Squamous cell carcinomas are the most common kind of oral and maxillofacial malignant tumour, accounting for more than 90% of all cases of oral cancer. Oral cancer is becoming more common across the globe, and despite significant advances in cancer therapy, it still has a high death rate and a low 5-year survival rate (Allavena, 2008) 1. When a person is diagnosed with oral cancer, the disease quickly spreads to other parts of the head and neck, threatening the patient's life and causing severe impairment in areas like swallowing, speech, pain, numbness, and breathing. Although tobacco use, betel nut chewing, and excessive alcohol use are known to increase the likelihood of developing oral squamous cell carcinoma, this only accounts for around 85% of all cases (Berkovits, 2016). 2

Helicobacter pylori was originally implicated as a causative factor in stomach cancer, and its carcinogenicity was shown in the 1990s. 3 Subsequently, other analyses examined the link between microorganisms and secondary malignancies. Specifically, it has been established that Epstein-Barr virus (EBV) infection is connected to both Burkitt lymphoma and nasopharyngeal carcinoma, that Salmonella typhi infection is associated with an increased risk of gallbladder cancer, and that HPV infection is linked to cervical cancer (Perera, 2018). 4 These results offer a foundation for future study of the link between microbes and oral cancers and point the way towards potential new avenues of inquiry. 

Oral bacteria have been linked to tumour formation in several studies; however, the exact mechanism is unclear. From culturing bacteria in the lab through PCR molecular techniques to next-generation sequencing for the identification of 16S rRNA genes and on to metagenomic sequencing, the research process has come a long way. 5 Alterations to the oral microbiota have been linked to an increased risk of cancer in certain studies, whereas in others no such link has been found. Increasing evidence suggests that the mucosal surface, tumour tissue, and saliva of patients with oral squamous cell carcinoma vary considerably from those of people with healthy oral cavities (Eckert, 2018). 6 In many circumstances, the aggressiveness of the cancer patient's medication may change the oral microbiota; the appearance of potential pathogens may result in opportunistic infections in immune-compromised people, increasing morbidity and death. Since only handful of comparative studies have been undertaken in the past to compare the bacterial profile in precancer, cancerous and cancer patients, so we aimed to carry out this research to correlate the bacterial isolates in these patients.

This Hospital based comparative type of cross-sectional study was conducted in the Bacteriology Lab, Department of Microbiology, SMS Medical College, and Jaipur. Data has been collected for about one and half year after taking approval from Research Review board i.e. from Jan 2021 to June 2022. A total of 200 oral swab samples were collected,100 each for both the study groups.

The study population was grouped as:

Group 1: Clinically and Histo-pathologically diagnosed patients with oral squamous cell carcinoma.

Group 2: Patients with clinically diagnosed oral premalignant lesions (Leukoplakia, orallichen planus, erythroplakia, oral submucous fibrosis) which were taken as control for this study.

Out of 200 samples studied from both the groups, most cases were studied in the control i.e Group 2 (32) with an average age of 41-50 years, followed by (31 cases) with an average age of 41-50 years. In the Group 1, a similar pattern was observed; the greatest number of cases (41) 41% were in the age range of 41-50 years, followed by (27) 27% in the age range of 51-60 years as shown in Table 1.

Table 2 shows the distribution of the cases according to sex. In group 2 there were 72 % males. Similar patterns were found in Group 1, with 79% of them being male and the 21% being female.

A total of 8.82% of those in the control (pre-cancer) group consumed BIDI/cigarettes, 70.59 % gutka, and 20.59% of them consumed supari, whereas 21.05 % of those in the cancer group consume BIDI/cigarettes, 73.68 % took gutka, and 22.81% of them consumed supari. Among the control group the maximum lesions (46.08%) were observed on the buccal mucosa, followed by 26% on the tongue. Similar patterns were observed in cancer group (group1); the buccal mucosa had the highest number of cases 42.11%, followed by the tongue 10.53% as depicted in Table 3.

Table 4 shows the distribution of bacteria in both the groups according to gram staining. In the cancer group; the Gram negative bacteria were (79) 69.30%, and the Gram positive bacteria were (35) or 30.7% while in the control (18) 17.65 % were Gram negative bacteria rest 84 (82.35%) were Gram positive bacteria. This observation was statistically significant.

In the cancer group the most common organism isolated was Pseudomonas (39.47%), followed by Klebsiella (22.81%), Escherichia coli (5.26%), and Acinetobacter (3.51%) while Staphylococcus (13.16%) was the most common pathogen found in Gram positive cocci followed by Streptococcus sp. (7.89%) in the control group. Streptococcus sp. (16.67%) was the most common pathogen found in Gram positive cocci followed by Staphylococcus sp. which were (11.76%.) Gram negative bacilli were statistically higher in cancer group cases as compared to control group as depicted by Table 5.

More than 700 different kinds of microbes call the oral cavity home, making it one of the most diverse and complicated microbial ecosystems in the human body. Ecological dysbiosis, often known as an imbalance in the microbiome, has been researched extensively in both human and animal subjects. These are characterized by a decline in the variety of microorganisms and an increase in the prevalence of harmful bacteria. Evidence suggests that ecological dysbiosis may play a role in carcinogenesis. Previous research has shown a link between oral cavity bacteria and other types of cancer.

In this study, most of the patients were males in both the study groups i.e 72% males in control and 79% males in cancerous group, similar results are also mirrored in previous studies of Anjali K et al. (2019) where 72.9% (70) of cancer patients were males and 27.1% (26 cases) were females, with a male-to-female ratio of 2.7:1. Also, the cancer group in previously conducted studied were mostly in the age –group between 41-50 years. Ashreen et al. (2020). Gutkha chewing habit is seen in both the study groups. This observation is similar to Ashreen et al. (2020) study. The distribution of predisposing factors in oral cancer patients has shown that almost 90% of patients had regular uptake of betel nut and betel leaf.

In this study Pseudomonas had a prevalence of 27.31% whereas a previous study of Ashreen et al. (2020) ⁷ reported Pseudomonas prevalence as 36%. Our findings are similar to another study conducted in western European hospitals where P. aeruginosa was one of the most common organisms, constituting 29% of all Gram-negative isolates. However, in regards to isolates from pre-operative patients, the percentage of Pseudomonas was higher in this study. Further studies are also required to rule out the potential of these pathogenic bacteria in role of carcinogenesis.

The microbial population and the host often maintain a state of dynamic equilibrium, but there are exceptions such as specific helpful bacteria that may efficiently combat foreign infections and boost tissues and the immune system. The metabolic activities and the uncontrolled growth of cells might both contribute to tumor development. A previous study has observed that Capnocytophaga gingivalis, Prevotella melaninogenica, and Streptococcus mitis counts were significantly increased among the oral cancer patients and these species could be used as diagnostic markers for oral cancers with 80% sensitivity and 82% specificity, respectively. 8 In cancerous patients an optimized antibiotic prophylaxis is one clinical focus to limit postoperative infections. A combination therapy is most often recommended, and broad-spectrum antibiotics are the drugs of choice because of increased pattern of resistance in these patients. ⁹

Microbial markers' significance in making the tumor-disease connection demonstrates their utility as a noninvasive diagnostic tool for oral cancers. ^{10, 11} Using such a small sample size is the study's main flaw. Oral malignant tumors have been linked to changes in the microbial microbiome, 12 and further testing will confirm their diagnostic utility. Also, we studied only the aerobic bacteria in both the study groups which is another limitation, further studies could be conducted in future studying complete microbiota so that their role in malignant transformation can be linked.

The oral microbiota of persons with oral cancer and precancerous lesions was shown to have significant morphological changes across a wide variety of species. Our research shows that metabolic pathway changes brought about by dysbiosis of the oral microbiota have a direct and detrimental effect on dental health. More research is needed to better understand the relationship between “oral microbiota imbalances and oral cancer”, so that new data may be gathered in support of using microbiome-targeted therapy for disease prevention.