European Journal of Cardiovascular Medicine

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral malignancies and represents a significant cause of cancer-related morbidity and mortality worldwide, particularly in India, where it contributes to a substantial public health burden [1,2]. Globally, OSCC constitutes approximately 1–4% of all malignancies and is associated with poor long-term survival due to late diagnosis, recurrence, and metastasis [2,3].

The etiology of OSCC has traditionally been linked to tobacco use, alcohol consumption, smoking, and betel nut chewing, which together account for nearly 85% of cases [4]. However, a growing proportion of OSCC occurs in individuals without identifiable risk factors, suggesting the involvement of alternative pathogenic mechanisms [5]. Infectious agents are increasingly recognized as contributors to carcinogenesis, with nearly 15% of human cancers attributed to microbial infections [6].

The oral cavity harbors one of the most complex microbial ecosystems in the human body, second only to the gastrointestinal tract, comprising more than 700 bacterial species across distinct ecological niches [7,8]. Under physiological conditions, the oral microbiome maintains mucosal homeostasis; However, disruption of this balance—termed microbial dysbiosis—has been implicated in chronic inflammation and malignant transformation [9,10].

Emerging evidence suggests that alterations in the oral microbiome may contribute to OSCC pathogenesis by promoting persistent inflammation, immune modulation, epithelial dysregulation, and the production of carcinogenic metabolites [11–13]. Several bacterial species, including Streptococcus anginosus, Fusobacterium nucleatum, Prevotella melaninogenica, Capnocytophaga species, and Streptococcus mitis, have been reported with increased frequency in OSCC tissues and saliva [14–18].

Despite increasing interest in the oral microbiome, data on intratumoral bacterial colonization—particularly from the Indian population—remains limited. Moreover, many studies rely on salivary or surface samples, which may not accurately reflect the tumor microenvironment [19,20]. Therefore, the present study aims to identify and compare the microbial flora of OSCC tissues and adjacent clinically normal oral  through biopsy samples followed by culture-based and molecular approaches.

Study Design and Ethical Considerations This observational cross-sectional study was undertaken to compare the microbial composition of oral squamous cell carcinoma (OSCC) tissues with that of adjacent clinically normal oral mucosa from the same individuals. The study protocol was reviewed and approved by the Institutional Ethics Committee. Written informed consent was obtained from all participants prior to enrollment. All procedures conformed to the ethical standards of the Declaration of Helsinki. Study Population Twenty adult patients (≥18 years) with newly diagnosed, untreated, histopathologically confirmed primary OSCC were recruited over a one-year period. Only immunocompetent individuals without concurrent oral mucosal pathology were included. Inclusion criteria comprised: (i) age ≥18 years; (ii) primary OSCC confirmed by histopathology; (iii) no prior surgical, chemotherapeutic, or radiotherapeutic intervention; and (iv) immunocompetent status. Exclusion criteria included recent antibiotic therapy (within three months), pregnancy or lactation, immunosuppressive disorders, secondary oral malignancies, prior oncologic treatment, and the presence of active inflammatory oral lesions. Demographic data, tumor site, relevant habits (tobacco and alcohol use), and clinical staging were recorded for all participants. Clinical Evaluation and Tissue Sampling A comprehensive intraoral examination was performed under standardized conditions. Two biopsy specimens (approximately 1 cm³ each) were obtained from each patient: one from a viable, non-necrotic region of the tumor and a second from adjacent clinically normal oral mucosa located at least 5 cm away from the lesion on the same side. To minimize contamination, tissue surfaces were disinfected with 0.5% povidone–iodine for three minutes, followed by rinsing with sterile phosphate-buffered saline (PBS). The final PBS rinse was cultured separately to confirm sterility. Each specimen was divided into two portions for microbiological culture and molecular analysis. Microbiological Analysis Sample Homogenization Biopsy specimens were homogenized aseptically in 0.5 mL sterile PBS to ensure uniform microbial dispersion and immediately processed for aerobic and anaerobic cultures. Aerobic Culture Homogenates were inoculated onto nutrient agar, blood agar, and MacConkey agar plates and incubated aerobically at 37°C for 24–48 hours. Bacterial identification was based on colony morphology, hemolysis patterns, Gram staining, and standard biochemical tests, including catalase, coagulase, oxidase, indole, urease, citrate utilization, and carbohydrate fermentation assays. Anaerobic Culture Anaerobic cultures were performed using CDC anaerobic blood agar, Bacteroides bile esculin agar, and Mitis salivarius agar. Anaerobic conditions were maintained using GasPak systems, with incubation at 37°C for 48–72 hours. Identification was based on colony morphology, Gram staining, fluorescence under ultraviolet light, odor, biochemical reactions, and antibiotic susceptibility patterns to kanamycin, vancomycin, and colistin. Gram Staining Gram staining was performed on tissue homogenates and isolated colonies to assess bacterial morphology, Gram reaction, and the presence of epithelial or inflammatory cells. Microscopic evaluation was conducted using a 100× oil immersion objective. Molecular Identification of Culture-Negative Samples DNA Extraction Culture-negative samples were subjected to molecular analysis. Genomic DNA was extracted using the Qiagen DNA Mini Kit following the manufacturer’s protocol. DNA purity and concentration were assessed spectrophotometrically, and samples were stored at −20°C until further analysis. 16S rRNA Gene Amplification and Sequencing Broad-range polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene was performed using universal primers. PCR products were visualized on 1.5% agarose gel electrophoresis, purified using the QIAquick Gel Extraction Kit, and sequenced using the ABI Dye Deoxy Terminator Cycle Sequencing Kit on an ABI 373A Genetic Analyzer. Bioinformatic Analysis Sequencing data were aligned and compared with reference sequences in the NCBI GenBank and EMBL databases using the FASTA algorithm. Sequence similarity was assessed using the Escherichia coli 16S rRNA gene as a reference standard. Statistical Analysis Data were compiled and analyzed descriptively. Microbial profiles of carcinomatous and adjacent normal tissues were compared qualitatively.

Demographic and Clinical Characteristics (Table 1)

Among the 20 participants, 35% were aged 61–70 years, 30% were 41–50 years, 25% were 51–60 years, and 10% were 71–80 years. OSCC was more common in males (60%) than females (40%). The buccal mucosa was the most frequently affected site (75%), followed by the tongue (15%) and floor of mouth/alveolus (10%) (Table 1).

Table 1. Demographic and Clinical Characteristics

Microbial Flora in Carcinomatous Tissue (Table 2)

Aerobic and anaerobic bacteria were isolated from OSCC tissues. Among aerobes, Streptococcus anginosus was the most prevalent (21.27%), followed by Capnocytophaga (19.15%), Streptococcus mitis (17.02%), and Streptococcus mutans (10.64%). Other aerobic species included Staphylococcus aureus, Corynebacterium, and Micrococcus luteus (8.5%), and Serratia marcescens (6.4%).

Anaerobic bacteria were more abundant than aerobes. Prevotella melaninogenica comprised 25% of isolates, while Peptostreptococcus and Propionibacterium each accounted for 22.5%. Fusobacterium constituted 20% and Lactobacillus 10% of the anaerobic isolates.

Table 2. Microbial Flora in Carcinomatous Tissue

Microbial Flora in Normal Tissue (Table 3)

In adjacent clinically normal mucosa, aerobes included Streptococcus mitis and Streptococcus salivarius (each 33.33%) and Staphylococcus (13.33%). Anaerobic isolates included Propionibacterium (29.16%), Peptostreptococcus (20.8%), and Actinomyces and Lactobacillus (12.5% each).

Table 3. Microbial Flora in Normal Tissue

Molecular Analysis by PCR and 16S rRNA Sequencing (Table 4)

Culture-negative samples were analyzed using 16S rRNA gene sequencing. In cancerous tissue, Streptococcus anginosus, Prevotella melaninogenica, and Capnocytophaga were identified with a sequence length of 310 bp. Fusobacterium and Micrococcus luteus had 302 bp, Propionibacterium 295 bp, Streptococcus mutans and Streptococcus mitis 290 bp, and Corynebacterium 284 bp.

In normal tissue, Streptococcus, Capnocytophaga, and Propionibacterium were detected with 300 bp, and Streptococcus and Lactobacillus with 280 bp.

Table 4. Bacterial Genera Identified by 16S rRNA Sequencing

This study demonstrates that oral squamous cell carcinoma (OSCC) tissues harbor distinct and viable microbial communities that differ substantially from those present in adjacent clinically normal oral mucosa. The predominance of aerobic and facultative anaerobic bacteria within carcinoma tissues supports the concept that microbial dysbiosis is closely linked to the altered tumor microenvironment, characterized by hypoxia, necrosis, immune modulation, and persistent inflammation. These findings add to the growing body of evidence suggesting that microorganisms may actively participate in tumor biology rather than merely colonize malignant tissues opportunistically (8,9,11).

Traditionally, OSCC has been associated with established risk factors such as tobacco use, alcohol consumption, and betel nut chewing, which account for approximately 85% of cases (3,4). However, the rising incidence of OSCC among younger individuals without these exposures highlights the potential contribution of nontraditional etiological factors. Infectious agents are estimated to be involved in nearly 15% of human cancers, and the identification of Helicobacter pylori in gastric carcinogenesis provided a paradigm for bacterially driven malignancies (5). Similar mechanisms—including chronic inflammation, immune dysregulation, and genotoxic stress—are increasingly implicated in oral carcinogenesis (8,10,11).

The oral cavity represents one of the most complex microbial ecosystems in the human body (7). Under physiological conditions, microbial homeostasis maintains mucosal integrity and immune balance. Disruption of this equilibrium results in dysbiosis, which has been linked to chronic inflammatory diseases and malignancy (8,11). In the present study, carcinoma tissues exhibited enrichment of bacteria known to induce inflammatory signaling, produce carcinogenic metabolites, and modulate host immune responses. These processes parallel inflammatory pathways implicated in systemic disorders, including cardiovascular disease, underscoring shared host–microbe mechanisms across organ systems (24,25).

Among aerobic isolates, Streptococcus anginosus was frequently detected in OSCC tissues. This organism has been repeatedly associated with oral and oropharyngeal cancers and is known to induce chronic inflammatory responses, generate reactive oxygen species, and metabolize ethanol into acetaldehyde, a recognized carcinogen (14,16,17). Activation of cyclooxygenase-2 (COX-2) and subsequent prostaglandin-mediated signaling may further promote tumor invasion and progression (15). The identification of Streptococcus mitis within carcinoma tissues reflects its dual role as a commensal organism and a potential contributor to carcinogenesis through acetaldehyde production, despite reported cytotoxic effects on tumor cells (14,15).

Facultative anaerobes such as Capnocytophaga species were also prevalent. These organisms are well adapted to hypoxic environments and produce lipopolysaccharides and tissue-degrading enzymes that may enhance epithelial–mesenchymal transition, immune evasion, and tumor invasiveness (14,18). Their consistent detection in OSCC across multiple studies supports their relevance as potential biomarkers of disease (14,17).

Anaerobic bacteria, including Prevotella melaninogenica and Fusobacterium species, were prominent in carcinomatous tissues. Prevotella species produce volatile sulfur compounds and promote oxidative stress, basement membrane degradation, and inflammatory signaling (14,15). Fusobacterium nucleatum, in particular, has been extensively implicated in OSCC progression through activation of β-catenin and NF-κB pathways, promotion of epithelial–mesenchymal transition, and modulation of antitumor immune responses (19,20,22). These mechanisms are analogous to those described in colorectal and pancreatic cancers, suggesting a conserved oncogenic role for this organism across tissues (19,23).

The detection of Lactobacillus, Propionibacterium, and Peptostreptococcus species further highlights the complexity of tumor-associated microbial ecosystems. These bacteria influence local pH, generate short-chain fatty acids, and modulate redox balance, which may exert context-dependent pro- or antitumor effects (8,10,15). Such metabolic interactions within the tumor microenvironment may affect epigenetic regulation, immune surveillance, and therapeutic response (11).

A notable finding of this study was the high proportion of culture-negative samples that yielded bacterial DNA upon 16S rRNA sequencing. This underscores the limitations of conventional culture techniques and emphasizes the importance of molecular approaches for accurate microbiome characterization (12,13,21). The integration of sequencing-based methodologies enables the detection of fastidious and uncultivable organisms, providing a more comprehensive understanding of intratumoral microbial diversity (9,21).

Comparative analysis revealed a shift from predominantly commensal species in normal mucosa to opportunistic and pathogenic bacteria in carcinomatous tissues. Similar microbiome transitions have been documented in other malignancies and are associated with activation of Toll-like receptor–mediated signaling pathways, including NF-κB and STAT3, which promote cellular survival, proliferation, and immune tolerance (11,18). Such inflammatory signaling pathways are also central to cardiovascular pathology, reinforcing the concept of shared molecular mechanisms linking microbial dysbiosis, chronic inflammation, and disease progression (24,25).

The clinical implications of these findings are significant. Microbial profiling may offer novel opportunities for early diagnosis, risk stratification, and personalized therapeutic interventions in OSCC (6,13). Noninvasive sampling approaches, such as saliva-based microbiome analysis, could facilitate screening in high-risk populations. Furthermore, modulation of the tumor-associated microbiome through targeted antimicrobial strategies or probiotics may enhance responsiveness to conventional oncologic therapies (15).

Several limitations warrant consideration. The small sample size and cross-sectional design preclude causal inference, and confounding factors such as oral hygiene, dietary habits, and tobacco exposure may have influenced microbial composition. Longitudinal studies incorporating metagenomic, metabolomic, and immune profiling are required to clarify temporal relationships and functional relevance (10,11,23).

Clinical Relevance

Understanding microbial involvement in OSCC has important diagnostic and therapeutic implications. Microbial profiling may aid early detection, risk stratification, and personalized treatment strategies. Modulating tumor-associated microbiota may enhance therapeutic response and reduce recurrence.

OSCC tissues harbor a dysbiotic microbiome enriched with anaerobic and pro-inflammatory bacteria capable of influencing tumor biology. These findings reinforce the emerging paradigm that cancer is a result of complex host–microbe interactions. Further longitudinal and functional studies are warranted to elucidate causal mechanisms and therapeutic potential.

1.             Tandon P, Dadhich A, Saluja H, Bawane S, Sachdeva S. The prevalence of squamous cell carcinoma in different sites of oral cavity at a rural health care centre in Maharashtra: a retrospective 10-year study. Contemp Oncol (Pozn). 2017;21:178–183.

2.             Bugshan A, Farooq I. Oral squamous cell carcinoma: metastasis, potentially associated malignant disorders, etiology and recent advancements in diagnosis. F1000Res. 2020;9:229.

3.             Johnson N. Tobacco use and oral cancer: a global perspective. J Dent Educ. 2001;65:328–339.

4.             Lalremtluangi R, Dangore-Khasbage S. Non-habit-related oral squamous cell carcinoma: a review. Cureus. 2024;16:e54594.

5.             Plummer M, de Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global burden of cancers attributable to infections in 2012. Lancet Glob Health. 2016;4:e609–e616.

6.             Perera M, Al-Hebshi NN, Speicher DJ, et al. Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria. J Oral Microbiol. 2016;8:32762.

7.             Minarovits J. Anaerobic bacterial communities associated with oral carcinoma: intratumoral, surface biofilm and salivary microbiota. Anaerobe. 2021;68:102300.

8.             Cao Y, Xia H, Tan X, et al. Intratumoural microbiota: a new frontier in cancer development and therapy. Signal Transduct Target Ther. 2024;9:15.

9.             Ma J, Huang L, Hu D, et al. The role of the tumor microbe microenvironment in the tumor immune microenvironment. J Exp Clin Cancer Res. 2021;40:327.

10.          Gao F, Yu B, Rao B, et al. The effect of the intratumoral microbiome on tumor occurrence, progression, prognosis and treatment. Front Immunol. 2022;13:1051987.

11.          Peterson J, Garges S, Giovanni M, et al. The NIH Human Microbiome Project. Genome Res. 2009;19:2317–2323.

12.          Talapko J, Erić S, Meštrović T, et al. The impact of oral microbiome dysbiosis on the aetiology, pathogenesis, and development of oral cancer. Cancers. 2024;16:2997.

13.          Wang J, Gao B. Mechanisms and potential clinical implications of oral microbiome in oral squamous cell carcinoma. Curr Oncol. 2024;31:168–182.

14.          Stasiewicz M, Karpiński TM. The oral microbiota and its role in carcinogenesis. Semin Cancer Biol. 2022;86:633–642.

15.          Marwick C. Helicobacter: new name, new hypothesis involving type of gastric cancer. JAMA. 1990;264:2724–2727.

16.          Mager DL, Haffajee AD, Devlin PM, Norris CM, Posner MR, Goodson JM. The salivary microbiota as a diagnostic indicator of oral cancer. J Transl Med. 2005;3:27.

17.          Tateda M, Shiga K, Saijo S, et al. Streptococcus anginosus in head and neck squamous cell carcinoma: implication in carcinogenesis. Int J Mol Med. 2000;6:699–706.

18.          Sasaki M, Yamaura C, Ohara-Nemoto Y, et al. Streptococcus anginosus infection in oral cancer and its infection route. Oral Dis. 2005;11:151–156.

19.          Pushalkar S, Mane SP, Ji X, et al. Microbial diversity in saliva of oral squamous cell carcinoma. FEMS Immunol Med Microbiol. 2011;61:269–277.

20.          Rai AK, Panda M, Das AK, et al. Dysbiosis of salivary microbiome and cytokines influence oral squamous cell carcinoma through inflammation. Arch Microbiol. 2021;203:137–152.

21.          Al-Hebshi NN, Nasher AT, Maryoud MY, et al. Inflammatory bacteriome featuring Fusobacterium nucleatum identified in association with oral squamous cell carcinoma. Sci Rep. 2017;7:1834.

22.          Harrandah AM, Chukkapalli SS, Bhattacharyya I, Progulske-Fox A, Chan EKL. Fusobacteria modulate oral carcinogenesis and promote cancer progression. J Oral Microbiol. 2020;13:1849493.

23.          Yang CY, Yeh YM, Yu HY, et al. Oral microbiota community dynamics associated with oral squamous cell carcinoma staging. Front Microbiol. 2018;9:862.

24.          Binder Gallimidi A, Fischman S, Revach B, et al. Periodontal pathogens promote tumor progression in an oral-specific carcinogenesis model. Oncotarget. 2015;6:22613–22623.

25.          Ganly I, Yang L, Giese RA, et al. Periodontal pathogens as a risk factor of oral cavity squamous cell carcinoma independent of tobacco and alcohol. Int J Cancer. 2019;145:775–784.