Introduction

The World Health Organization highlights in its “Report on the Global Tobacco Epidemic” the harmful effects of all tobacco products, including heated tobacco products and also smokeless tobacco [1]. Likewise, a recent policy statement issued by the International Association for Dental, Oral, and Craniofacial Research (IADR) and the American Association for Dental, Oral, and Craniofacial Research (AADOCR) highlights the substantial and well-documented public health risks associated with all forms of tobacco use and emphasizes the importance of independent, methodologically transparent research in this domain [2].

The tobacco epidemic is estimated to be one of the biggest threats to public health and global poverty due to nicotine’s highly addictive effect, especially in low- and middle-income countries [3]. As of 2023, with 80% of 1.3 billion tobacco users globally these are accountable for over 7 million global deaths annually in addition to other long-term health harms [4]. It is estimated, that 300 million people in at least 127 countries regularly use some kind of smokeless tobacco whose ingredients differ quite significantly from each other [5–9].

The most commonly used form of smokeless tobacco in Sudan is Toombak produced from Nicotiana rustica, a tobacco plant grown in the northwest of Sudan [10]. This tobacco plant is known for its high nicotine and nornicotine content resulting in relatively high nicotine dry weight in the smokeless tobacco product content of 8–102 mg/g compared to 3–15 mg/g in commonly used chewing tobacco in the USA [7, 11–13]. It is used by approximately 34% of the Republic of the Sudans adult population, a prevalence doubled compared to that of USA, Canada, Sweden, Norway or Nigeria [14–19]. Toombak is used by rolling a portion of 1–10 g in one‘s palm to a ball-like portion called „Saffa“, placing it in the oral vestibular region of lips or cheeks for a duration of 10 to 15 min and not chewing but rather sucking on it for several minutes to hours until it is displaced and the mouth is rinsed with water [7, 12]. Depending on the manner of consumption the Saffa is either placed in the labial vestibulum of the lower jaw or the cheeks and different amounts of the insoluble tobacco-saliva-mixture are swallowed or spit [7, 12, 20]. It has been reported, that on average, an amount of 10–30 „Saffa“ are consumed per day and person [21].

The cancerogenic effects of Toombak have been investigated thoroughly [7, 11, 20, 22–26]. The high content of tobacco-specific nitrosamines, which are up to 100-fold elevated compared to US- and Swedish chewing tobacco and their effective extraction rate of 80% by sucking on the Saffa result in a significantly higher saliva concentration comparable to that used to induce cancer in cheeks and palate of rats [20, 27, 28].

However, although the microbiota of Toombak itself [29–31] and its association with intraoral pathology like gingival recession and periodontal disease have been investigated previously [32], the evidence on potential effects of Toombak on the oral microbial composition is scarce [33]. Only few studies have examined the microbiological characteristics of Toombak or its potential effects on the oral microbiota. A study by Abakar et al. indicated that Toombak users may exhibit shifts in selected viable bacterial taxa, although methodological constraints limited the interpretability of these findings [33]. More recently, metagenomic sequencing demonstrated broader alterations of the oral microbiome in Sudanese Toombak users, including shifts in community composition and functional microbial potential [26]. In addition, analyses of smokeless tobacco products have revealed substantial microbial diversity and batch-to-batch variability, suggesting that product-associated microbiota may contribute to user exposure [29]. The available literature remains heterogeneous and limited, underscoring the need for further exploratory studies that assess viable bacteria at the habitual site of Toombak placement. Current High-throughput sequencing studies have substantially expanded current knowledge of the diversity, biogeography, and functional complexity of the human oral microbiome in health and disease [34]. Therefore, the aim of this culture technique pilot study was to investigate the impact of regular Toombak consumption on the oral microbial composition, its potential antibacterial effects, and the microbial profile of Toombak itself.