Mentholation affects the cigarette microbiota by selecting for bacteria resistant to harsh environmental conditions and selecting against potential bacterial pathogens

Abstract

Background: There is a paucity of data regarding the microbial constituents of tobacco products and their impacts on public health. Moreover, there has been no comparative characterization performed on the bacterial microbiota associated with the addition of menthol, an additive that has been used by tobacco manufacturers for nearly a century. To address this knowledge gap, we conducted bacterial community profiling on tobacco from user- and custom-mentholated/non-mentholated cigarette pairs, as well as a commercially-mentholated product. Total genomic DNA was extracted using a multi-step enzymatic and mechanical lysis protocol followed by PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene from five cigarette products (18 cigarettes per product for a total of 90 samples): Camel Crush, user-mentholated Camel Crush, Camel Kings, custom-mentholated Camel Kings, and Newport Menthols. Sequencing was performed on the Illumina MiSeq platform and sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package.

Results: In all products, Pseudomonas was the most abundant genera and included Pseudomonas oryzihabitans and Pseudomonas putida, regardless of mentholation status. However, further comparative analysis of the five products revealed significant differences in the bacterial compositions across products. Bacterial community richness was higher among non-mentholated products compared to those that were mentholated, particularly those that were custom-mentholated. In addition, mentholation appeared to be correlated with a reduction in potential human bacterial pathogens and an increase in bacterial species resistant to harsh environmental conditions.

Conclusions: Taken together, these data provide preliminary evidence that the mentholation of commercially available cigarettes can impact the bacterial community of these products.

Keywords: 16S rRNA; Cigarette; Menthol; Microbiota; Pathogens; Tobacco.

Fig. 1

Heat map showing the relative abundances of the most dominant bacterial genera identified (>1%) in tested cigarette products. Samples pooled by product type: Camel Crush (CC), mentholated Camel Crush (CCM), Camel Kings (CK), custom-mentholated Camel Kings (CKM), and Newport Menthol Box (NMB). Hierarchical clustering of the pooled samples is represented by the dendrogram at the top and inside the color key shows a histogram of the count of the individual values

Fig. 2

Box plots showing alpha diversity (Chao1 richness estimator and Shannon Index) variation across samples on non-rarefied data (a) and with data rarefied to the minimum sampling depth (b). Bars are colored by mentholation status: red bars—non-mentholated; green bars—user mentholated; blue bars—custom-mentholated; purple bars—commercially-mentholated

Fig. 3

PCoA analysis plots of Bray-Curtis computed distances between cigarette products. a Points colored by brand: purple—Newport Menthol (NMB); green—mentholated Camel King (CKM); blue—mentholated Camel Crush (CCM); orange—Camel Kings (CK); red—Camel Crush (CC) (ANOSIM R value = 0.35, p value = 0.0001), (b) Points colored by mentholation status: green—non-mentholated; purple—user mentholated; blue—commercially-mentholated; red—custom mentholated. (ANOSIM R = 0.43, p = 0.0001)

Fig. 4

Overview of relative abundances of bacterial OTUs that were statistically significantly different (p < 0.05) between custom-mentholated Camel Kings (CKM) and non-mentholated Camel Kings (CK). OTUs are colored by Phylum and differentiated by Gram negative (a) and Gram positive (b) classification. A positive log2-fold change value denotes an OTU that is significantly higher in custom-mentholated Camel Kings, while a negative log2-fold change indicates an OTU that is significantly higher in non-mentholated Camel Kings. The dotted line and arrows highlight the conversion in log2-fold change from negative to positive values

Fig. 5

Overview of relative abundances of OTUs that were statistically significantly different (p < 0.05) between mentholated Camel Crush (CCM) and non-mentholated Camel Crush (CC). OTUs are colored by Phylum and differentiated by Gram negative (a) and Gram positive (b) classification. A positive log2-fold change value denotes an OTU that is significantly higher in mentholated Camel Crush, while a negative log2-fold change indicates an OTU that is significantly higher in non-mentholated Camel Crush. The dotted line and arrows highlight the conversion in log2-fold change from negative to positive values