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, 104 (2), 751-763

An Exploration of Smokeless Tobacco Product Nucleic Acids: A Combined Metagenome and Metatranscriptome Analysis

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An Exploration of Smokeless Tobacco Product Nucleic Acids: A Combined Metagenome and Metatranscriptome Analysis

R E Tyx et al. Appl Microbiol Biotechnol.

Abstract

Smokeless tobacco (ST) products are used worldwide and are a major public health concern. In addition to harmful chemicals found in these products, microbes found in ST products are believed to be responsible for generating harmful tobacco-specific nitrosamines (TSNAs), the most abundant carcinogens in ST. These microbes also contribute endotoxins and other pro-inflammatory components. A greater understanding of the microbial constituents in these products is sought in order to potentially link select design aspects or manufacturing processes to avoidable increases in harmful constituents. Previous studies looked primarily at bacterial constituents and had not differentiated between viable vs nonviable organisms, so in this study, we sought to use a dual metatranscriptomic and metagenomic analysis to see if differences exist. Using high-throughput sequencing, we observed that there were differences in taxonomic abundances between the metagenome and metatranscriptome, and in the metatranscriptome, we also observed an abundance of plant virus RNA not previously reported in DNA-only studies. We also found in the product tested, that there were no viable bacteria capable of metabolizing nitrate to nitrite. Therefore, the product tested would not be likely to increase TSNAs during shelf storage. We tested only a single product to date using the strategy presented here, but succeeded in demonstrating the value of using of these methods in tobacco products. These results present novel findings from the first combined metagenome and metatranscriptome of a commercial tobacco product.

Keywords: 16S; Metagenome; Metagenomics; Metatranscriptome; Microbial communities; Microbiome; Smokeless; Tobacco.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Cladogram representing taxonomic groups and relative abundance in the a metagenome and b the metatranscriptome of selected smokeless tobacco product. Raw counts were output from IMG/M system using Radial Tree. Data was processed by adjusting all abundances relative to a maximum relative abundance of 1, and then taking the square root of that number, to better illustrate lower abundance taxons. Twenty of the most abundant families are highlighted. Low-abundance phylogeny in either the metagenome or metatranscriptome was excluded and grouped into a category labeled “Others.” The “D.” in the center represents the domain level of taxonomy. GraPhlAn was used to create the cladogram (Asnicar et al. 2015)
Fig. 2
Fig. 2
Distribution of the Firmicutes phylum families, highlighting the differences between metagenome and metatranscriptome. Using the data from the IMG/M-ER system (“Radial Tree” function), we constructed graphs highlighting the change in abundances between the metagenome and metatranscriptome for families present in the Firmicutes phylum
Fig. 3
Fig. 3
Graph of Functional Gene Content (as COGS) of metagenome vs metatranscriptome. Metagenome (orange bars) and metatranscriptome (blue bars) gene hits with COG functional annotation. These tables were combined from individual table outputs using the “with COG” link from “Metagenome Statistics” portion of the Genome Overview in IMG/M-ER. Relative percentages were from the “% of Total” column
Fig. 4
Fig. 4
Targeted Categorical Gene Content heatmaps. Number of estimated gene copies of various COGS representing markers of a nitrogen cycle genes, b antimicrobial resistance genes, c gene transfer, and d phylogenetic marker COGS (as reference). Generated using “Functional Profile” tool in IMG/M-ER

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