Tobacco, Microbes, and Carcinogens: Correlation Between Tobacco Cure Conditions, Tobacco-Specific Nitrosamine Content, and Cured Leaf Microbial Community

Abstract

Tobacco-specific nitrosamines are carcinogenic N-nitrosamine compounds present at very low levels in freshly harvested tobacco leaves that accumulate during leaf curing. Formation of N-nitrosamine compounds is associated with high nitrate levels in the leaf at harvest, and nitrate is presumed to be the source from which the N-nitrosation species originates. More specifically, nitrite is considered to be a direct precursor, and nitrite is linked with N-nitrosation in many environmental matrices where it occurs via microbial nitrate reduction. Here, we initiate work exploring the role of leaf microbial communities in formation of tobacco-specific nitrosamines. Leaves from burley tobacco line TN90H were air cured under various temperature and relative humidity levels, and 22 cured tobacco samples were analyzed for their microbial communities and leaf chemistry. Analysis of nitrate, nitrite, and total tobacco-specific nitrosamine levels revealed a strong positive correlation between the three variables, as well as a strong positive correlation with increasing relative humidity during cure conditions. 16S rRNA gene amplicon sequencing was used to assess microbial communities in each of the samples. In most samples, Proteobacteria predominated at the phylum level, accounting for >90 % of the OTUs. However, a distinct shift was noted among members of the high tobacco-specific nitrosamine group, with increases in Firmicutes and Actinobacteria. Several OTUs were identified that correlate strongly (positive and negative) with tobacco-specific nitrosamine content. Copy number of bacterial nitrate reductase genes, obtained using quantitative PCR, did not correlate strongly with tobacco-specific nitrosamine content. Incomplete denitrification is potentially implicated in tobacco-specific nitrosamine levels.

Differences in the Bacteriome of Smokeless Tobacco Products with Different Oral Carcinogenicity: Compositional and Predicted Functional Analysis

“…Obviously then, the levels of TSNAs in ST products differ not only as function of total microbial load but probably also as a function of microbial community composition. Indeed, a very recent study has found the TSNA concentrations in tobacco leaves to correlate positively with the proportions of Firmicutes and inversely with those of Proteobacteria [20]. Interestingly, Firmicutes was the predominant phylum in the American, Sudanese and Yemeni products in this study, while…”

“…Early reports, mostly by investigators of the tobacco industry, performed identification and quantification of bacteria and fungi in fresh and processed tobacco, using cultivationbased methods [15]. Recently, 16S rRNA-based techniques including random fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP) and sequencing have been used to characterize bacterial communities in fresh and cured tobacco leaves as well as those associated with tobacco fermentation process [16][17][18][19][20]. These studies revealed great deal of diversity and differences in the composition of microbiota associated with the different forms of tobacco.…”

Temporal Variations in Cigarette Tobacco Bacterial Community Composition and Tobacco-Specific Nitrosamine Content Are Influenced by Brand and Storage Conditions

“…Many of the members of the core microbiome were also present in the core microbiome defined for air-cured burley tobacco including Pantoea , Pseudomonas , Sphingomonas , and Bacillus (Law et al, 2016). Despite this agreement in core members between products, our results showed there was some divergence in bacterial community composition between brands of cigarettes.…”

“…This is suggested to be, in part, due to certain nitrate and nitrite reducing bacterial species present on or in the tobacco leaves (Atawodi and Richter, 1996). High temperatures and relative humidities have been shown to be key factors that contribute to increasing levels of TSNAs throughout curing (Burton et al, 1989b; Law et al, 2016) and storage (Burton et al, 1989a; Shi et al, 2013) of tobacco. TSNA levels in smokeless tobacco brands have also been shown to be influenced by storage conditions, with high levels of TSNAs associated with storage for 4 weeks at room and high temperatures (>37°C), but not low temperature (4°C; Djordjevic et al, 1993).…”

Differences in the Bacteriome of Smokeless Tobacco Products with Different Oral Carcinogenicity: Compositional and Predicted Functional Analysis

“…Early reports, mostly by investigators of the tobacco industry, performed identification and quantification of bacteria and fungi in fresh and processed tobacco using cultivation-based methods [15]. Recently, 16S rRNA-based techniques including random fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP), and sequencing have been used to characterize bacterial communities in fresh and cured tobacco leaves as well as those associated with tobacco fermentation process [16,17,18,19,20]. These studies revealed great deal of diversity and differences in the composition of microbiota associated with the different forms of tobacco.…”