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      Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis

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          Abstract

          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.

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          Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases.

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            New and traditional smokeless tobacco: comparison of toxicant and carcinogen levels.

            Declining cigarette use and spreading bans on smoking in public places in the United States are encouraging the U.S. cigarette industry to turn to another tobacco category, smokeless tobacco products. Currently, a number of new brands are being test marketed, including Taboka, Marlboro Snus, Camel Snus, and Skoal Dry. We report here levels of tobacco-specific nitrosamines (TSNAs), alkaloids, anions, polycyclic aromatic hydrocarbons (PAH), and volatile aldehydes in these products, and compare them to the most popular traditional moist snuff brands. Total TSNAs averaged 1.97 microg/g dry weight tobacco in Taboka, Marlboro Snus, and Camel Snus, 4.54 microg/g tobacco in Skoal Dry, and 7.42 microg/g tobacco in traditional brands. The amounts of unprotonated nicotine averaged 0.961 mg/g tobacco in Taboka, Marlboro Snus, and Skoal Dry, 7.22 mg/g tobacco in Camel Snus, and 7.57 mg/g tobacco in traditional brands. Levels of minor tobacco alkaloids were relatively high in Taboka, Marlboro Snus, and Skoal Dry, as compared to other products analyzed here. Levels of nitrite and nitrate in new U.S. smokeless tobacco products and the Swedish snus General were lower than those in the other products. Remarkably high levels of chloride and some PAH were observed in the traditional moist snuff. Crotonaldehyde levels were about five times higher in Taboka and Marlboro Snus than in traditional products. The large variation in the levels of some toxicants and carcinogens analyzed here indicates that more effort is required from the U.S. tobacco industry to further reduce their amounts in new and traditional smokeless tobacco products.
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              Human Pathogens Abundant in the Bacterial Metagenome of Cigarettes

              Background Many studies have evaluated chemical, heavy metal, and other abiotic substances present in cigarettes and their roles in the development of lung cancer and other diseases, yet no studies have comprehensively evaluated bacterial diversity of cigarettes and the possible impacts of these microbes on respiratory illnesses in smokers and exposed nonsmokers. Objectives The goal of this study was to explore the bacterial metagenomes of commercially available cigarettes. Methods A 16S rRNA-based taxonomic microarray and cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Normalized microarray data were compared using principal component analysis and hierarchical cluster analysis to evaluate potential differences in microbial diversity across cigarette brands. Results Fifteen different classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples. Most notably, we detected Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia in ≥ 90% of all cigarette samples. Other pathogenic bacteria detected included Campylobacter, Enterococcus, Proteus, and Staphylococcus. No significant variability in bacterial diversity was observed across the four different cigarette brands. Conclusions Previous studies have shown that smoking is associated with colonization by pathogenic bacteria and an increased risk of lung infections. However, this is the first study to show that cigarettes themselves could be the direct source of exposure to a wide array of potentially pathogenic microbes among smokers and other people exposed to secondhand smoke. The overall public health implications of these findings are unclear at this time, and future studies are necessary to determine whether bacteria in cigarettes could play important roles in the development of both infectious and chronic respiratory diseases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                19 January 2016
                2016
                : 11
                : 1
                : e0146939
                Affiliations
                [1 ]Division of Laboratory Sciences at the Centers for Disease Control and Prevention, Atlanta, GA, United States of America
                [2 ]Division of Reproductive Health, Center for Disease Control and Prevention, Atlanta, GA, United States of America
                [3 ]Oak Ridge Institute of Science and Education, Oak Ridge, TN, United States of America
                [4 ]Battelle Analytical Services, Atlanta, GA, United States of America
                University Paris South, FRANCE
                Author notes

                Competing Interests: The commercial affiliation of authors (LMK) does not alter their adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: RET SBS LMK AJR GAS CHW. Performed the experiments: RET LMK AJR GAS. Analyzed the data: RET SBS LMK AJR GAS. Contributed reagents/materials/analysis tools: RET SBS GAS CHW. Wrote the paper: RET SBS LMK AJR GAS CHW.

                Article
                PONE-D-15-47985
                10.1371/journal.pone.0146939
                4718623
                26784944
                5195db4e-6fd0-41ee-89cc-afe4a22e04f5

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 2 November 2015
                : 27 December 2015
                Page count
                Figures: 6, Tables: 3, Pages: 21
                Funding
                Battelle Analytical Services, through a contract with U.S. Centers for Disease control and Prevention, provided support in the form of salaries for authors (LMK), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Custom metadata
                Sequence Read Archive (SRA), SRR2157163. BioSample, SAMN03956429. BioProject, PRJNA291915.

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                Uncategorized

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