Oral cancer is the most common malignant tumor of the oral and maxillofacial region, and more than 90% of cases are squamous cell carcinomas (SCC) (Tandon et al., 2017). The incidence of oral cancer is increasing worldwide, and despite the remarkable progress in cancer treatment, the incidence and mortality rates of oral cancer remain high, with the 5-year survival rate staying at approximately 50% (Overman, 2006). Oral cancer mostly involves the whole tongue, larynx, jaws, important blood vessels, and functional nerves of the neck and skull base, as well as other important areas, which threatens the patient’s life and is accompanied by obvious dysfunction such as swallowing, speech, pain, numbness, and breathing problems, which greatly reduces the quality of life. The main risk factors of OSCC include smoking, betel nut chewing, and heavy alcohol consumption, but approximately 15% of oral squamous carcinomas cannot be explained by these main risk factors (Perera et al., 2018), and other potential risk factors need to be explored.

In the 1990s, researchers first demonstrated the pathogenic role of Helicobacter pylori in gastric cancer, linking carcinogenicity to the bacteria (Marwick, 1990). Subsequently, many studies evaluated the relationship between bacteria and cancers of other organs. For example, EBV was determined to be associated with Burkitt lymphoma and nasopharyngeal carcinoma (Allavena et al., 2008), an increased risk of gallbladder cancer was found to be associated with Salmonella typhi infection (Scanu et al., 2015), and HPV infection was linked to cervical cancer. These findings provide reference and new research directions for the relationship between microorganisms and oral tumors. Some studies have found (Rajeev et al., 2020) that oral microorganisms might be related to the development of oral tumors, but the mechanism is not clear. The research methods range from simple bacterial culture to PCR molecular techniques and next-generation sequencing-16S rRNA gene detection (Perera et al., 2016), and these have now progressed to metagenomic sequencing techniques. Microbial species variation associated with oral cancer is variable owing to differences in study results. Accumulating evidence suggests that the bacterial composition of the mucosal surface, inside tumor tissue, and in saliva of patients with oral squamous cell carcinoma is very different from that of the oral cavities of healthy individuals (Berkovits et al., 2016). The abundance of opportunistic pathogens in the saliva of oral cancer patients was found to be remarkably higher than that in healthy people (Pushalkar et al., 2011). Schmidt et al. (2014), using the 16SrDNA method, found that the abundance of Streptococcus and Actinomycetes in oral squamous cell carcinoma and precancerous lesions was significantly reduced, whereas the abundance of Bacteroides was significantly increased, indicating that the change in oral microbiota occurred in the early stage of cancer and that cancer progressed along with the development of the tumor. Yang et al. (2018) showed that the oral bacterial microbiota was different between healthy participants and patients with oral squamous cell carcinoma and that it changed during tumor progression. Yost et al. (2018) conducted a microbial macrotranscriptomic study on 15 samples and found that the microbial community in tumor sites of patients with oral squamous cell carcinoma changed significantly compared with that of the matching sites of healthy participants. Therefore, squamous cell carcinoma can be efficiently screened by detecting the oral microbiota. Changes in the oral microbial community structure reveal that these microorganisms might have a pivotal role in the prevention and early diagnosis of oral cancer.

Most studies now focus on tumor tissues and adjacent and contralateral normal tissues, but the distribution of microbiota in tissues is small and unstable. In our study, we collected samples of gargle fluid with relatively stable microbiota. The present study used metagenomic sequencing technology to detect the presence and distribution characteristics of oral microbiota in patients with oral cancer, precancerous lesions, and normal populations and explored the relationship between the occurrence of oral cancer and the oral microbial microbiota. This has implications for further mechanistic exploration and can be used as a biomarker to predict OSCC with high diagnostic accuracy.

The oral microbiome is one of the most complex microbial communities in the human body, with the oral cavity comprising more than 700 microorganisms (Vogtmann et al., 2017). Ecological imbalances in microbial communities, or ecological dysbiosis, have been extensively studied in human and animal models (Meurman et al., 2009). These are characterized by the loss of beneficial microorganisms, expansion of pathogenic microorganisms, and a general loss of microbial diversity (Xuan et al., 2014). It is becoming increasingly evident that ecological dysbiosis might contribute to cancer development (Schwabe and Jobin, 2013). Various beneficial or pathogenic microorganisms colonize the oral cavity, eventually leading to good health or diseases (Lamont et al., 2018). Previous studies (Fan et al., 2018; Ren et al., 2019; Ren et al., 2020) have verified the relationship between bacteria and cancer in various parts of the body, including the oral cavity. In recent years, researchers (Sheflin et al., 2014) have found that the role of a single species is transferred to the role of the microbial community. The oral cavity includes the hard teeth and the soft mucosal epithelium, making the oral environment relatively complex. In general, 50–1,000 different microbial species are simultaneously present in the oral cavity, that is, in the saliva, gingival sulcus, dental plaque, and tongue (Dewhirst et al., 2010). Chronic and persistent inflammation is a leading cause of cancer (Rewell, 1965). Previous studies have also reported an association between bacterial infection and carcinogenesis (Helicobacter et al., 2001; Littman, 2005). H. pylori plays a pivotal role in the occurrence and progression of gastric cancer (Kim et al., 2011; Moss, 2017). Furthermore, microbes and their products such as endotoxin (lipopolysaccharide), enzymes (protease, collagenase, fibrinolysis enzyme, and phospholipase A), and metabolic byproducts (hydrogen sulfide, ammonia, and fatty acids) exhibit toxicity to their surrounding cells and might directly induce mutations in tumor suppressor genes and proto-oncogenes or alter the signaling pathways associated with epithelial cell proliferation. Exogenesis can also be stimulated by indirect mechanisms that activate the peripheral inflammatory cells, exposing epithelial cells to a mutagenic microenvironment. Herein, microbes and their products were found to activate granulocytes, macrophages, monocytes, lymphocytes, fibroblasts, and epithelial cells to produce reactive oxygen species (hydrogen peroxide and oxygen free radicals), reactive nitrogen species (nitric oxide), lipid metabolites, and matrix metalloproteinases. These substances can induce DNA damage in epithelial cells and might also lead to the production of cytokines, chemokines, growth factors, and other signals for cell survival, proliferation, migration, angiogenesis, and apoptosis inhibition (Karin et al., 2006). The microbial community and the host mostly maintain a dynamic balance; some beneficial bacteria can effectively counter the invasion of external pathogens and enhance the tissues and immune system. Moreover, poor eating habits, lifestyle, and a weak immune system might lead to imbalances and could cause the overgrowth of pathogenic bacteria, thereby triggering various oral diseases, such as tooth decay, periodontal disease, oral leukoplakia, compressed moss, and oral cancer (Suwannakul et al., 2010; Valm et al., 2011).

In the present study, we aimed to determine the relationship between oral microbiota characteristics and oral squamous cell carcinoma. Through our analysis, we found significant changes in the oral microbiota among the three groups. In terms of the oral microbial composition, Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, were the four main phyla in the oral cavity. At the family level, Erysipelotrichaceae, Peptostreptococcaceae, Prevotellaceae, and Carnobacteriaceae were mainly enriched in the oral cancer group. Among these, the genera Dialister, Peptostreptococcus, Prevotella, and Granulicatella were enriched in the oral cancer group. Further, S. cristatus, G. elegans, P. intermedia, and P. stomatis were significantly increased in abundance at the species level. These are present in the oral mucosa as commensal bacteria but might be opportunistic pathogens of potential relevance in relation to oral squamous cell carcinoma. Simultaneously, the precancerous lesion group was included in this study to represent a transition between the oral cancer group and the healthy control group.

Fusobacterium nucleatum, a gram-negative bacillus, is one of the important pathogenic bacteria of periodontal disease. It was found that infection with periodontal pathogens and the development of oral cancer are closely related (Tezal et al., 2007). F. nucleatum is widely found in the gastrointestinal tract as an oral colonizing bacterium and has been shown to be virulent or pathogenic and an opportunistic pathogen (Abe et al., 2011; Brennan et al., 2018). In the present study, the abundance of F. nucleatum increased sequentially in the normal (6.48%), precancerous (15.07%), and cancerous (40.88%) groups. This is in agreement with the findings of Ganly et al. (2019) in 2019. Meanwhile, one study detected F. nucleatum in the saliva, dental plaque, oral exfoliated cells, cancerous tissues, and paracancerous tissues of patients with oral squamous carcinoma by quantitative PCR and 16S rRNA, with the highest abundance, and manifestation as an inflammatory microorganism, in cancerous tissues (Al-hebshi et al., 2017). F. nucleatum can also promote the progression of oral squamous carcinoma and is associated with poor prognosis with this disease. (Yang et al., 2018; Zhang et al., 2019) F. nucleatum is an oral symbiotic bacterium (Brennan et al., 2018), which has the ability to colonize, adhere to, and invade tissues, promote the proliferation and migration of host and tumor cells, and also interact with other microbiota, thus affecting the body’s immune function and promoting the formation of inflammatory and tumor microenvironments (Kostic et al., 2013; Nosho, 2016).

P. intermedia and P. stomatis were relatively abundant in the oral cancer groups. Prevotella is closely associated with chronic inflammatory diseases and is present in biofilms of gingivitis and periodontal disease (Dahlen, 1993; Berezow et al., 2011). The mechanism involves neutrophil recruitment and the expression of proinflammatory cytokines and metalloproteinases that mediate the destruction of connective tissue and alveolar bone. Presently, most studies on this mechanism have focused on the role of Porphyromonas gingivalis (a gram-negative anaerobic member of Bacteroides) (Hajishengallis, 2014). P. stomatis is a gram-positive anaerobic bacterium and an obligate parasite of the oral cavity, mucous membrane, and intestinal tract of mammals; it might also play a pivotal role in suppurative infection and is closely related to the occurrence of periodontal disease (Rams et al., 1992). Previous studies have reported that chronic periodontal disease is associated with the development of head and neck cancer, particularly oral squamous cell carcinoma. For every millimeter of alveolar bone loss, the risk of head and neck squamous cell carcinoma increases four-fold. In addition, those with periodontal disease are at a higher risk of developing poorly differentiated cancers (Tezal et al., 2009; Javed et al., 2016). As periodontal disease is a type of microbial disease, it provides a foundation for studying the etiology of oral cancer with respect to microorganisms.

We should also not ignore taxa with low abundances but significantly increased in OSCC, as these taxa could be considered “key” microorganisms and might be more virulent and therefore have an increased role in the development of cancer. G. elegans is a fastidious gram-positive coccus (pairs, chains) and is part of the nutritionally variant streptococci (NVS) group. Like other constituents of the NVS, it can cause bacteremia and infective endocarditis, which are associated with significant morbidity and mortality. D. pneumosintes (formerly named Bacteroides pneumosintes) is a non-fermentative, anaerobic, gram-negative rod. D. pneumosintes has been recovered from deep periodontal pockets, but little is known about the relationship between this organism and destructive periodontal disease (Doan et al., 2000).

The present study revealed that at the phylum level, the relative abundance of Bacteroidetes in the oral cancer and precancerous lesion groups was markedly higher than that in healthy subjects. Schmidt et al. (2014) used the 16S rDNA method and found that the abundance of Streptococcus and Actinomycetes in oral squamous cell carcinoma and precancerous lesions was markedly reduced, whereas that of Bacteroides was remarkably increased, thereby indicating that the change in oral microbiota occurred during the early stage of cancer and might indicate the progression of cancer along with tumor development. This finding is consistent with the results of the present study. Yost et al. (2018) conducted a microbial macrotranscriptome study on 15 samples and found that compared with that of the matching sites of healthy participants, the microbial community in tumor sites of oral squamous cell carcinoma patients changed remarkably; Streptococcus and Haemophilus were highly abundant in the healthy control group, which was also consistent with the results of this study.

There is a wide variety of inflammatory biomarkers, which are of great importance for the diagnosis and treatment of clinical diseases, as well as for prognosis. The neutrophil ratio, C-reactive protein (CRP), procalcitonin (PCT), white blood cells (WBCs), and IL-6 have been extensively studied as markers of long-term prognosis in many cancers. Most studies have demonstrated that the postoperative survival of cancer patients correlates with the systemic inflammatory response (Ethier et al., 2017). In the early stages of tumor proliferation and metastasis, systemic inflammatory markers can signify pro-cancer inflammation in the tumor microenvironment (Choi et al., 2016). Multiple cellular interactions with inflammatory factors further promote tumor formation due to an extremely immunosuppressive microenvironment (Kummar et al., 2007). Neutrophils play an essential role as they exert various potential effects in cancer, including promoting metastasis. In the inflammatory response to cancer, neutrophils can act directly with circulating tumor cells, acting as reservoirs of circulating vascular endothelial growth factors and promoting metastasis. In addition, the metabolites of neutrophils, such as reactive oxygen species and proteases, have been shown to have clear and specific roles in regulating tumor cell proliferation, angiogenesis, and metastasis. Recently it has been shown that neutrophils can have cytotoxic effects on tumor cells (Jensen et al., 2009). Elevated CRP is a marker of systemic inflammation and is considered a predictor of poor survival in patients with various cancers (Crumley et al., 2006). PCT is an inactive pro-peptide substance of serum calcitonin and is barely detectable in blood under healthy physiological conditions (<0.1 ng/mL). Some studies have shown that PCT is significantly increased with fungal and virus infections (Clementi et al., 2017). IL-6 promotes cancer development by activating endothelial cells, increasing adhesion molecule expression, and inducing matrix metalloproteinase production. In conclusion, there is a close relationship between malignancy and inflammatory markers.

In this study, the MATECYC database was used to retrieve results. Coenzyme A biosynthesis, phosphopantothenic acid biosynthesis, inosine 5’-phosphate degradation, and riboflavin biosynthesis participate in various metabolic activities in the body, such as DNA synthesis, respiration, and oxidative metabolism. These metabolic activities are related to cell proliferation and might further lead to the development of tumors. The metabolic pathways of the normal control group included lactose and galactose degradation, L-methionine biosynthesis, tetrahydrofolate biosynthesis, L-tryptophan biosynthesis, and thiamine phosphate formation, wherein the oral cancer group and the healthy control group revealed significant differences in the metabolic pathways between related microbiota. The decrease in the phosphotransferase system and glycolysis and galactose metabolism might reflect a community response to reduced sugar sources on the tumor surface, as increased glucose uptake is critical for OSCC cell survival (Eckert et al., 2018). Notably, the function of O-antigen biosynthesis in microorganisms was increased in precancerous lesion samples. Lipopolysaccharide is composed of fat and polysaccharides containing an O-antigen, outer core, and inner core connected by covalent bonds. Therefore, whether the increase in the microbial O-antigen biosynthesis function is related to an increase in lipopolysaccharide biosynthesis remains to be elucidated.

Previously, major research on oral disease microorganisms was limited to simple bacterial culture technology. Owing to the limitations of bacterial culture, some microorganisms are difficult or impossible to cultivate. Metagenomic sequencing technology has the advantages of unbiasedness, wide coverage, and rapid detection. Research on the abundance and diversity of key bacterial groups through metagenomic sequencing can increase the clinical diagnostic value of oral malignancy. The diagnostic value of tumors reveals the potential of microbial markers as a noninvasive diagnostic tool for oral malignancies, which is conducive to the early diagnosis and treatment of oral malignancies. The limitation of this study is that the sample size was small. In future research, we will verify the diagnostic potential of oral microbial microbiota changes in oral malignant tumors using more samples.

In summary, we have found significant structural changes in the oral microbiota of patients with oral cancer, patients with precancerous lesions, and healthy controls at different species levels. Our research showed that dysbiosis of the oral microbiota changes certain metabolic pathways to affect oral health. Future studies are still warranted to investigate the correlation between oral microbiota imbalances and oral cancer, providing new evidence for microbiota-targeted approaches for disease prevention.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author. The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0002274 http://db.cngb.org/cnsa/project/CNP0002274/reviewlink/.

This study was approved by the Ethics Committee from the First Affiliated Hospital of Zhengzhou University (KY-2019-LW007). Informed consent was obtained from all participants.

ZL and QS conceived and designed the experiments. ZL, GC, PW, JF, MS, and AL performed the experiments. AL and ZL analyzed the data. ZL wrote the paper and edited the manuscript. The final manuscript was read and approved by all authors.

This present work was funded by the grants of the Henan Natural Science Foundation project (21300410391), Henan Province Young and Middle-aged Health Science and Technology Innovative Talents (Outstanding Youth) Project (YXKC2020030), Henan Province Medical Science and Technology Research (Provincial and Ministry Joint Construction Key) Project (SBGJ202002071), Henan Medical Science and Technology Research Program (JointLy Built, LHGJ20200270), and National Natural Science Foundation of China Youth Fund (81702979). Henan Provincial Science and Technology Department Key Research and Development and Promotion Special (212102310592), Overseas Talent Program of Henan Provincial Health Care Commission (2018048).

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.

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