Differences in the Bacteriome of Smokeless Tobacco Products with Different Oral Carcinogenicity: Compositional and Predicted Functional Analysis

Smokeless tobacco (ST) products vary significantly in their oral carcinogenicity. Much is known about the differences in the chemical, but not the bacterial, constituents of these products. In this study, we explored the composition and function of the bacteriome in ST products from four countries using quantitative polymerase chain reaction (qPCR) and 16S rRNA-based next generation sequencing. The bacterial load (16S rRNA copies/gram) was lowest in Swedish snus (3.4 × 10⁶) and highest in Yemeni shammah (6.6 × 1011). A total of 491 species-level taxa, many of which are potentially novel, belonging to 178 genera and 11 phyla were identified. Species richness and diversity were highest for Swedish snus and lowest for Yemeni shammah. Bacillus, Paenibacillus, and Oceanobacillus spp. were the most abundant in American snuff; species of Pseudomonas, Massilia, Propionibacterium, Puniceispirillum, and Gloeothece predominated in Swedish snus. In Sudanese toombak, Facklamia, Desemzia, Atopostipes, and Lysinibacillus spp. accounted for the majority of the bacteriome. Yemeni shammah exclusively contained Bacillus spp. Functional prediction by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that genes encoding cadmium/zinc and nickel transport systems were enriched in the presumptively "high carcinogenicity" products. The bacteriome of ST products thus differed qualitatively, quantitatively, and functionally. The relevance of these differences, particularly with respect to nickel and cadmium, to oral carcinogenesis warrants further investigation.