The Sudabiome: oral and gut microbiome parameters of the Sudanese population including dietary and cultural [Toombak] metagenomics
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Date
2022-12-15
Authors
Sami, Amel
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University College Cork
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Abstract
The Sudabiome is a unique metagenomic project focusing on several critical areas of the Sudanese population. It highlights a pivotal framework in understanding novel aspects of Sudanese health that include oral and gut health, cultural metagenomics, nutritional trends, migratory travel impacts, and the susceptibility or protection against diseases.
Toombak is a fermented smokeless tobacco produced from the Nicotiana Rustica tobacco plant, utilised predominantly by Sudanese males. The Toombak is ‘dipped’ in the oral cavity and replaced several times a day. The microbiome, mycobiome, phylogenetics and metabolomics of 21 pre-prepared and ready to buy Toombak samples purchased from different regions in Khartoum city were assessed, as well as the pH, chemical and microscopical composition of the product. The heavy metals, chromium, cobalt, and copper were high in the pre-prepared form of Toombak, while iron, tobacco-specific nitrosamines [TSNAs], formaldehyde and acetaldehyde were considerably elevated in both types compared to EU regulated smokeless tobacco products.
The phyla, Firmicutes and Actinobacteria dominated all samples of Toombak. Virgibacillus was found to be significantly increased in the pre-prepared form [q=3.6314e-8] compared to the ready to buy forms, while Corynebacterium casei [q=1.6417e-5], Atopococus tabaci [p=0.05], and Staphylococcus gallinarum [p=0.01] were the genera abundant in the ready to buy Toombak in comparison to the pre-prepared form. The mycobiome of the ready to buy Toombak was found to be enriched with Aspergillus [85.54%] with the species, Aspergillus heterocaryoticus [43.27%] and austwikii [39.48%] dominating the samples. Furthermore, utilising PICRUSt, the ready to buy samples were found to harbour an increased activity of metal transport systems [K02006, K02013 and K02015] and an antibiotic transport system [K09687]. The Toombak had a large non-homogenous and irregular particle distribution with increased sodium, while the pH of samples was in the alkaline range [8.73-9.92]. TSNA, formaldehyde and acetaldehyde levels observed in Toombak were found to be some of the highest compared to other smokeless tobaccos from around the world such as snus from Sweden and moist smokeless tobacco from the USA. These findings highlight that the final composition of Toombak is affected by its preparation method, with Toombak use having the potential to impart many negative consequences on the health of its users.
Dependency on Toombak use was examined through the utilisation of the Fagerstrom test for nicotine dependence smokeless tobacco questionnaire. Nineteen chronic Toombak users had an 85% dependence score. In addition, stress level over a 3-month retrospective period was evaluated through ‘scalp-side’ hair cortisol analysis. Mean cortisol levels were significantly lower [p=0.023] amongst Toombak users [9.7 pg/ml] compared to non-users [19.4 pg/ml]. While use of Toombak may initially bring anxiolytic effects to its users, blunting of the hypothalamic-pituitary axis chronically ensues in Toombak users, affecting cortisol release. This is likely due to the continued high levels of nicotine exposure and toxic effects that many Toombak compounds cause to body organs, including the adrenal glands.
The Sudan has a rich heritage of food fermentation that involves a vast array of raw starting materials and methodologies of production that have been preserved for centuries. Forty-six Sudanese fermented foods were sourced from six food categories that included, crop [sorghum and millet], plant [Cassia obtusifolia and sesame], fish, animal, and dairy sources to establish these foods metagenomic composition, some of which were explored for the first time. 16S rRNA sequencing was undertaken, while a subset of foods underwent internal transcriber [ITS] sequencing for mycobiome analysis. Beta diversity [p=0.001] was significantly varied between the categories of Sudanese fermented foods. Animal, fish, and plant-based foods exhibited the highest alpha diversity richness [p=8.7229e-07] while fish and plant-based foods had an elevated Shannon index [p=0.001]. Crop-based foods [sorghum and millet] were found to be enriched in the genus Acetobacter [96.89%], plant-based foods in Aeriscardovia [99.14%], dairy-based foods in Enhydrobacter [85.03%], animal-based foods in Bacteroides [98.22%] and fish-based foods in Sporosarcina [99%].
A loss of Lactobacillus abundance was observed between the preparatory [26.91%] and final [4.38%] stages of sorghum and millet fermented foods. The Kawal food, obtained from the fermentation of the Cassia obtusifolia plant was found to harbour a comprehensive microbiome [Bacillus, Lactobacillus, Pediococcus, Citrobacter and Bifidobacterium] and mycobiome [Candida, Aspergillus, Cladosporium and Malassezia] which could potentially harbour unknown but novel probiotic benefits to consumers. Sudanese fermented foods were however also found to be a source for pathogenic bacteria that included Escherichia-Shigella found abundantly in the plant-based foods [46.8%] and Wohlfahrtiimonas which were found to be abundant in animal-based foods [79.07%]. Intra-food categorical analysis found Lactobacillus to be enriched in the dairy food Mish or deep yoghurt [86%] compared to Gergosh or the dairy/legume biscuit [24%] and Garis or fermented camel milk [10%].
Extensive screening of the oral microbiome of users and non-users of Toombak was undertaken through 415 samples detecting salivary [n=72], plaque [n=71], tongue, buccal cheek, floor of the mouth and palatal mucosal [n=272] microbiome variations. In a subset of salivary samples, the mycobiome [ITS] was also assessed. The microbiome of oral squamous cell carcinoma and premalignant samples [n=46] from formalin-fixed paraffin-embedded tissue was further evaluated from another cohort of users and non-users of Toombak. Alpha diversity [richness] was low for tongue microbiome compared to other mucosal locations [p=3.6849e-34] and Beta diversity was found to be significant between the four oral mucosal locations [p<0.001].
Fusobacteria and Patescibacteria in saliva and Actinomyces in the saliva [p=0.0045], tongue [p=0.013] and palate [q=0.001], were correlated with Toombak use. Skin-associated microbiome were increased amongst the oral cavity of Toombak users and included Staphylococcaceae [q=0.037] in the saliva and Cutibacterium [q=0.04] in the buccal cheek and floor of the mouth. In Toombak users, utilising LEfSe plotting, the genus Peptostreptococcus was most distinct for Toombak use. Non-users of Toombak were found to have abundances in Prevotella [buccal, p=0.04] and Bifidobacterium [tongue, q=0.0049] while Scardovia was discriminant of non-user’s tongue microbiome. Virulent strains such as Prevotella nigrescens and Streptococcus equinus [tongue, p=0.043] were found in the oral microbiome of Toombak users while Prevotella salivae and Streptococcus sobrinus [tongue, q=0.023] were found in non-users of Toombak. There was a three-fold enrichment of oral Aspergillus [78.93%] in the saliva of Toombak users compared to non-users [21.07%] with reduced Candida abundance in Toombak users [4.33%] compared to non-users [95.67%]. Virulent fungal species were also found in smokeless tobacco users [Candida tropicalis] compared to non-users [Candida albicans].
Premalignant lesion microbiome composition included Rothia [p=1.5e-10] and Peptostreptococcus [p=6.5e-07] agreeing with the literature. The oral cancer microbiome in Toombak users harboured genera favouring a poor survival and metastasis that included Stenotrophomonas, [p=0.043] and Schlegelella, [p=0.048], compared to genera found in oral cancer samples from non-users of Toombak such as Lactobacillus, [p=0.024]. The genus Corynebacterium_1 was found to be common to the Toombak product, the oral cavity [saliva, p=0.0054, floor of the mouth, p=0.0028] and oral cancer samples [p=0.044] of Toombak users. These findings highlight that several oral distinctions follow Toombak use, allowing for a microbiome that could potentiate disease and destabilise the normal oral microbial flora while our findings also harbour the potential for microbiome biomarkers to be employed in the future screening of oral carcinoma in Sudan.
In 141 participants, an inter-continental metagenomics study was achieved on residents from Africa [Sudan] and Europe [Ireland] concerning diet frequency, microbiome composition and metabolome activity of stool samples. The European Prospective Investigation into Cancer and Nutrition Norfolk Food Frequency Questionnaire [EPIC - Norfolk FFQ] was modified and employed to compare food groups, nutrient and energy intakes between three cohorts, the Irish living in Ireland [Irel], the Sudanese living in Ireland [SudIrel] and the Sudanese living in Sudan [Sud]. Cereals and cereal products, fats and oil, fish and fish products, fruits and vegetables, meat and meat products, sugar preserves and snacks were consumed at a reduced intake in the Sud cohort with a high strength of association and large effect size [eta2 = >0.14]. Egg and egg products [eta2 =0.04], nuts and seeds [eta2=0.05] and non-alcoholic beverages [eta2=0.108] were also consumed highly in Sud, albeit with low and medium effect sizes. The average energy intake [measured in kcal/day] between groups was statistically different [p <0.001] and was found to be lowest in Sud [1077 kcal/day], compared to SudIrel [2729 kcal/day] and Irel [3442 kcal/day]. The diet of the Sud cohort was thus found to be low in calcium [p=0.05], carbohydrates, fibre, protein, fat, potassium, sodium, and vitamins A, C, B1, B2, B6, B12 and D [p<0.001] but high in vitamin B5 [pantothenic acid] and intermediate in Vitamin K levels. These results highlight the variations of dietary consumption between the two populations; Sudan and Ireland and helps set a fundamental previously unmet base for future research to evaluate dietary trends in the Sudanese population, including children.
Microbiome Beta diversity plotting [q=0.001] highlighted a distinct ‘sandwiched’ presentation to the cohort SudIrel compared to Irel and Sud groups, with R2 = 63%. Alpha diversity measures however were found to be non-significant between groups [Shannon index or evenness, p=0.74]. Microbiome genera such as Prevotella, [q=3.169e-06], Megasphaera [q=4.61e-05], Klebsiella [q=3.9067-e], Bifidobacterium [q=7.023e-03] and Lactobacillus [q=2.918e-09] were found to be depleted in the Irel cohort compared to both Sud and SudIrel while in the Irel cohort, the microbiome was instead enriched in Lachnospira [q=1.623e-09] and Bacteroides [q=3.056e-05]. Collinsella was found to be four times more abundant in Sud and SudIrel [44%] compared to Irel [11%].
Principal coordinate analysis of metabolomic compounds from stool samples highlighted 27 metabolites to be significantly varied between Irel and Sud including proline [p=0.003], adipic acid [0.04], and phosphocholine [p=0.02], 21 metabolites to be significantly varied between Sud and SudIrel including sugar alcohol [p=0.04] and N-acetylneuraminic acid [p=0.01] and no significant metabolomic variations between Irel and SudIrel. The short-chain fatty acids, isobutyric [p=0.0] and isovaleric acid [0.002] were significantly lower in the stool samples from those in the Irel cohort. LEfSe plotting of PICRUSt KEGG orthology pathways indicated multi-drug resistance activity pathways in the Irel cohort [K09686], mineral and heavy metal transport activity in Sud [K06199] and in SudIrel, carbohydrate metabolism pathways were prominent [K02794, K02796, K02775, K02773, K02774]. From these results, a migratory impact on the traditional microbiome of the Sudanese population occurs that has not been previously explored which may allow for both the susceptibility and prevention of disease in this population. Those who migrate from the Sudan to Ireland [SudIrel], carry both tradition microbiome preservation [i.e., continued Prevotella abundance] and modern microbial carriage [i.e., Bacteroides enrichment].
Through these studies, there has been a novel and in depth uncovering of human and environmental microbiome features associated for the first time with the Sudanese population in comparison to Irish controls. Through fermentation techniques for example, cultural [Toombak] and dietary [fermented foods] produces may carry health benefits but also numerous predicaments such as the increased risk of oral cancer development and systemic cortisol imbalance from Toombak use and the contraction of food-borne diseases from contamination during Sudanese fermented food production. The studies on Sudanese human oral and gut health have further provided insight into how the oral and gut microbiome may respond to modern challenges such as migration and economic, dietary and cultural influences.
This project further give light on the merging of next generation sequencing science with economic and population challenges. Continued microbiome research in Sudan should continue to be geared towards sharper preventative disease strategies, management, and treatment outcomes.
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Keywords
Sudabiome , Sudan , Toombak , 16S rRna sequencing , Fermented foods of Sudan , Stress response Toombak , Oral microbiome and mycobiome , Gut microbiome of Sudanese
Citation
Sami, A. 2022. The Sudabiome: oral and gut microbiome parameters of the Sudanese population including dietary and cultural [Toombak] metagenomics. PhD Thesis, University College Cork.