Microbial communities and gene contributions in smokeless tobacco products.
ABSTRACT: Smokeless tobacco products (STP) contain bacteria, mold, and fungi due to exposure from surrounding environments and tobacco processing. This has been a cause for concern since the presence of microorganisms has been linked to the formation of highly carcinogenic tobacco-specific nitrosamines. These communities have also been reported to produce toxins and other pro-inflammatory molecules that can cause mouth lesions and elicit inflammatory responses in STP users. Moreover, microbial species in these products could transfer to the mouth and gastrointestinal tract, potentially altering the established respective microbiotas of the consumer. Here, we present the first metagenomic analysis of select smokeless tobacco products, specifically US domestic moist and dry snuff. Bacterial, eukaryotic, and viral species were found in all tobacco products where 68% of the total species was comprised of Bacteria with 3 dominant phyla but also included 32% Eukarya and 1% share abundance for Archaea and Viruses. Furthermore, 693,318 genes were found to be present and included nitrate and nitrite reduction and transport enzymes, antibiotic resistance genes associated with resistance to vancomycin, β-lactamases, their derivatives, and other antibiotics, as well as genes encoding multi-drug transporters and efflux pumps. Additional analyses showed the presence of endo- and exotoxin genes in addition to other molecules associated with inflammatory responses. Our results present a novel aspect of the smokeless tobacco microbiome and provide a better understanding of these products' microbiology. KEY POINTS: • The findings presented will help understand microbial contributions to overall STP chemistries. • Gene function categorization reveals harmful constituents outside canonical forms. • Pathway genes for TSNA precursor activity may occur at early stages of production. • Bacteria in STPs carry antibiotic resistance genes and gene transfer mechanisms.
Project description:Use of smokeless tobacco products (STPs) is associated with oral cavity cancer and other health risks. Comprehensive analysis for chemical composition and toxicity is needed to compare conventional and newer STPs with lower tobacco-specific nitrosamines (TSNAs) yields. Seven conventional and 12 low-TSNA moist snuff products purchased in the U.S., Sweden, and South Africa were analyzed for 18 chemical constituents (International Agency for Research on Cancer classified carcinogens), pH, nicotine, and free nicotine. Chemicals were compared in each product using Wilcoxon rank-sum test and principle component analysis (PCA). Conventional compared to low-TSNA moist snuff products had higher ammonia, benzo[a]pyrene, cadmium, nickel, nicotine, nitrate, and TSNAs and had lower arsenic in dry weight content and per mg nicotine. Lead and chromium were significantly higher in low-TSNA moist snuff products. PCA showed a clear difference for constituents between conventional and low-TSNA moist snuff products. Differences among products were reduced when considered on a per mg nicotine basis. As one way to contextualize differences in constituent levels, probabilistic lifetime cancer risk was estimated for chemicals included in The University of California's carcinogenic potency database (CPDB). Estimated probabilistic cancer risks were 3.77-fold or 3-fold higher in conventional compared to low-TSNA moist snuff products under dry weight or under per mg nicotine content, respectively. In vitro testing for the STPs indicated low level toxicity and no substantial differences. The comprehensive chemical characterization of both conventional and low-TSNA moist snuff products from this study provides a broader assessment of understanding differences in carcinogenic potential of the products. In addition, the high levels and probabilistic cancer risk estimates for certain chemical constituents of smokeless tobacco products will further inform regulatory decision makers and aid them in their efforts to reduce carcinogen exposure in smokeless tobacco products.
Project description:Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 ?g/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA's proposed product standard to be consistently compliant.
Project description:There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 × 10<sup>6</sup> CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 10<sup>1</sup> CFU/g STP) and some chewing tobacco products (average of 2.54 × 10<sup>5</sup> CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs.<h4>Importance</h4>It is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the potential microbiological risks associated with STP use and provide a baseline microbiological profile of STPs. Several bacterial species were identified that are of possible concern due to their potential to cause opportunistic infections. In addition, some species have abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. Overall, this report provides a microbiological baseline to help fill knowledge gaps related to the microbiological risks of STPs and to inform potential regulations regarding the manufacture and testing of STPs.
Project description:Smokeless tobacco products (STP) contain diverse microbial communities that contribute to the formation of harmful chemical byproducts. This is concerning since 300 million individuals around the globe are users of smokeless tobacco. Significant evidence has shown that microbial metabolic activities mediate the formation of carcinogens during manufacturing. In recent years, studies have revealed a series of additional health impacts that include lesions and inflammation of the oral mucosa and the gastrointestinal tract, as well as alterations of the endogenous microbiota. These findings are due to recent developments in molecular technologies that allowed researchers to better examine the microbial component of these products. This new information illustrates the scale of the STP microbiota and its diversity in the finished product that is sold for consumption. Additionally, the application of metagenomics and metatranscriptomics has provided the tools to look at phylogenies across bacterial, viral, and eukaryotic groups, their functional capacities, and viability. Here we present key examples of tobacco microbiology research that utilizes newer approaches and strategies to define the microbial component of smokeless tobacco products. We also highlight challenges in these approaches, the knowledge gaps being filled, and those gaps that warrant further study. A better understanding of the microbiology of STP brings vast public health benefits. It will provide important information for the product consumer, impact manufacturing practices, and provide support for the development of attainable and more meaningful regulatory goals. KEY POINTS: Newer technologies allowed quicker and more comprehensive identification of microbes in tobacco samples, encapsulating microorganisms difficult or impossible to culture. Current research in smokeless tobacco microbiology is filling knowledge gaps previously unfilled due to the lack of suitable approaches. The microbial ecology of smokeless tobacco presents a clearer picture of diversity and variability not considered before.
Project description:The major components of 70 brands of smokeless tobacco products (STPs) from Sweden and the US were determined to provide greater understanding of the general chemical composition of these products. Various styles of STPs were examined: loose and portion snus from Sweden, and chewing tobacco, dry snuff, moist snuff, hard pellet, soft pellet and plug from the US. The components analysed were major STP components such as water, nicotine, sugars, humectants, sodium ions, chloride ions and ash. The relative quantities of the components varied significantly between different styles of STP. The major component of moist snuff and Swedish loose snus is water. With Swedish portion snus water and pouch material comprise more than half of the product mass; with chewing tobaccos water and sugars comprise around 60% of the products. With these STPs, tobacco was a minor component (30-35%) of the product mass. By way of contrast, tobacco comprised the majority (around 70-90%) of the product mass with dry snuff, hard pellet and soft pellet products. Additives such as sugars, propylene glycol, glycerol, and sodium chloride comprised up to around 12% of the STPs, except for plug and chewing tobaccos where sugars comprised 15-30% by mass of the STP on average. Significant disagreements were found amongst alternative methods of determining water/moisture content for STPs. In particular the oven method, commonly used to determine moisture in tobacco, gave significantly higher values than the Karl Fischer water method when propylene glycol was present. Smaller but similar differences were found using the Near-Infrared method. Choice of measurement technique has important consequences for accuracy of toxicant levels when reporting on a dry-weight basis, a commonly used parameter in smokeless tobacco research and emerging regulatory standards. Conversion to a DWB was also found to produce a preferential bias between and within different STP categories in favour of drier products. These data provide greater understanding of differences in the compositions of contemporary smokeless tobacco products, and demonstrate challenges associated with conversion of actual product contents to dry weight basis values.
Project description:Nicotine, the psychoactive ingredient in tobacco, is metabolically inactivated by CYP2A6 to cotinine. CYP2A6 also activates procarcinogenic tobacco-specific nitrosamines (TSNA). Genetic variation in CYP2A6 is known to alter smoking quantity and lung cancer risk in heavy smokers. Our objective was to investigate how CYP2A6 activity influences tobacco consumption and procarcinogen levels in light smokers and smokeless tobacco users. Cigarette smokers (n = 141), commercial smokeless tobacco users (n = 73) and iqmik users (n = 20) were recruited in a cross-sectional study of Alaska Native people. The participants' CYP2A6 activity was measured by both endophenotype and genotype, and their tobacco and procarcinogen exposure biomarker levels were also measured. Smokers, smokeless tobacco users and iqmik users with lower CYP2A6 activity had lower urinary total nicotine equivalents (TNE) and (methylnitrosamino)-1-(3)pyridyl-1-butanol (NNAL) levels (a biomarker of TSNA exposure). Levels of N-nitrosonornicotine (NNN), a TSNA metabolically bioactivated by CYP2A6, were higher in smokers with lower CYP2A6 activities. Light smokers and smokeless tobacco users with lower CYP2A6 activity reduce their tobacco consumption in ways (e.g. inhaling less deeply) that are not reflected by self-report indicators. Tobacco users with lower CYP2A6 activity are exposed to lower procarcinogen levels (lower NNAL levels) and have lower procarcinogen bioactivation (as indicated by the higher urinary NNN levels suggesting reduced clearance), which is consistent with a lower risk of developing smoking-related cancers. This study demonstrates the importance of CYP2A6 in the regulation of tobacco consumption behaviors, procarcinogen exposure and metabolism in both light smokers and smokeless tobacco users.
Project description:BACKGROUND:How carcinogen exposure varies across users of different, particularly noncigarette, tobacco products remains poorly understood. METHODS:We randomly selected 165 participants of the Golestan Cohort Study from northeastern Iran: 60 never users of any tobacco, 35 exclusive cigarette, 40 exclusive (78% daily) waterpipe, and 30 exclusive smokeless tobacco (nass) users. We measured concentrations of 39 biomarkers of exposure in 4 chemical classes in baseline urine samples: tobacco alkaloids, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), and volatile organic compounds (VOC). We also quantified the same biomarkers in a second urine sample, obtained 5 years later, among continuing cigarette smokers and never tobacco users. RESULTS:Nass users had the highest concentrations of tobacco alkaloids. All tobacco users had elevated TSNA concentrations, which correlated with nicotine dose. In both cigarette and waterpipe smokers, PAH and VOC biomarkers were higher than never tobacco users and nass users, and highly correlated with nicotine dose. PAH biomarkers of phenanthrene and pyrene and two VOC metabolites (phenylmercapturic acid and phenylglyoxylic acid) were higher in waterpipe smokers than in all other groups. PAH biomarkers among Golestan never tobacco users were comparable to those in U.S. cigarette smokers. All biomarkers had moderate to good correlations over 5 years, particularly in continuing cigarette smokers. CONCLUSIONS:We observed two patterns of exposure biomarkers that differentiated the use of the combustible products (cigarettes and waterpipe) from the smokeless product. Environmental exposure from nontobacco sources appeared to contribute to the presence of high levels of PAH metabolites in the Golestan Cohort. IMPACT:Most of these biomarkers would be useful for exposure assessment in a longitudinal study.
Project description:The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.
Project description:The use of smokeless tobacco products (STPs) can cause many serious health problems. The oral microbiota plays important roles in oral and systemic health, and the disruption in the oral microbial population is linked to periodontal disease and other health problems. To assess the impact of smokeless tobacco on oral microbiota in vivo, high-throughput sequencing was used to examine the oral microbiota present in Syrian Golden hamster cheek pouches. Sixteen hamsters were divided into four groups and treated with the STP Grizzly snuff (0, 2.5, 25, or 250?mg) twice daily for 4 weeks. After 0, 1, 2, 3, and 4 weeks of treatment, bacterial genomic DNA was extracted from oral swabs sampled from the cheek pouches of the hamsters. The oral bacterial communities present in different hamster groups were characterized by sequencing the hypervariable regions V1-V2 and V4 of 16S rRNA using the Illumina MiSeq platform. Fifteen phyla, 27 classes, 59 orders, 123 families, and 250 genera were identified from 4,962,673 sequence reads from the cheek pouch samples. The bacterial diversity and taxonomic abundances for the different treatment groups were compared to the non-treated hamsters. Bacterial diversity was significantly decreased after 4 weeks of exposure to 2.5?mg, and significantly increased by exposure to 250?mg STP. Treatment with 250?mg STP significantly increased Firmicutes, transiently increased Cyanobacteria and TM7, and decreased Bacteroidetes and Fusobacteria compared to the control group. At the genus level, 4 weeks of administration of 250?mg STP significantly increased Granulicatella, Streptococcus, Oribacterium, Anaerococcus, Acidaminococcus, Actinomyces, Eubacterium, Negativicoccus, and Staphylococcus, and decreased Bacteroides, Buleidia, Dialister, and Leptotrichia, and transiently decreased Arcanobacterium compared to the control group. For the first time, an animal model was used for evaluating the effects of STP on oral microbiota by metagenomic sequencing. Our results provide a view of the shift of the oral microbiota in response to STP exposure in Syrian Golden hamster. Our findings indicate that the use of smokeless tobacco significantly disrupts the oral microbiota.