Exploratory Assessment of Oral Microbiome Dysbiosis Associated with Chronic Smokeless Tobacco Use: A Sibling-Based Study

Smokeless tobacco (SLT) consumption is a significant risk factor for oral dysbiosis and oral cancer in South Asian countries, including India. However, its impact on oral microbial ecology, functional restructuring, and sex-specific microbial responses remains unclear. The present study aimed to evaluate tobacco-associated oral microbiome dysbiosis across different subject groups (addicted male/AM, addicted female/AF, non-addicted male/NAM, and non-addicted female/NAF) using a controlled sibling-based pooled temporal representative framework. To reduce short-term temporal variability and generate representative oral microbiome profiles, oral gargle samples were collected daily for seven days and pooled within each study group prior to DNA extraction and 16S rRNA gene sequencing. Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla in all samples. Addicted users showed a higher Firmicutes/Bacteroidetes ratio, fewer cultivable species, and more pathobionts, along with lower Shannon diversity in tobacco addiction and a partial separation in beta diversity between addicted and non-addicted samples. The enrichment of Streptococcus, Gemella, Neisseria, Veillonella, Acinetobacter, and Pseudomonas in tobacco-associated microbiomes and Comamonas, Chryseobacterium, Delftia, Flavobacterium, and related taxa in non-addicted individuals were consistently identified by all three analyses, that is, differential abundance, STAMP, and SIMPER. A shared core oral microbiome and unique “tobacco-associated” microbial signatures were observed. Orotype mapping showed that the transition in the microbial community to a Streptococcus-dominated (S-type) dysbiotic state was driven by tobacco addiction, introducing a novel conceptual framework, the Tobacco-Associated Dysbiosis Index (TDI), which showed higher values in addicted participants. Furthermore, functional prediction analysis revealed increased enrichment of ammonia oxidation, dehalogenation, sulfate reduction, nitrite reduction, chitin degradation, and xenobiotic metabolism pathways, highlighting microbial adaptation towards detoxification. In summary, chronic exposure to smokeless tobacco is linked to ecological imbalance, pathobiont enrichment, loss of microbial diversity, functional dysbiosis, and metabolic reprogramming, suggesting descriptive ecological patterns that may be relevant to chronic inflammation and oral carcinogenesis.