The Bacterial Communities of Little Cigars and Cigarillos Are Dynamic Over Time and Varying Storage Conditions

Abstract

Despite their potential importance with regard to tobacco-related health outcomes, as well as their hypothesized role in the production of tobacco-specific N-nitrosamines, bacterial constituents of tobacco products lack characterization. Specifically, to our knowledge, there has been no comprehensive characterization of the effects of storage conditions on the bacterial communities associated with little cigars and cigarillos. To address this knowledge gap, we characterized the bacterial community composition of the tobacco and wrapper components of the following four products: Swisher Sweets Original; Swisher Sweets, Sweet Cherry; Cheyenne Cigars Full Flavor 100's; and Cheyenne Menthol Box. Each product was stored under three different conditions of temperature and relative humidity to mimic different user storage conditions: room (20°C 50% RH), refrigerator (5°C 18% RH) and pocket (25°C 30% RH). On days 0, 5, 9 and 14, subsamples were collected, the wrapper and tobacco were separated, and their total DNA was extracted separately and purified. Resulting DNA was then used in PCR assays targeting the V3 V4 region of the bacterial 16S rRNA gene, followed by sequencing using Illumina HiSeq 300bp PE. Resulting sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package, followed by analyses in R using the Phyloseq and Vegan packages. A single bacterial phylum, Firmicutes, dominated in the wrapper subsamples whereas the tobacco subsamples were dominated by Proteobacteria. Cheyenne Menthol Box (CMB) samples were characterized by significant differential abundances for 23 bacterial operational taxonomic units (OTUs) in tobacco subsamples and 27 OTUs in the wrapper subsamples between day 0 and day 14 under all conditions. OTUs from the genera Acinetobacter and Bacillus significantly increased in the CMB tobacco subsamples, and OTUs from Bacillus, Streptococcus, Lactobacillus, and Enterococcus significantly increased in the CMB wrapper subsamples over time. These initial results suggest that the bacterial communities of little cigars and cigarillos are dynamic over time and varying storage conditions.

Keywords: 16S rRNA gene sequencing analysis; bacterial exposure; bacterial microbiota; cigarillos; little cigars; tobacco components.

FIGURE 1

Heatmap displaying the normalized relative abundance of the top 20 operational taxonomic units (OTUs) in each tobacco and wrapper components across the little cigar brands tested. OTU taxonomic assignments were performed at the species level whenever possible, or a higher taxonomic level whenever species-level assignment was not possible. Numbers between parentheses represent OTU numbers.

FIGURE 2

Alpha-diversity analysis of little cigar brands and components over time and under different storage conditions. Alpha-diversity was measured for tobacco (red) and wrapper (blue) components using the Observed richness metric and compared using ANOVA with Tukey’s HSD (honestly significant difference) post hoc test. Significance levels: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. The letters over the boxes denote statistical significance assessed by Tukey’s HSD test comparing time points for each tobacco component: time points with different letters are significantly different (p < 0.05) in their Observed richness, whereas time points with the same letter indicate no significant differences.

FIGURE 3

Changes in beta-diversity over time and under different storage conditions. Beta-diversity was calculated using principal coordinates analysis (PCoA) of Bray–Curtis dissimilarity, and averages of PCoA Axis 1 values were plotted over time for each brand and component (T: tobacco; W: wrapper) of little cigars stored under different conditions. PCoA axis 1 was chosen as it captures most of the variation in dissimilarity in the dataset (40.7% of variation explained by Axis 1 – Supplementary Figure S2A) and with the highest eigenvalues (Supplementary Figure S1). The microbiota from the tobacco component of Cheyenne Menthol Box little cigars fluctuated significantly (p < 0.05 Tukey HSD test) between day 0 and days 5, 9, and 14 for refrigerator and day 0 and day 9 for pocket conditions.

FIGURE 4

Operational taxonomic unit differential abundance analysis for the microbiota of Cheyenne Menthol Box tobacco and wrapper components. Differential abundance plots showing the significant (assessed using DESeq2 at level p < 0.05) log2 fold changes of OTUs under all storage conditions between day 0 to day 14 for both Cheyenne Menthol Box tobacco (A) and wrapper component (B). Colors indicate the storage conditions. Numbers between parenthesis for the taxa shown on the Y axis represent OTU.

FIGURE 5

Interaction plot showing shared core and biomarker OTUs identified across the different little cigar brands and tobacco components. Shared core OTUs were identified as the OTUs present in all the tobacco brands, components, time points, and storage conditions. Biomarker OTUs were defined as the OTUs present in 100% of the samples from combination of tobacco brands, components, time points, and storage condition samples compared and absent from all others.