Characterization of the bacterial community associated with smokeless tobacco reference products under different storage conditions
In this study we characterize the bacterial communities associated with smokeless tobacco reference products (STRP), including loose leaf chewing tobacco (3S1), moist snuff (3S3), American snus (1S5), and Swedish snus (1S4) during a 1-year storage period. All products were stored under three temperatures: frozen (FR -20°C), cold room (CR 4°C), and room temperature (RT 21°C), and were sampled seven times (initial, 1-month, 2-month, 3-month, 6-month, 9-month, and 12-month). We performed culture-dependent colony counting to assess the bacterial and fungal load and used Illumina Miseq sequencing of 16S rRNA gene V4 region to profile the bacterial community structures of our tobacco samples. The results showed that bacteria, not fungi, were the dominant microbes and the bacterial loads were different among products: 3S3 > 3S1 > 1S5 > 1S4. The culturable populations significantly decreased at the 12-month sampling point for all products. Diversity and composition of the bacterial communities shifted under different sampling time and different storage condition. The most abundant phyla were Firmicutes and Proteobacteria for all products and the top 10 operational taxonomic units (OTUs) of 3S1 and 3S3 were the same. The top 10 OTUs for snus samples were different within 1S4 and 1S5, as well as between snus samples and moist snuff and loose leaf. Our results will be correlated with physiochemical parameters (e.g., moisture, pH, nicotine, and tobacco-specific nitrosamines).