Discussion

The aim of this culture-based pilot study was to characterize the cultivable microbiota of Toombak, to assess its potential antibacterial effects, and to evaluate its impact on the cultivable oral microbial composition compared with individuals without a history of Toombak use. For this purpose, samples from a Sudanese population were collected and analyzed. Only male participants were enrolled, reflecting the higher prevalence of Toombak use among men and the sociocultural tendency of female users to conceal consumption, which precluded reliable inclusion [19].

Culturomics has substantially expanded the range of detectable oral microorganisms and has demonstrated that traditional culture-dependent workflows capture only a fraction of the taxonomic and functional diversity present within the oral cavity [37]. Although large-scale metagenomic and amplicon sequencing efforts have provided comprehensive insights into oral microbial communities across niches, culture-based methods remain essential for assessing viability and phenotypic characteristics [34]. Recent metagenomic analyses have reported alterations of the oral microbiome in Sudanese Toombak users, including shifts in microbial community structure; however, differences in study design, analytical methodology, and sampling sites limit direct comparability with the present culture-based investigation [26]. Another important limitation relates to the use of culture-dependent techniques, which capture only the cultivable fraction of the oral microbiota and therefore do not reflect the full taxonomic diversity present in the oral cavity. However, this methodological approach was deliberately chosen, as it enables the assessment of viable microorganisms and allows conclusions regarding potential toxic or inhibitory effects of Toombak on bacterial growth.

Analyses were conducted in a descriptive and hypothesis-generating manner. Consequently, no formal adjustment for multiple comparisons was applied. All p-values should therefore be interpreted cautiously and not as confirmatory evidence of group differences. The observed statistical signals are intended to guide future, adequately powered studies rather than to support definitive inferences. The relatively small sample size of the present study reflects the exploratory pilot character of the investigation and is comparable to other experimental studies in oral microbiota research using culture-based approaches. For example, Vach et al. conducted a longitudinal analysis of oral biofilm composition with 11 participants and emphasized that such study designs primarily aim to identify overall patterns and relationships within the microbiota rather than to provide confirmatory statistical evidence [38]. In line with these considerations, the substantial interindividual variability of the oral microbiota represents a fundamental challenge, which cannot be fully compensated by moderate increases in sample size. Consequently, results from small-scale studies should be interpreted as hypothesis-generating and descriptive rather than confirmatory. This approach is consistent with previous methodological work in the field, which highlights that even under controlled experimental conditions, definitive statistical inferences remain difficult to justify in small cohorts.

S. mitis and S. oralis were the only bacterial species consistently detected in every sample. Furthermore, these bacteria exhibited the highest mean bacterial counts in smokeless tobacco users and in the control group. Similar findings have been reported in other studies, where S. mitis and S. oralis were isolated from nearly all niches of the oral cavity and across individuals [39, 40] ranking among the most frequently identified species [41]. Liu et al. demonstrated that high concentrations (50 mg/mL) of smokeless tobacco extracts from the United States could reduce the growth of S. mitis and S. oralis [42].

Representatives of the salivarius group identified in this study included S. salivarius and S. vestibularis. Tappuni and Challacombe demonstrated that S. salivarius remains a dominant bacterial species in the oral cavity from childhood through adulthood [43]. Along with S. mitis and S. oralis, it was among the most frequently isolated streptococcal species, collectively accounting for 83% of all recovered streptococci also representing the most frequently isolated streptococci with the highest mean CFU/ml. Falkler et al. showed that smokeless tobacco extracts could promote the growth of S. salivarius, suggesting that specific components may serve as nutrients for [44]. However, in a non–peer-reviewed preprint study by Abakar et al. a reduced abundance of viridans streptococci in buccal swabs of Toombak users was observed [33].

Sch. odontolytica was detected only in the control group in four individuals. It is associated with root caries and is also common in the oral microbiome of children below the age of 2 [45–47]. Liu et al. reported reduction of growth and cell viability through aqueous extracts of smokeless tobacco [42]. This species showed a statistically significant difference in detection between groups. As Gram-positive bacteria could be more sensitive to the possible toxic effects of consuming Toombak, this might have toxic effects on Sch. odontolytica in the oral cavity. Sch. odontolytica is considered a common member of the healthy oral microbiota and has been detected across multiple intraoral habitats in healthy individuals, including tongue surfaces and tooth-associated biofilms, as demonstrated in comprehensive molecular surveys of the healthy oral cavity [39]. While generally regarded as a health-associated commensal, this species has also been linked to early colonization on tooth surfaces and has occasionally been associated with root caries in ecological studies. In light of this background, the lower relative abundance observed in Toombak users in our study should be interpreted cautiously and primarily as an exploratory ecological signal rather than as evidence of clinical impairment.

The significantly reduced abundance of Gram-positive rods, and aerobic Gram-positive rods in Toombak users observed in this study might be caused by the relatively high nicotine and TSNA content of Toombak [11, 13, 20]. Gram-positive rods like Lactoaseibacillus spp. are known to have beneficial effects on periodontal health, possibly highlighting a potentially increased risk for inflammation in Toombak users [48, 49]. Additionally, as mentioned above, these results suggest that Gram-positive bacteria are more sensitive to Toombak than Gram-negative bacteria, which have an additional outer membrane that prevents toxic substances from diffusing into the cells.

The present investigation identified B. licheniformis, B. subtilis, and B. thermoamylovorans in samples from Toombak users and B. subtilis in the control group. Bacillus spp. have before been detected on the buccal mucosa and in gingival crevicular fluid [50, 51] and can survive unfavorable environmental conditions and nutrient deprivation by forming endospores [52], which are highly resistant to heat, UV and gamma radiation, desiccation, toxic chemicals such as hydrogen peroxide, and hydrolytic enzymes [53]. Additionally, they are capable of biofilm formation [54, 55]. Abakar et al. observed an increased abundance of Bacillus spp. and Aspergillus spp. in oral swabs of Toombak users [33]. To date, no reports exist of isolation of B. thermoamylovorans from the oral cavity. Certain Bacillus spp., including B. pumilus, B. licheniformis, and B. subtilis, possess nitrate-reducing activity [56], forming nitrites as an important precursors of tobacco-specific nitrosamines (TSNAs) [57, 58]. Approximately 90% of the bacteria isolated from smokeless tobacco products belong to the genus Bacillus, including B. brevis,_ B. licheniformis_,_ B. megaterium_, B. pumilus, B. safensis, and B. subtilis [59, 60]. These findings are consistent with the present study, in which only B. licheniformis, B. subtilis, and B. pumilus were extracted from Toombak samples. Given a reported average consumption of 10–30 saffas per day residual Toombak in the samples from users may have contributed to an increased presence of Bacillus spp [21].

F. nucleatum was the second most frequently isolated species in the smokeless tobacco group. F. nucleatum is known to play an important role in periodontal diseases but is also found in healthy individuals and is considered part of the physiological oral microbiota [39, 61, 62]. Smoking has been shown to increase the subgingival prevalence of this taxon in periodontitis patients [63], whereas Indian smokeless tobacco reduced its occurrence [64]. Fusobacterium spp. have previously been found in American smokeless tobacco products [60]. Aqueous extracts of U.S. smokeless tobacco exerted inhibitory effects on the growth of F. nucleatum [42].

To date, the influence of Sudanese chewing tobacco on the oral microbiota has scarcely been investigated. In an aforementioned preprint, non-peer-reviewed publication by Abakar et al. an inhibitory effect of Toombak on viridans streptococci and an increased abundance of Bacillus spp. and Aspergillus spp. in oral swabs was investigated [33]. In our study however, no inhibitory effects of Toombak itself could be found. It can be hypothesized that the antimicrobial effects of Toombak under in vivo conditions may differ substantially from those observed in in vitro assays. In the oral cavity, Toombak is continuously exposed to saliva, which contains a complex mixture of proteins, enzymes, and antimicrobial peptides that may modulate microbial viability and community dynamics. In contrast, laboratory-based antimicrobial testing relies on aqueous extracts and standardized growth media, which do not reflect these physiological conditions. Furthermore, habitual Toombak use involves repeated and long-term exposure of the oral mucosa and microbiota, potentially resulting in cumulative ecological effects that cannot be reproduced by single, short-term in vitro experiments. Consequently, the absence of inhibitory activity observed in vitro does not preclude indirect or long-term microbiological effects under real-life exposure conditions.

The duration and frequency of chewing tobacco use in this study did not significantly affect the total bacterial load in the samples. However, other types of chewing tobacco, have demonstrated an effect on bacteria of the human oral microbiota: Liu et al. demonstrated that aqueous extracts of American chewing tobacco had a concentration-dependent effect on the growth and cell viability of human oral bacteria, promoting growth at concentrations of 1–50 mg/mL while suppressing growth and viability of most bacteria above 50 mg/mL [42]. These applied concentrations corresponded to realistic exposure conditions [65, 66]. As the production process of Toombak is neither an industrially standardized procedure nor governmentally regulated, a broad variance of pH values of 8–11 and nicotine contents of 8–102 mg/g may result [20, 67]. Toombak is either prepared domestically or purchased in numerous small shops [12, 21]. The final processing step is typically performed by the vendors themselves by mixing four parts of coarse dried Toombak leaf powder with one part of an aqueous sodium bicarbonate concentrate. The mixture is vigorously kneaded by hand, and its taste is repeatedly assessed by the vendors until the product reaches a moist and sticky consistency. The final mixture is then stored in airtight metal containers for approximately two hours before it is ready for consumption or sale [7, 12, 19]. Yet, since the content of toxic substances such as nicotine and TSNAs is in general higher in Sudanese chewing tobacco with TSNA concentrations of up to 100 times more than in American chewing tobacco [11, 13, 20], it is reasonable to assume that the influence on oral microbiota could be more pronounced.

The observed trend of a lower total bacterial count among chewing tobacco users could be indicative of a slightly inhibitory effect on the growth of oral bacteria. This would align with the significantly lower percentage of Gram-positive rods and aerobic Gram-positive rods in the chewing tobacco group. Nevertheless, differences in total bacterial counts did not reach statistical significance, except for Sch. odontolytica. The largely comparable cultivable microbiota observed in the present study should therefore not be interpreted as evidence against the reported association between Toombak use and periodontal disease, as the present investigation was not designed to assess clinical periodontal outcomes. However, while Liu et al. (2016) examined the direct effect of chewing tobacco extracts on cultured oral bacteria in vitro, the present study analyzed the oral microbiota based on swab samples, taking into account interbacterial interactions, the semi-planctonic state of oral bacteria and other environmental factors [42].

Furthermore, it remains unclear which specific components of chewing tobacco are primarily responsible for potential effects on the oral microbiota. Most chewing tobaccos with high sugar content are capable of promoting the growth S. mutans, S. sanguinis, and Lacticaseibacillus casei in vitro [68, 69] while Falkler et al. showed promoted growth of S. mutans, S. salivarius, and S. sanguinis even in the absence of detectable sucrose [44]. Nicotine may inhibit the activity of neutrophils and monocytes, particularly against F. nucleatum, suggesting a potential shift in oral microbial composition [70], which aligns with more frequent detection of F. nucleatum in the chewing tobacco group, although the difference was not statistically significant. Fluoride as an ingredient in some chewing tobacco products [71], is known for its caries-preventive and antimicrobial effects on bacteria such as S. mutans by influencing enzymatic and metabolic pathways [72–74]. Moreover, unknown components in chewing tobacco have been shown to influence bacterial metabolism e.g. in Capnocytophaga sputigena [75] or affecting oral microbiota indirectly by modulating host response and stimulating secretion of inflammatory mediators like prostaglandin E2 and interleukin-1β from monocytes [76], activating lymphoid tissue [77], suppressing natural killer cell activity [78], or enhancing IgA secretion [79]. It must also be considered that chewing tobacco itself can contain bacteria. Recent sequencing-based analyses have demonstrated that smokeless tobacco products harbor distinct and diverse bacterial communities, which may vary substantially between products and are influenced by production and processing conditions [30]. Another important limitation relates to the analysis of the microbial composition of Toombak itself. Toombak is not a standardized industrial product but is typically prepared domestically or by local vendors, resulting in substantial variability in raw materials, preparation methods, storage conditions, and overall composition. Consequently, the microbial profile observed in the present study may not be representative of all Toombak products and should be interpreted with caution. Rather than providing a definitive characterization, the present findings should be understood as an exploratory assessment of the microbiological properties of Toombak under real-life conditions of use. Future studies using standardized or systematically sampled products would be required to better characterize the variability and generalizability of these findings. In the present study, Bacillus spp. were detected in Sudanese chewing tobacco. Bacillus spp. have also been found in chewing tobacco products from the U.S. and South Africa [59, 80]. Additional studies identified bacteria of the genera Tetragenococcus, Carnobacterium, Lacticaseibacillus Geobacillus, and Staphylococcus, as well as 12 fungal species, with Bacillus spp. being predominant [60, 81]. Because tobacco is not combusted during use, as it is in smoking, the impact of viable microorganisms present in the product may be greater [60]. In the present study, three Bacillus species, namely B. licheniformis, B. subtilis, and B. thermoamylovorans, were detected in the chewing tobacco group, while only B. subtilis was found in the control group. Prevotella spp. and Fusobacterium spp., which were also found in U.S. chewing tobacco [60], were more frequently isolated from the chewing tobacco group than in the control group.

Although major confounding factors such as smoking and periodontal pockets ≥ 4 mm were controlled through exclusion criteria, no standardized clinical oral health indices were recorded. Therefore, residual or subclinical variability cannot be entirely excluded. The relatively broad age range of the participants represents another potential source of confounding, since age may influence both periodontal health and the oral microbiota. This aspect should be addressed in future studies with larger cohorts. Furthermore, since caries experience, tooth loss, and restorative treatment may influence the oral microbiota, residual confounding related to dental status cannot be excluded. Future studies should therefore include standardized assessment of the DMFT index. In addition, as local mucosal alterations may influence the composition of the oral microbiota, a potential confounding effect cannot be excluded. Future studies should therefore combine comprehensive clinical assessment of the oral mucosa with microbiological analyses. Although all specimens from both groups were processed using the same standardized storage protocol at − 80 °C prior to microbiological cultivation, freezing may have affected the recovery of viable microorganisms and therefore influenced absolute bacterial counts. Consequently, the present findings should be interpreted with this potential limitation in mind. Further research is needed to assess the influence of Toombak on total salivary microorganisms in a larger cohort analyzing the microbiome by next-generation sequencing methods. Unfortunately, continued recruitment of participants at the original study site is currently not feasible due to an ongoing armed conflict in Sudan, restricting clinical research activities. Validation of the present findings will therefore depend on future studies once research conditions permit. Nevertheless, toxic effects on oral bacteria on the site of Toombak placement can only be investigated using culture techniques. In the present study, potentially disease-associated bacteria were isolated from Toombak and the control group. Future research should genetically compare these isolates in order to clearly identify the transmission route, as described before for bacterial species originating from food [82]. Furthermore, it would be interesting to investigate potential effects of Toombak on the transcriptomic level of these taxa [83].