Associations between microbial communities and key chemical constituents in U.S. domestic moist snuff

Abstract

Background Smokeless tobacco (ST) products are widely used throughout the world and contribute to morbidity and mortality in users through an increased risk of cancers and oral diseases. Bacterial populations in ST contribute to taste, but their presence can also create carcinogenic, Tobacco-Specific N-nitrosamines (TSNAs). Previous studies of microbial communities in tobacco products lacked chemistry data (e.g. nicotine, TSNAs) to characterize the products and identify associations between carcinogen levels and taxonomic groups. This study uses statistical analysis to identify potential associations between microbial and chemical constituents in moist snuff products. Methods We quantitatively analyzed 38 smokeless tobacco products for TSNAs using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nicotine using gas chromatography with mass spectrometry (GC-MS). Moisture content determinations (by weight loss on drying), and pH measurements were also performed. We used 16S rRNA gene sequencing to characterize the microbial composition, and additionally measured total 16S bacterial counts using a quantitative PCR assay. Results Our findings link chemical constituents to their associated bacterial populations. We found core taxonomic groups often varied between manufacturers. When manufacturer and flavor were controlled for as confounding variables, the genus Lactobacillus was found to be positively associated with TSNAs. while the genera Enteractinococcus and Brevibacterium were negatively associated. Three genera (Corynebacterium, Brachybacterium, and Xanthomonas) were found to be negatively associated with nicotine concentrations. Associations were also investigated separately for products from each manufacturer. Products from one manufacturer had a positive association between TSNAs and bacteria in the genus Marinilactibacillus. Additionally, we found that TSNA levels in many products were lower compared with previously published chemical surveys. Finally, we observed consistent results when either relative or absolute abundance data were analyzed, while results from analyses of log-ratio-transformed abundances were divergent.

What Can We Learn about the Bias of Microbiome Studies from Analyzing Data from Mock Communities?

“…To create the ZMC + Matrix samples, a 75 µL volume of the ZMC solution was pipetted directly onto a 0.5 g snus tobacco sample and vortexed for 30 s. Before continuing with extractions, a 10 min incubation at room temperature was executed to allow complete absorption of ZMC microbes onto the snus substrate, to better simulate a real tobacco product. Sample processing of the tobacco samples is as described in Tyx et al (2022) [ 10 ].…”

“…For the ZMC, ZMC + Matrix and Swedish Snus data we performed four replicate extractions for each protocol; thus, these sample sets each have 16 observations. The smokeless tobacco samples comprise six different smokeless tobacco products that were purchased in 2016 by Lab Depot (Atlanta, GA, USA) and shipped to CDC [ 10 ]. The four extraction protocols were run in triplicate for each of the six products for a total of samples.…”

“…All replicates from all four sample sets were then subject to 16S rRNA sequencing as described in [ 10 ]. We did not observe any significant or consistent trends in recovered DNA using the various modifications of our extraction process; for additional information see Supplementary Text S1 .…”

“…The motivation for this work stems from surveys of the bacterial content in smokeless tobacco products sold in the US [ 10 ]. Cell wall degrading enzymes are commonly used in microbial nucleic acid extraction procedures to obtain more consistent and complete recovery of bacterial nucleic acids.…”

Microbial community and functions involved in smokeless tobacco product: a metagenomic approach

“…STPs contain a diverse microbial community that can metabolize the chemical constituents of the STP and synthesize various carcinogens. The TSNAs were detected during the curing and senescence of a tobacco product after microbial conversion of nitrate to nitrite and its abiotic reaction with the tobacco alkaloid molecules (Tyx et al 2022 ). Previous studies conducted on STPs have largely employed culture-based methods that may have skewed data results and inaccurately depicted community diversity estimates (Saleem et al 2018 ; Shetty and Hegde 2015 ).…”