Gastric cancer (GC) constitutes the third highest cause of cancer mortality worldwide (Bray et al., 2018), and the 5-year survival rates are 27.4 and 32% in China and the USA, respectively. The occurrence and development of gastric carcinogenesis is a complex pathogenic process involving multiple factors, multi-stage changes and polygenic alterations (Massarrat and Stolte, 2014; Goral, 2016). A late-stage presentation is common in most GC cases, because symptoms in early stages of the disease are usually vague and non-specific. As early detection leads to better outcomes, there is a critical need for new avenues of prevention, risk stratification, and early detection for GC.

Microbes in the upper digestive tract have been shown to facilitate carcinogenesis by contributing inflammatory processes via activation of Toll-like receptors pathway (Kauppila and Selander, 2014), or protect against carcinogenesis by providing barriers to pathogen invasion (Yang et al., 2014). Chronic infection with Helicobacter pylori is a well-established risk factor for gastric carcinogenesis. Lines of evidence demonstrated that the process of Correa’s cascade of gastric carcinogenesis initiated by H. pylori involves multiple virulence factors, host genetic make-up, and nutritional factors (Warren and Marshall, 1983; Polk and Peek, 2010; Engstrand and Lindberg, 2013; Plummer et al., 2015). Nevertheless, only about 3% of those infected with H. pylori will eventually develop into gastric cancer, and the eradication of H. pylori does not completely prevent the occurrence of GC (Peek and Crabtree, 2006). These lines of evidence suggest that non-H. pylori microorganisms colonizing the stomach may represent an additional modifier of gastric cancer risk (Sung et al., 2020). The enrichment of some bacteria in the gastric mucosa has been associated with the progression of gastric cancer, including Peptostreptococcus stomatis, Streptococcus anginosus, Parvimonas micra, Slackia exigua, and Dialister pneumosintes (Coker et al., 2018). Our recent study suggested that a reduction of nitrite-oxidizing Nitrospirae taxa in the gastric mucosa may contribute to gastric neoplastic progression via nitrate accumulation (Wang et al., 2020).

Most of the microbial sources in the stomach are believed from the external environment. The oral cavity contains a large number of microorganisms, including bacteria, viruses, fungi, mycoplasma, and chlamydia (Aas et al., 2005; Wade, 2013; He et al., 2015). The oral microbiota can enter the downstream digestive tract from the oral cavity through saliva and can also migrate to various parts of the body to cause infections and local inflammatory reactions in corresponding sites (Han and Wang, 2013), oral microbes are closely correlated with several systemic diseases such as the oral tumors, type 2 diabetes, cardiovascular disease, urinary systemic diseases and rheumatoid arthritis (Seymour, 2010; Ahn et al., 2012; Salazar et al., 2012; Whitmore and Lamont, 2014; Gao et al., 2018). Recently, oral microbiota has been suggested to play a role in the etiology of esophageal cancer, colorectal cancer (CRC), and pancreatic cancer (Michaud and Izard, 2014; Peters et al., 2017; Flemer et al., 2018). Interestingly, a higher incidence of GC was found among people with worse oral hygiene (Watabe et al.), indicating a potential link between the oral microbiota and the occurrence/development of gastric cancer. In this study, we characterized the microbial compositional and ecological changes in salivary microbiota of patients with GC and non-malignant gastric lesions including superficial gastritis (SG) and atrophic gastritis (AG). We demonstrated the possibility of using salivary microbes as biomarkers GC detection, and explored the potential mechanisms of oral microbiota in the pathogenesis of GC.

Previous studies have shown that periodontal disease and poor oral health status were associated with increased incidence of malignant diseases (Dizdar et al., 2017; Michaud et al., 2018), pointing to a potentially oncogenic role of oral microorganisms in the development of cancer. Some species have been identified that correlate strongly with oral squamous cell carcinoma (OSCC), such as Capnocytophaga gingivalis, Prevotella melaninogenica, and Streptococcus mitis (Mager et al., 2005), which were suggested as diagnostic markers since they predicted 80% of cancer cases. Oral microbes are also detected in tumors distant to the oral cavity. For example, many works have shown that the oral periopathogens Fusobacterium nucleatum and Porphyromonas gingivalis are essential in the development of colorectal and pancreatic cancer, respectively (Rubinstein et al., 2013; Fan et al., 2018a). The oral microbiota was shown to reflect an inflammatory status of the stomach in patients with H. pylori infection (Zhao et al., 2019), and could detect GC with a high accuracy (Coker et al., 2018; Wu et al., 2018). In this study, we demonstrated that the salivary microbiota could identify GC among patients with non-malignant gastric diseases including SG and AG, yielding a high accuracy (AUC of 91%). With a cohort consisting of 37 GC patients and 13 healthy individuals, a previous study also showed a high sensitivity rate (AUC of 97%) of using oral microbiota screening gastric cancer (Sun et al., 2018), further enhancing the diagnostic potential of the oral bacteria for gastric malignancy. The microbiome is exclusive to the individual and influenced by lifestyle and phenotypic and genotypic determinants. For example, alcohol consumption and tobacco usage have been shown to influence the oral microbiome composition (Wu et al., 2016; Fan et al., 2018b). Therefore, lifestyle should be considered as confounding factors when identifying diagnostic microbial markers from the oral microbiome. Multivariate analysis method revealed that enrichment of Faecalibacterium was negatively associated with smoking, and no significant correlation was found between salivary bacteria and alcohol or HP-infection. Thus, the potential biomarkers identified based on our data seemed not affected by the recorded lifestyle and HP infection.

Our data showed that alpha diversity of the salivary microbiota was similar among patients with different gastric histological types, which was consistent with another study (Kageyama et al., 2019). One previous study found that the microbial diversity of saliva and dental plaque significantly increased in GC patients (Sun et al., 2018), whereas another one suggested that the microbiota diversity significantly reduced in the tongue coating of GC patients (Cui et al., 2019). Taken together, the microbial diversity of oral microbiota seems not strongly associated with the development of GC.

Data from our recent study (Wang et al., 2020) as well as others (Dicksved et al., 2009; Castano-Rodriguez et al., 2017; Chen et al., 2019) showed that commensals of the oral cavity including Fusobacterium, Peptostreptococcus, Prevotella, Streptococcus, and Veillonella were found to have higher relative abundances in the gastric mucosa of GC patients. Notably, these genera are also commensals of oral cavity, but their translocation and expansion may be involved in the onset and development of multiple diseases including cancers. One possible mechanism of oral microbiota participating carcinogenesis is enrichment of pro-inflammatory oral bacterial species. For example, Streptococcus bovis has been shown to promote the development of colon cancer by enhancing the inflammation (Abdulamir et al., 2011). We observed that Streptococcus genus was enriched in the saliva microbiota of GC patients, which agrees with the findings in a recent study (Sun et al., 2018). Interestingly, an enrichment of Streptococcus spp. was also reported across several types of cancer such as colorectal adenocarcinomas (Abdulamir et al., 2011). Taken together, these results indicate a potential of some strains of Streptococcus being involved in gastric carcinogenesis. In addition, Corynebacterium genus was also found to be enriched in the saliva of GC patients, which was in line with (Wu et al., 2018) a higher level Corynebacterium; this genus was found higher in the tongue coating microbiota community of GC patients than that of the healthy controls. Species of Corynebacterium are widely distributed in the microbiota of human skin, and most of them are innocuous while some species are known to cause infection such as C. diphtheria. In recent years, they have been increasingly reported as emerging opportunistic pathogens in immunocompromised patients with cancer, hematologic malignancy, and critical condition (Chen et al., 2012). Thus, a higher level of Corynebacterium spp., which appeared in the oral cavity, may reflect immune deficiency in cancer patients. Altogether, an enrichment of proinflammatory bacteria in the oral cavity is likely an import factor contributing to the development and progression of GC.

Several bacterial taxa were found reduced in the salivary microbiota of GC patients, including Bulleidia, Fusobacterium, Haemophilus, Lachnoanaerobaculum, Neisseria, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Intriguingly, a decreased carriage of Bulleidia was also captured in the oral cavity of patients with esophageal squamous cell carcinoma (Chen et al., 2015). Moreover, some taxa of these genera were found to be enriched in tumor and stool samples of colorectal cancer patients, such as Fusobacterium nucleatum, Parvimonas micra, Porphyromonas asaccharolytica, and Peptostreptococcus stomatis, Prevotella intermedia (Ternes et al., 2020). And we recently found a higher load of Fusobacterium in the gastric mucosa of GC patients compared to SG (Wang et al., 2020). In the present study, a low level of these genera was observed in the oral cavity of GC patients as compared with SG and/or AG. In fact, bacterial abundance is majorly regulated by nutrient availability and antimicrobial signals specific to their environmental conditions. Thus, albeit these bacteria colonize and expanded in the tumor site (such as gut of patients with colorectal cancer), they may not overgrow in their original localization such as the oral cavity.

Network analysis revealed that Prevotella was negatively correlated with a variety of oral bacteria in the oral cavity of all three histological stages, and the number of its negative relationships decreased in AG and GC groups. In the GC patients, the abundance of Prevotella in the salivary cavity was lower than that of SG and AG groups, which is opposite to the findings in Sun et al.’s study (Sun et al., 2018). This discrepancy at the genus level may be explained by increasing the phylogenetic resolution via metagenomic sequencing and identifying the specific species/strains that related to gastric cancer.

We previously found that patients with intraepithelial neoplasia had higher relative abundances of Haemophilus parainfluenzae and Nitrospirae family in the gastric mucosa, which decreased in that of GC patients (Wang et al., 2020). Both Haemophilus and Nitrospirae are nitrate-reducing bacteria, which convert nitrate to nitrite, and also to nitric oxide (NO), which can be absorbed through the blood vessels in the oral cavity or through being swallowed into the gastrointestinal system. Accumulation of N-nitroso compounds in the gastrointestinal tract is likely to increase the risk of carcinogenesis (Forsythe and Cole, 1987; Bryan et al., 2012). Thus, the decreased abundances of Haemophilus in the salivary microbiota may contribute to the formation of gastric tumor.

Functional analysis based on the PICRUSt2-predicted pathways suggested that metabolic functions of salivary microbiota changed along with the disease progression in the stomach. In particular, pathways involved in isoleucine and valine biosynthesis were highly expressed by the salivary microbiota in GC patients compared to the non-malignant stages. Interestingly, an upregulation of amino acids including isoleucine and valine was also detected in human gastric tumor tissues (Jung et al., 2014; Wang et al., 2016). Higher levels of most amino acids and their primary derivatives in gastric tumor tissues were thought to be related to two main sources: the degradation of extracellular matrix by matrix metalloproteinases and the autophagic degradation of intracellular proteins (Hirayama et al., 2009). The production of amino acid from microbes in the oral cavity and gastrointestinal tract has not been quantified and deserves further investigation in terms of proliferation and survival of gastric cancer cells.

There were several limitations in this study. Firstly, we didn’t collect samples from healthy individuals as control. Secondly, amplicon sequencing of 16S rRNA has limited resolution in determining the bacterial species or strains, and it is therefore difficult to access the functions of specific bacteria involved in gastric cancer development and progression. In addition, the 16S rRNA gene V1–V3 region has been shown to provide superior taxonomic resolution for the bacterial microbiota of the human oral and respiratory tracts compared to the V3–V4 region (Zheng et al., 2015; Escapa et al., 2018). Thus, some other oral taxa with diagnostic potential for gastric cancer might not have been detected in the present data. Finally, independent clinical cohorts from multiple centers are required to evaluate the diagnostic value of the identified GC-associated saliva bacteria.

We demonstrated, with a large cohort, that the salivary microbiota can be used to predict GC as well as its non-malignant stages. The contributions of the oral microbiota in the pathogenesis of GC include an accumulation of proinflammatory bacteria and a decline in those reducing carcinogenic N-nitroso compounds.

The data presented in the study are deposited in European Nucleotide Archive (https://www.ebi.ac.uk/ena/submit/sra/#studies), accession number PRJEB42657.

The studies involving human participants were reviewed and approved by the ethics committee of the Chinese PLA General Hospital (No. S2016-057-02). The patients/participants provided their written informed consent to participate in this study.

YY conceived the study and revised the manuscript. KH, LW, BY, ZW, XZ, LP, JY, and GS performed the subject’s enrolment and sample collection. XG and KH analyzed clinical and sequencing data. KH and XG wrote this manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.