Compositional profile of mucosal bacteriome of smokers and smokeless tobacco users

Abstract

Introduction: Smoked, and especially smokeless, tobacco are major causes of oral cancer globally. Here, we examine the oral bacteriome of smokers and of smokeless tobacco users, in comparison to healthy controls, using 16S rRNA gene sequencing.

Methods: Oral swab samples were collected from smokers, smokeless tobacco users, and healthy controls (n = 44). Microbial DNA was extracted and the 16S rRNA gene profiled using the Illumina MiSeq platform. Sequencing reads were processed using DADA2, and taxonomical classification was performed using the phylogenetic placement method. Differentially abundant taxa were identified using DESeq2, while functional metagenomes based on KEGG orthology abundance were inferred using LIMMA.

Results: A significantly higher microbial diversity was observed in smokeless tobacco users and smokers relative to controls (P < 0.05). Compositional differences in microbial communities were observed in all comparisons with healthy controls (PERMANOVA P < 0.05) but not between smokers and smokeless tobacco users. Levels of Fusobacterium spp., Saccharibacterium spp., and members of Shuttleworthia were elevated in smokers when compared to controls (BH adj P < 0.01). In addition, the relative abundance of three bacterial taxa belonging to genera Fusobacterium spp., Catonella, and Fretibacterium spp. was significantly increased in smokeless tobacco users relative to controls (BH adj P < 0.01). Major functional pathways significantly increased in smokeless tobacco users relative to both controls, and smokers were similar and involved amino acid metabolism including glutamate and aspartate biosynthesis and degradation (log FC > 1.5; BH adj P < 0.01).

Conclusions: A distinct taxonomic and functional profile of oral microbiome in smokers and smokeless tobacco users as compared to healthy controls implicates a significant role of microbes and their metabolites in diseases associated with tobacco use including oral cancer.

Clinical relevance: Future efforts in preventive, diagnostic, curative, and prognostic strategies for diseases associated with tobacco use in smokers and smokeless tobacco users could incorporate the oral microbiome.

Keywords: Microbiome; Microbiota; Oral microbiome; Smokeless tobacco; Smoking; Tobacco.