Fungal Community Composition and Function Associated with Loose Smokeless Tobacco Products.

Smokeless tobacco products (STPs) contain several microbial communities which are responsible for the formation of carcinogens, like tobacco-specific nitrosamine (TSNAs). A majority of STPs are sold in loose/unpackaged form which can be loaded with a diverse microbial population. Here, the fungal population and mycotoxins level of three popular Indian loose STPs, Dohra, Mainpuri Kapoori (MK), and loose leaf-chewing tobacco (LCT) was examined using metagenomic sequencing of ITS1 DNA segment of the fungal genome and LC-MS/MS, respectively. We observed that Ascomycota was the most abundant phylum and Sterigmatomyces and Pichia were the predominant fungal genera in loose STPs. MK displayed the highest α-diversity being enriched with pathogenic fungi Apiotrichum, Aspergillus, Candida, Fusarium, Trichosporon, and Wallemia. Further, FUNGuild analysis revealed an abundance of saprotrophs in MK, while pathogen-saprotroph-symbiotroph were abundant in Dohra and LCT. The level of a fungal toxin (ochratoxins A) was high in the MK product. This study caution that loose STPs harbor various harmful fungi that can infect their users and deliver fungal toxins or disrupt the oral microbiome of SLT users which can contribute to several oral pathologies.

Taxonomic and functional profiling of Indian smokeless tobacco bacteriome uncovers several bacterial-derived risks to human health.

Smokeless tobacco (ST) consumption keeps human oral health at high risk which is one of the major reasons for oral tumorigenesis. The chemical constituents of the ST products have been well discussed; however, the inhabitant microbial diversity of the ST products is less explored especially from south Asian regions. Therefore, the present investigation discusses the bacteriome-based analysis of indigenous tobacco products. The study relies on 16S amplicon-based bacteriome analysis of Indian smokeless tobacco (ST) products using a metagenomic approach. A total of 59,15,143 high-quality reads were assigned to 34 phyla, 82 classes, 176 orders, 256 families, 356 genera, and 154 species using the SILVA database. Of the phyla (> 1%), Firmicutes dominate among the Indian smokeless tobacco followed by Proteobacteria, Bacteroidetes, and Actinobacteria (> 1%). Whereas, at the genera level (> 1%), Lysinibacillus, Dickeya, Terribacillus, and Bacillus dominate. The comparative analysis between the loose tobacco (LT) and commercial tobacco (CT) groups showed no significant difference at the phyla level, however, only three genera (Bacillus, Aerococcus, and Halomonas) were identified as significantly different between the groups. It indicates that CT and LT tobacco share similar bacterial diversity and poses equal health risks to human oral health. The phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt 2.0) based analysis uncovered several genes involved in nitrate/nitrite reduction, biofilm formation, and pro-inflammation that find roles in oral pathogenesis including oral cancer. The strong correlation analysis of these genes with several pathogenic bacteria suggests that tobacco products pose a high bacterial-derived risk to human health. The study paves the way to understand the bacterial diversity of Indian smokeless tobacco products and their putative functions with respect to human oral health. The study grabs attention to the bacterial diversity of the smokeless tobacco products from a country where tobacco consumers are rampantly prevalent however oral health is of least concern.

Delineating the Bacteriome of Packaged and Loose Smokeless Tobacco Products Available in North India.

Smokeless tobacco product (STP) consumption is a significant public health threat across the globe. STPs are not only a storehouse of carcinogens and toxicants but also harbor microbes that aid in the conversion of tobacco alkaloids to carcinogenic tobacco-specific nitrosamines (TSNAs), thereby posing a further threat to the health of its consumers. The present study analyzed the bacterial diversity of popular dry and loose STPs by 16S rRNA gene sequencing. This NGS-based investigation revealed four dominant phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria and identified 549 genera, Prevotella, Bacteroides, and Lactobacillus constituting the core bacteriome of these STPs. The most significantly diverse bacteriome profile was displayed by the loose STP Mainpuri kapoori. The study further predicted the functional attributes of the prevalent genera by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) algorithm. Genes encoding for nitrate and nitrite reduction and transport enzymes, antibiotic resistance, multi-drug transporters and efflux pumps, secretion of endo- and exotoxin, and other pro-inflammatory molecules were identified. The loose STPs showed the highest level of nitrogen metabolism genes which can contribute to the synthesis of TSNAs. This study reveals the bacteriome of Indian domestic loose STPs that stagger behind in manufacturing and storage stringencies. Our results raise an alarm that the consumption of STPs harboring pathogenic genera can potentially lead to the onset of several oral and systemic diseases. Nevertheless, an in-depth correlation analysis of the microbial diversity of STPs and their elicit impact on consumer health is warranted. KEY POINTS: • Smokeless tobacco harbors bacteria that aid in synthesis of carcinogenic nitrosamines. • Most diverse bacteriome profile was displayed by loose smokeless tobacco products. • Pathogenic genera in these products can harm the oral and systemic health of users.

An exploration of smokeless tobacco product nucleic acids: a combined metagenome and metatranscriptome analysis.

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.

Microbiological safety of areca nut-containing, ready-to-eat chewing substances common among Pakistani paediatric population: A pilot study.

OBJECTIVE: To evaluate microbiological contamination of areca nut-containing, ready-to-eat chewing substances easily accessible to vulnerable paediatric population. METHODS: A pilot study was conducted at the Aga Khan University Medical College from June to October 2016 on twelve samples of areca nut-containing chewing substances (four supari, paan masala and gutka each) collected from various localities of Karachi. These were evaluated individually for total colony counts, hygiene indicator organisms, pathogenic organisms, and levels of aflatoxin. Microbial contamination was analysed using pour-plate method. Fungal aflatoxin levels were measured by enzyme-linked immunosorbent assay (ELISA).. RESULTS: Wet gutka preparations were contaminated by Escherichia coli and Enterobacteriacaea. High levels of fungal aflatoxin (range: 0.43-1.84 mg/kg), a proven carcinogen, were identified in all the 12(100%) products. No sample contained pathogenic bacteria. However, 1(8.33%) sample did not meet hygiene criteria cut-off. CONCLUSIONS: Habitual use of unhygienic chewing substances containing fungal toxins is a public health concern that needs to be addressed through a preventative, behaviour-changing strategy..

Fungal contamination in smokeless tobacco products traditionally consumed in Pakistan.

OBJECTIVE: To isolate potential pathogenic fungi from smokeless tobacco products. METHODS: The study was conducted from January 2015 to February 2017 during which samples of smokeless tobacco products such as Mainpuri, Tambako, Khiwam, Gutkha, Naswar and Mawa etc. were collected from different cities of Pakistan. The samples were tested for fungal contamination by spread plate method. Different strains of fungi were isolated and identified on the basis of their macroscopic as well as microscopic characteristics. The fungal strains isolated were also screened for their susceptibility to commonly used antifungal drugs by disc diffusion method. RESULTS: Of the 600 samples collected, 300(50%) were from Sindh, 70(11.7%) Balochistan, 74(12.3%) from Khyber Pakhtunkhwa, 105(17.5%) from Punjab and 51(8.5%) from Azad Kashmir. In terms of products, there were 404(67.3%) samples of Naswar, 69(11.5%) Patti, 40(6.6%) Khiwam, 35(5.8%) Mawa, 32(5.3%) Gutkha, and 20(3.3%) Mainpuri samples. Different species of Aspergillus were predominantly isolated followed by Penicillium, Mucor, Sepedonium and Trichophyton. The isolated strains of Aspergillus also revealed resistance against many commonly-used anti-fungals such as Amphotericin B and Itraconazole.. CONCLUSIONS: There was high prevalence of opportunistic fungi in study samples, posing a threat for human health which requires prompt notice and management.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 107 CFU g-1. Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Restoring the Duality between Principal Components of a Distance Matrix and Linear Combinations of Predictors, with Application to Studies of the Microbiome.

Appreciation of the importance of the microbiome is increasing, as sequencing technology has made it possible to ascertain the microbial content of a variety of samples. Studies that sequence the 16S rRNA gene, ubiquitous in and nearly exclusive to bacteria, have proliferated in the medical literature. After sequences are binned into operational taxonomic units (OTUs) or species, data from these studies are summarized in a data matrix with the observed counts from each OTU for each sample. Analysis often reduces these data further to a matrix of pairwise distances or dissimilarities; plotting the first two or three principal components (PCs) of this distance matrix often reveals meaningful groupings in the data. However, once the distance matrix is calculated, it is no longer clear which OTUs or species are important to the observed clustering; further, the PCs are hard to interpret and cannot be calculated for subsequent observations. We show how to construct approximate decompositions of the data matrix that pair PCs with linear combinations of OTU or species frequencies, and show how these decompositions can be used to construct biplots, select important OTUs and partition the variability in the data matrix into contributions corresponding to PCs of an arbitrary distance or dissimilarity matrix. To illustrate our approach, we conduct an analysis of the bacteria found in 45 smokeless tobacco samples.

Bacterial Populations Associated with Smokeless Tobacco Products.

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 × 106 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 101 CFU/g STP) and some chewing tobacco products (average of 2.54 × 105 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. IMPORTANCE: 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.

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

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.