EVALUATING THE BACTERIAL DIVERSITY OF SMOKELESS TOBACCO PRODUCT USING SHOTGUN METAGENOMIC ANALYSIS

. Naswar is a smokeless tobacco product, commonly consumed in Pashtun population of Pakistan and Afghanistan. Despite being an established risk factor for oral cancer, not much is known about microbiological constituents of Naswar. Objectives: The objective of the study was to explore bacterial diversity in Naswar using shotgun metagenomic sequencing. Methodology: DNA was extracted from twelve (n = 12) famous brands of Naswar samples using an enzymatic and mechanical lysis method followed by next generation sequencing on Illumina Miseq platform. Results: Naswar samples exhibits differential abundance profile at all taxonomic levels with Khamar brand having the highest species richness and diversity indices. Of the total 22 phyla identified, phylum Proteobacteria was the most abundant (88.4%) followed by Bacteroidetes (4.71%) and Firmicutes (3.73%). At genus level, 234 genera (range: 32 – 132) were identified. Metagenomic sequencing also revealed presence of 455 (range: 44 – 210) different bacterial species in Naswar samples. The most common species were Pesudomonas aurgenosa , Yersinia enterocolitica , Salmonella enterica , Xanthomonas campestris , and E. coli . Some of the species are of concern due their ability to cause infections in humans. Conclusions: Naswar products harbor diverse bacterial microbiota with differential abundance at phylum, genus and species level. These findings may have several implications in terms of further research to explore microbiological associated health risks and provide scientific evidence to inform potential regulations regarding manufacturing and testing of Naswar in Pakistan.