The ultra-structural, metabolomic and metagenomic characterisation of the sudanese smokeless tobacco 'Toombak'

Abstract

Toombak is a smokeless tobacco produced from the Nicotiana rustica tobacco plant from Sudan. Pre-prepared and ready to buy Toombak samples were analysed using mass spectrometry (heavy metals), gas and liquid chromatography (metabolomics), 16S rRNA metagenomic sequencing (microbiome) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) and pH analysis. Chromium, cobalt, and copper were high in the pre-prepared form of Toombak while iron, tobacco specific nitrosamines (TSNAs), formaldehyde and acetaldehyde were high in both types. Firmicutes and Actinobacteria dominated Toombak. Samples of ready to buy Toombak showed inter-variational differences depending on place of purchase. We found Virgibacillus were increased in the pre-prepared form while Corynebacterium casei, Atopococus tabaci, Atopostipes suicloacalis, Oceanobacillus chironomi and Staphylococcus gallinarum were the most abundant species in the ready to buy forms. PICRUSt analysis highlighted increased activity of metal transport systems in the ready to buy samples as well as an antibiotic transport system. SEM-EDX highlighted large non-homogenous, irregular particles with increased sodium, while pH of samples was in the alkaline range. The final composition of Toombak is affected by its method of preparation and the end product has the potential to impart many negative consequences on the health of its users. TSNA levels observed in Toombak were some of the highest in the world while the micro-environment of Toombak supports a distinct microbiota profile.

Keywords: Composition; Heavy metal; Metabolome; Microbiome; Scanning electron microscopy; Smokeless tobacco; Sudan; Toombak; pH.

Graphical abstract

Fig. 1

A: the smokeless tobacco, Toombak locally known as ‘sultan alkeef’ in Sudan is sold in common polythene bags from local vendors around the country. B: Toombak is of a brown coarse structure ranging from finely ground to macro-sized particles. C: a user placing the Toombak dip in the upper and D: the lower mucolabial folds.

Fig. 2

Multivariate score plot: PCA model describing two dimensional LC/MS data of Toombak. Data have been auto scaled. S01 = pre-prepared Toombak, S02 – S11 = Khartoum North samples and S12 –S21= Omdurman samples. Distinct variation between S01 and the ready to buy samples in the plot suggests handmade preparation of Toombak greatly modifies the final product. No major variation was found between the ready to buy groups.

Fig. 3

Bar graph: hexose monosaccharide in Toombak samples was found to be significantly elevated in the pre-prepared sample S01, compared to the remaining ready to buy samples.

Fig. 4

A: Microbiome Analyst R programming of abundant phyla within Toombak samples. Firmicutes are the most abundant phylum, followed by Actinobacteria. Pre-prepared samples harboured increased Cyanobacteria; due to the less degraded plant composition of the pre-prepared form. B: Stacked bar chart representing 16S rRNA compositional data of families within Toombak. Corynebacteriaceae and Carnobacteriaceae are predominant in the ready to buy forms while in the pre-prepared Toombak; Bacillaceae are markedly increased. C: Stacked bar chart of genera composition amongst Toombak samples. Corynebacterium_1, Atopostipes and Atopococcus are the most abundant genera in Khartoum North samples. Atopostipes and Alloiococcus were found to be abundant amongst Omdurman samples while Virgibacillus was the most abundant genus in the pre-prepared form. Oceanobacillus, Staphylococcus, Yaniella and Aerococcus are other genera identified in the ready to buy Toombak samples.

Fig. 5

α Diversity as measured by Chao1 (p = 0.46), observed richness (p = 0.16), and Shannon diversity index (p = 0.25) comparing ready to buy samples of Toombak (pink = Khartoum North, blue = Omdurman). Non-significant α diversity highlights that the microbial ecological niche is the same amongst all ready to buy Toombak and is attributed to OTU feature domination. Shannon scores > 2 indicate an increased number of rare species.

Fig. 6

PCoA model highlighting β diversity of ready to buy Toombak in relation to place of purchase at feature level (OTUs). Predominant inter-group microbiome variation of Toombak samples (dependant variable) due to location of purchase (independent variable). Samples from Khartoum North exhibit intra – group commonality compared to those from Omdurman. This variation is statistically significant (p <0.025). R²= 0.61, thus 61 % of the microbiome in Toombak ready to buy samples can be related to place of purchase most likely affected by multi-local handlers and the social and geographical considerations that are unique between Khartoum North and Omdurman.

Fig. 7

Classic univariate analysis (Anova), log scaled, highlights significant variation in microbiome between the two Toombak ready to buy groups; FDR adjusted p values or q values. Staphylococcus (q = 0.002), Sinibacillus (q = 0.028), Terribacillus (q = 0.028), Brachybacterium (q = 0.046), Enterococcus (q = 0.032), Glutamibacter (q = 0.025), Lysinibacillus (q = 0.021), Saccharomonospora (q = 0.025) and Devosia (q = 0.004) were the most abundant genera in Toombak obtained from Khartoum North. Shrub contamination (Solanum_incanum) in the final ready to buy Toombak from Khartoum North was also found (q = 0.006) and reflects the varied production techniques of Toombak. Pink = Khartoum North, blue = Omdurman.

Fig. 8

Deseq2 was used to assess differentially abundant OTUs amongst Omdurman samples. Deseq2 determines the mean expression of various shrinkage estimates and fold changes to improve final interpretability, while also correcting dispersion estimates that may be too low through modelling or under sampling. OTU_3 was the most significantly aligned species utilising BLASTn and was found to be 98.49 % identical to Atopostipes suicloacalis. Omdurman samples also contained increased abundances of Alkalibacterium iburiense, Serratia quinivorans and the genus Alloiococcus. Pink = Khartoum North, blue = Omdurman.

Fig. 9

PICRUSt data assimilation of the top 20 KEGG pathways in pre-prepared and ready to buy Toombak with visualisation of abundance activity using heatmap profiling. Increased peptide/nickel (K02035), cobalt/ nickel (K02006), and iron transport systems (K02013 and K02015) amongst ready to buy Toombak as well as active antibiotic transport systems (K09687) amongst all samples were identified.

Fig. 10

A: pre-prepared Toombak before handmade preparation with large, wooden and leaf like components at 35x magnification. B: ready to buy Toombak highlighting a non-homogenous large particle size product that is more closely packed together and may be quite abrasive to the oral mucosa (35x). C: evenly dispersed European snuff particles at 35x and D: ready to buy Toombak with abundance of bacterial rods and cocci at 4000x magnification.