacter, Tannerella, and Granulicatella decreased. Russo et al. [21] analyzed the saliva samples from 10 patients with colorectal cancer and 10 healthy controls using similar sequencing techniques and revealed an upregulation of Actinobacteria, Saccharibacteria, Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. These findings imply that oral microbial alterations may serve as non-invasive biomarkers for the early detection and prevention of gastrointestinal diseases. However, the precise mechanisms linking these oral microbial alterations and gastrointestinal diseases remains unclear.
Gastrointestinal disease-associated oral microbial alterations
Enrichment of oral microbiota in gastrointestinal cancer tissues
Gastrointestinal cancer tissues are highly colonized by oral microbiota [22-24], suggesting a reciprocal influence between these microbes and gastrointestinal cancer development via direct interaction (Table 2). To identify specific oral microbiota enriched in gastric cancer sites, gastric mucosal samples were collected from patients according to disease stages (20 gastric cancer, 17 intestinal metaplasia, 23 atrophic gastritis, and 21 superficial gastritis) to analyze the distribution of oral microbiota. The results revealed that the relative abundance of Streptococcus anginosus, Parvimonas micra, Dialister pneumosintes, Slackia exigua, Fusobacterium nucleatum, Prevotella intermedia, Catonella morbi, and Peptostreptococcus stomatis was significantly higher in samples from gastric cancer than in samples from all other stages [24], all of which belonged to the Human Oral Microbiome Database (HOMD, https://homd.org), developed through the Human Microbiome Project (Table 2). Similar findings have been observed in colorectal cancer tissues, which were also colonized by oral microbiota. Tran et al. [23] compared the microbial compositions of colorectal malignant tumors and non-malignant polyps and found that the compositional percentages of oral microbiota, including Gemella, Peptostreptococcus, F. nucleatum, Leptotrichia, Selenomonas sputigena, and Campylobacter rectus, were significantly higher in malignant tumors than in non-malignant polyps. Additionally, Loftus et al. [22] identified that oral microbiota, including F. nucleatum, P. stomatis, Gemella morbillorum, and P. micra, were differentially abundant in fecal samples from 74 colorectal cancer patients than in 178 healthy controls.
The enrichment of oral microbiota in gastrointestinal cancers
Interaction and relationship between oral microbiota and gastrointestinal microbiota
You et al. [25] elucidated the interaction between oral microbiota and gastrointestinal tumoral microbiota through simultaneous 16S rRNA metagenomic analysis of saliva and gastric juice samples from 141 patients with different gastric cancer stages (58 low-grade dysplasia, 33 high-grade dysplasia, and 41 gastric cancer) and 9 healthy controls. As shown in Table 3, several oral-origin bacteria were significantly enriched in the gastric juice samples of the disease groups than in the healthy controls. Notably, Gemella haemolysans was significantly enriched in both the oral cavity and stomach of the disease groups than in the control group, suggesting that the presence of G. haemolysans in the oral cavity could serve as a non-invasive marker for predicting gastric diseases. A key finding was the increased similarity in microbial compositions between the oral cavity and stomach with disease progression, indicating a potential role for oral-gastric bacterial interactions in gastric cancer development.
Salivary and gastric microbiota in gastric disease patients and healthy controls
In addition to gastric cancer, the interactions between oral and stomach microbiota contribute to early-stage intramucosal esophageal squamous carcinoma (EIESC) [26]. Chen et al. [26] compared the 16S rRNA metagenome results of both saliva and gastric antrum tissues from 31 patients with EIESC with those obtained from 21 healthy controls and revealed a significant enrichment of Streptococcus in the gastric samples of the EIESC group than in the control group, whereas the salivary levels of Streptococcus did not differ between the two groups. Notably, Porphyromonas endodontalis was identified as a potential biomarker for differentiating EIESC patients from healthy controls. The higher abundance of P. endodontalis in the saliva samples of EIESC patients could aid in early diagnosis, although the gastric P. endodontalis did not differ between the two groups. These findings highlight the importance of the combined analysis of both oral and gastric microbiota for improving diagnosis and potential treatment strategies for EIESC.
Modulating effects of oral pathogens on gastrointestinal tumorigenesis
Role of S. anginosus in gastric cancer pathogenesis
Although Helicobacter pylori is a major risk factor for gastric adenocarcinoma [27], the involvement of non-H. pylori pathogens in gastric tumorigenesis has been consistently suggested because of a weak association between the gastric abundance of H. pylori and gastric cancer development [28]. Alternatively, stomach-colonizing oral pathogens have recently emerged as modulating factors of gastric tumorigenesis. Among these, five oral microbial genera, including Streptococcus, Prevotella, Haemophilus, Veillonella, and Neisseria, the abundances of which were ranked high in the HOMD, accounted for a considerable proportion of the gastric microbiota. Collectively, these genera represent 17.6% and 11.6% of the gastric microbiome in Mexicans and Chinese populations, respectively [29].
Recently, Lei et al. [30] highlighted the spatially distinct correlation of H. pylori and stomach-colonizing oral pathogens with gastric cancer development. A total of 223 gastric tumoral tissues and their matched non-tumoral tissue samples were collected from three different sites of gastric tumors, including 76, 33, and 114 samples from the upper, middle, and lower third, respectively. Subsequent 16S rRNA metagenome sequencing revealed that the relative abundance of H. pylori was significantly higher in the lower-third of tumor tissues, whereas oral-origin bacteria, including Veillonella parvula, Streptococcus oralis, S. anginosus, and P. intermedia, were significantly enriched in the upper-third sites of tumor tissues. Therefore, identifying the specific oral-origin pathogens contributing to the development of gastric tumorigenesis could lead to a breakthrough in the diagnosis and intervention of H. pylori-independent gastric cancer.
Notably, Fu et al. [31] investigated the role of S. anginosus in the onset and development of gastric tumorigenesis, using an N-methyl-N-nitrosourea-induced gastric cancer mouse model, divided into three experimental groups: S. anginosus-infected, H. pylori-infected, and non-infected groups. The results indicated a higher tumor incidence rate in the S. anginosus-infected group (92%) than in the other two groups (67% and 31% in H. pylori- and non-infected groups, respectively) [31], underscoring the causative role of S. anginosus in promoting gastric tumorigenesis. Further investigation revealed that the downstream pathway underlying S. anginosus-mediated gastric tumorigenesis involves activation of the mitogen-activated protein kinase signaling, followed by S. anginosus colonization of the gastric mucosa via the interaction between S. anginosus surface protein Treponema pallidum membrane protein C (TMPC) [32] and gastric epithelial receptor Annexin 2 [31]. These findings suggest that targeting TMPC or Annexin 2 could be a therapeutic strategy for mitigating gastric cancer development.
Role of F. nucleatum in colorectal cancer pathogenesis
F. nucleatum overabundance was observed in both gastric and colorectal cancer tissues (Table 2), indicating that gastrointestinal tract-colonizing F. nucleatum is closely associated with gastrointestinal tumorigenesis. Yamamura et al. [33] conducted an interesting study to determine whether F. nucleatum is also distributed in extra-gastrointestinal cancer tissues, using various cancer tissues collected from patients with esophageal, gastric, colorectal, pancreatic, and liver cancers. The results demonstrated that F. nucleatum was exclusively colonized in the gastrointestinal tumor tissues (esophageal, gastric, and colorectal cancer) and not extra-gastrointestinal tumor tissues (pancreatic and liver cancer), highlighting its specific association with gastrointestinal cancers.
Given the increased abundance of F. nucleatum in gastrointestinal cancer tumors, its causative role in the onset and progression of gastrointestinal cancer has been extensively investigated. For instance, experiments were conducted using ApcMin/+ mice, a specific animal model useful for colonic tumorigenesis [34], in which the mice were fed F. nucleatum or Streptococcus to analyze the number of colonic tumors and inflammatory responses using dissected colon tissues [35]. Compared with Streptococcus-fed ApcMin/+ mice, F. nucleatum-fed ApcMin/+ mice exhibited an increased number of colonic tumor tissues and upregulated expression of proinflammatory genes in colonic tumor tissues. Furthermore, F. nucleatum was enriched in colonic tumor tissues but not in the adjacent normal tissues of F. nucleatum-fed ApcMin/+ mice. These results suggest that therapeutic interventions targeting F. nucleatum can be used to prevent or treat colonic cancer.
To delineate the molecular mechanism underlying F. nucleatum-mediated colorectal tumorigenesis, Brennan et al. [36] focused on pro-inflammatory gene expressions in ApcMin/+ mice neonatally inoculated with F. nucleatum, which revealed significant upregulation of interleukin-17 (Il-17) expression in both the colonic epithelium and colonic lamina propria. The immune-modulating roles of F. nucleatum were further examined ex vivo by Kim et al. [37] using tumor-infiltrating lymphocytes to elucidate the role of F. nucleatum. Lymphocytes isolated from tumors were infected with F. nucleatum, resulting in higher FoxP3+ regulatory T cells and lower CD3+, CD8+, and CD45RO+ T lymphocytes, which are associated with poor prognosis [38] and low survival rates [39].
In particular, compared with healthy individuals, cancer patients are more susceptible to F. nucleatum infection due to the anti-tumor immune evasion, which is accompanied by the upregulation of immune checkpoint inhibitory receptors, including programmed cell death-1 (PD-1) and T cell immunoglobulin and ITIM domains (TIGIT). The enhanced interaction between the PD-1 receptor of T lymphocytes and programmed death-ligand 1 in colorectal tumors suppresses the efficacy of cancer immunotherapy [40]. Additionally, the binding of the TIGIT receptor on T cells to the Fap2 adhesin protein of F. nucleatum inactivates T cells, allowing cancer cells to evade the immune system [41,42].
Reassessment of association between colorectal cancer and F. nucleatum at a subspecies-level
Recent studies have highlighted that F. nucleatum subsp. animalis (Fna) is exclusively colonized in inflammatory environmental niches, such as odontogenic abscesses [43] and intra-colorectal tumor [44], whereas other F. nucleatum subspecies, including F. nucleatum subsp. polymorphum (Fnp), F. nucleatum subsp. nucleatum (Fnn), and F. nucleatum subsp. vincentii, did not. Zepeda-Rivera et al. [44] further classified Fna into two distinct clades, C1 and C2, and observed that Fna C2 was more prevalent in the colorectal cancer niche, which was validated using Fna C2-treated ApcMin/+ mice, which showed a higher number of colonic adenomas than those of Fna C1-treated mice. An in-depth understanding of taxonomic profiles and their functions in relation to F. nucleatum requires a reassessment of previous experimental findings. Russo et al. [21] performed quantitative polymerase chain reaction (qPCR) using a forward primer (5'-CTTAGGAATGAGACAGAGATG-3') to compare the amounts of the amplified 16S rRNA fragment of F. nucleatum in the oral cavity of colorectal cancer patients and healthy controls. Their results showed no significant difference in F. nucleatum abundance between the two groups. However, we aligned sequences between the primer and the 16S rRNA of the four F. nucleatum subspecies and observed that the primer amplified the 16S rRNA of Fna, Fnp, and Fnn, except for Fna, due to the presence of mismatched sequences between the primer and the 16S rRNA of Fna [21]. This suggests the need for more additional qPCR experiments targeting each F. nucleatum subspecies-specific primer to re-evaluate the differences in abundance of F. nucleatum at the subspecies level between the experimental groups.
ORAL MICROBIOTA AND EXTRA-GASTROINTESTINAL DISEASES
Oral microbial alterations associated with extra-gastrointestinal diseases
Park et al. [6] reported oral microbial composition is closely associated with extra-gastrointestinal diseases, including atherosclerotic cardiovascular disease, diabetes, and Alzheimer’s disease. In this review, we focused on lung cancer, a malignant disease with low survival rates. Most lung cancer patients are often diagnosed at an advanced stage because of the lack of early-stage symptoms [45]. A comparative study of salivary microbial taxa between 46 lung cancer patients and 45 healthy controls revealed an abundance of Rothia, Granulicatella, Parvimonas, Abiotrophia, and Eubacterium in lung cancer patients [46]. Furthermore, the relative abundance of Capnocytophaga, Selenomonas, and Veillonella in salivary samples was substantially higher in lung cancer patients (n=20) than in healthy controls (n=10), and all participants were heavy smokers for >10 years [47]. In particular, Veillonella dispar was identified as a potential biomarker for smoking-associated oral microbial disturbances contributing to lung cancer development, as demonstrated in two different studies: V. dispar was highly abundant in the buccal swab samples from 55 heavy smokers than in 50 non-smokers [48], and V. dispar was elevated in salivary samples obtained from three lung cancer patients (smokers) than in five healthy individuals (non-smokers) [49]. Moreover, oral microbial alterations were observed in non-smokers, with an increased abundance of salivary Sphingomonas and Blastomonas in 75 lung cancer patients than in 172 healthy controls [50]. Taken together, these findings suggest that lung cancer-associated oral microbial compositions can be used for the early and non-invasive detection of lung cancer, potentially lowering mortality rates (Table 4).
Lung cancer-associated oral microbial alterations
Enrichment of oral microbiota in lung cancer tissues
Lungs were previously believed to be sterile because of the inability to culture microbes from lower airway samples using traditional microbial culture systems [51]. However, advancements in metagenomic sequencing have contributed to the understanding that the lungs are commonly colonized by diverse microorganisms, including oral microbiota such as Prevotella, Streptococcus, Veillonella, and Neisseria [52]. Changes in the lung microbiota are closely associated with the deterioration of pulmonary disorders, such as lung cancer (Table 5). Lee et al. [53] showed that the abundance of oral microbiota, including Veillonella, Megasphaera, Atopobium, and Selenomonas, was significantly higher in the bronchoalveolar fluids of lung cancer patients (n=20) than in those with benign mass-like lesions (n=8). Further investigations based on the smoking history of 20 lung cancer patients showed that Streptococcus and Porphyromonas were significantly elevated in 12 ever-smokers than in 8 never-smokers [53]. Another study reported an overabundance of Streptococcus and Veillonella in the biopsy samples of malignant lung lesions than in benign pulmonary nodules [54]. Microbial elevation is mechanistically associated with the activation of phosphoinositide 3-kinase (PI3K) and extracellular signal–regulated kinase (ERK) signaling [54], an early pathogenetic event in lung cancer development that regulates cell proliferation, survival, and tissue invasion [55]. Taken together, these findings suggest that the ectopic colonization of oral microbiota in the lungs plays a key role in the onset and progression of lung cancer, which is associated with disturbances in host molecular signaling pathways related to cellular growth and proliferation.
Enrichment of oral microbiota in lung cancers
Modulating effects of an oral pathogen on lung tumorigenesis
Role of Veillonella parvula in lung cancer pathogenesis
As shown in Table 5, two independent research groups have identified Veillonella as a malignant lung tumor-enriched taxon, suggesting its potential as a biomarker for diagnosing and treating lung cancer. To validate this, Tsay et al. [56] investigated whether V. parvula played a role in the development and progression of lung cancer using lung cancer mouse model (genotypically manipulated as KRASLSL-G12D;p53flox/flox, KP mice). The results demonstrated that KP mice inoculated with V. parvula exhibited increased tumor growth and upregulated PI3K/ERK pathways, whereas wild-type mice inoculated with V. parvula showed no such changes [56,57]. This could be attributed to the exaggerated inflammatory responses, marked by increased recruitment of T helper 17 cells, leading to elevated Il-17 production and PD-1 receptor expression [56]. Jin et al. [58] demonstrated that eliminating lung-resident bacteria from KP mice reduced tumor size and number by lowering Il-17-producing T cells, indicating that therapeutic interventions targeting both oral microbiota dysbiosis and Il-17 can ameliorate the risk of lung cancer development.
Zeng et al. [59] developed a different lung cancer mouse model by administrating Lewis lung carcinoma cells (LLC) into either the trachea or subcutaneous tissues of C57 BL/6J mice, followed by inoculation with V. parvula. The results showed that V. parvula promoted tumorigenesis induced by both LLC-intratracheal instillation and LLC-subcutaneous graft, as reflected by the increased number and size of tumors in LLC + V. parvula than in the LLC only model. Mechanistically, V. parvula alters the immune microenvironment by inhibiting the recruitment of tumor-infiltrating T lymphocytes and affecting the distribution of CD3+ and CD4+ T lymphocytes in the peripheral immune environment [59].
CONCLUDING REMARKS
This review highlights the association between oral microbiota and non-oral diseases, including gastric, colorectal, esophageal, and lung cancers. Cancer-associated microbial taxonomic alteration in the oral cavity can serve as early and non-invasive biomarkers for assessing disease. The enrichment of oral microbiota in malignant tissues emphasizes the role of oral microbiota in systemic disease spread via the esophagus, larynx, and bloodstream (Fig. 1) [60,61]. The specific oral microbiota can play a causative role in the development of diverse types of cancers by modulating host immune and inflammatory responses. In particular, regulation of IL-17 production is crucial for a balanced adjustment between its protective roles against extrinsic pathogens [62] and its detrimental roles in the development of various types of cancers [63,64].
Malignant tumor-enriched oral microbiota according to different kinds of cancers.
Notes
Availability of Data and Material
All data generated or analyzed during the study are included in this published article.
Conflicts of Interest
The authors have no financial conflicts of interest.
Funding Statement
This work was supported in part by the Korea Biobank Network Program run by the Korea Disease Control and Prevention Agency (KBN4-A04-03).
Authors’ Contribution
Conceptualization: Do-Young Park. Data curation: Do-Young Park. Formal analysis: Do-Young Park. Funding acquisition: Inseong Hwang. Investigation: Young-Youn Kim. Methodology: Do-Young Park. Project administration: Do-Young Park. Resources: Chang Kee Kim. Software: Do-Young Park. Supervision: Do-Young Park. Validation: Do-Young Park. Visualization: Do-Young Park. Writing—original draft: Do-Young Park. Writing—review & editing: Do-Young Park, Jeong-Hoo Lee, Jiyoung Hwang, Ju-Yeong Hwang. Approval of final manuscript: all authors.
Acknowledgements
None