Results

The microbiological findings for the chewing tobacco users and the control group were categorized into four groups which are depicted in the supplementary tables (Tables S1 to S4). All enrolled participants fulfilled the predefined eligibility criteria, including the absence of periodontal probing depths ≥ 4 mm and other tobacco use.

Comparison of colony forming units

Figure 1 shows the mean colony forming units (CFU) in the samples from chewing tobacco users and the control group, expressed as log₁₀ CFU/mL. The mean overall CFUs for the chewing tobacco group was 7.74 CFU/mL, while for the control group it was 8.23 CFU/mL.

Fig. 1: Comparison of the mean CFUs of the Chewing Tobacco Group and the Control Group expressed as Log10 divided into overall CFU, aerobic, anaerobic, Gram-positives and Gram-negative bacteria

The mean values of aerobic bacteria were 7.70 CFU/mL in the chewing tobacco group compared to 8.18 CFU/mL in the control group. The mean values of anaerobic bacteria were 5.94 CFU/mL compared to 6.30 CFU/mL in the control group. The mean values of Gram-positive bacteria were 7.64 CFU/mL compared to 8.18 CFU/mL in the control group. The mean values of Gram-negative bacteria were 6.56 CFU/mL compared to 6.59 CFU/mL in the control group. Overall, only a marginal trend toward slightly higher total bacterial counts in the control group was observed. However, the differences in total bacterial counts between the two groups were not statistically significant.

Figure 2 illustrates the relative proportions of aerobic, anaerobic, Gram-positive and Gram-negative bacteria in relation to the total bacterial count for both the chewing tobacco users and the control group. The proportion of aerobic and Gram-positive bacteria was higher in the chewing tobacco group compared to the control group, whereas the proportion of anaerobic and Gram-negative bacteria was higher in the control group. These differences ranged approximately between 5 and 10%. Overall, aerobic and Gram-positive bacteria were clearly dominant among the viable species in both groups. However, the percentage differences in total bacterial counts between the two groups were not statistically significant.

Fig. 2: Relative comparison of CFUs of the Chewing Tobacco Group and the Control Group, divided into aerobic, anaerobic, Gram-positives and Gram-negative bacteria

Comparison of species detected per participant and group

Figure 3 presents a comparison of the mean number of different bacterial species per participant between the chewing tobacco users and the control group. On average, 10.2 different species were detected in samples from chewing tobacco users, compared to 10.4 species in the control group. Further subdivision into aerobic, anaerobic, Gram-positive and Gram-negative species also revealed no statistically significant differences between the groups. In some categories, such as aerobes, the control group exhibited a slightly higher species count, whereas in others, such as Gram-negative bacteria, chewing tobacco users showed a marginally higher number of species. Overall, no clear trend or significant difference in species count could be identified between the two groups.

Fig. 3: Comparison of the mean number of species per participant of the Chewing Tobacco Group and the Control Group divided into overall number of different detected species, aerobic, anaerobic, Gram-positives and Gram-negative bacteria

Comparison of absolute distributions of bacterial groups

Figure 4 shows the distributions for all different detected bacterial species, including aerobic species and anaerobic species, categorized into Gram-positive cocci, Gram-positive rods, Gram-negative cocci and Gram-negative rods. Aerobic Gram-positive rods were identified 36 times in the control group compared to 29 times in the chewing tobacco group. Gram-positive species were detected 31 to 38 times and therefore more frequently than Gram-negative species which were detected 14 to 22 times. This trend was even more pronounced among aerobic bacteria, where Gram-positive organisms were observed 29 to 36 times, compared to only 8 to 11 detections of Gram-negative aerobes. In contrast, among anaerobes, Gram-negative cocci and rods were detected 6 to 11 times compared to Gram-positive anaerobes which were only detected 0 to 2 times. The differences in the absolute distribution of Gram-positive rods in Fig. 4 reached statistical significance when expressed as percentages (p = 0.0137).

Fig. 4: Stacked Bar Graph comparison of the distribution of different detected aerobic and anaerobic species in the Chewing Tobacco Group and the Control group divided into Gram-positives (Gr+) and Gram-negatives (Gr-) as well as cocci and rods

Comparison of anaerobic species

Figure 5 presents the frequency of different detected anaerobic bacterial species. Most species were detected only sporadically. Only V. parvula showed higher prevalence, being identified 9 times in the chewing tobacco group and 7 times in the control group. A more notable difference was observed for F. nucleatum, which was detected in 4 individuals in the chewing tobacco group but only once in the control group. In terms of the mean number of different species per participant, the chewing tobacco group showed a slightly higher value of 2.3 compared to 2 species in the control group. The total number of species detected was also similar between groups with 11 species in the chewing tobacco group compared to 13 in the control group. Statistical analysis of this data was not reasonably feasible.

Fig. 5: Comparison of the number of different detected anaerobic species in the Chewing Tobacco Group and the Control Group

Relationship between colony forming units and duration and frequency of chewing tobacco Use

The relationship between CFU and both the duration in years and frequency per day of chewing tobacco use were examined. Duration was recorded as the number of years each participant had used chewing tobacco, while frequency per day reflected the average number of times per day the individual consumed chewing tobacco (Fig. 6a and b). Although participant No. 9 displayed a comparatively lower aerobic and overall bacterial count despite a particularly long history and high daily frequency of chewing tobacco use, this pattern was not observed consistently across the other participants. However, a reduction of 1 log count presents a possibly noteworthy reduction. Statistical analysis of this data was not reasonably feasible.

Fig. 6: a: Correlation of aerobic CFU per ml (left y-Axis) and duration in years and frequency per day (right y-Axis) per participant. b: Correlation of anaerobic CFU per ml (left y-Axis) and duration in years and frequency per day (right y-Axis) per participant

Comparison of spore-forming bacteria

Table 1 compares both the number of different Bacillus spp. and the number of participants harboring Bacillus spp. Additionally, mean bacterial counts of Bacillus spp. were compared between chewing tobacco users and the control group by dividing the total CFU number of Bacillus spp. by the number of participants within each group. Bacillus spp. were detected in three samples from each group. However, three distinct Bacillus spp. were identified among the chewing tobacco users, whereas only one species was found in the control group. Furthermore, the mean bacterial count was approximately one log order higher in the chewing tobacco group, with 6.8 × 10⁴ CFU/mL compared to 1.7 × 10³ CFU/mL in the control group with no statistically significant difference between groups.

 | Number of different Bacillus spp. | Participants with Bacillus spp. | Mean CFU Bacillus spp.
Chewing Tobacco Group | 3 | 3 | 6.80E+04
Control Group | 1 | 3 | 1.70E + 03

Species level analysis

S. mitis and S. oralis were grouped together and detected in all samples. S. salivarius, N. macacae and mucosa, A. oris and viscosus, R. mucilaginosa, R. aeria, and V. parvula were detected in 10 samples. Among the anaerobic bacteria, V. parvula was the most frequently isolated species. The species A. oris and A. viscosus as well as N. macacae and N. mucosa were grouped together for analytical purposes.

S. sanguinis, G. haemolysans, N. flavescens, R. dentocariosa, B. subtilis, C. gingivalis, and F. nucleatum were identified in five to nine samples each. S. gordonii, representatives of the S. anginosus group (not further specified), S. parasanguinis, A. naeslundii, Sch. odontolytica, C. sputigena, and P. histicola were found in three to four samples each. S. odontolytica was detected exclusively in the control group. All other bacterial species were detected in ≤ 2 samples only.

Statistically significant differences on species level were observed for Schaalia odontolytica as well as for the percentage representation of all Gram-positive rods and aerobic Gram-positive rods as depicted in Table 2. Sch. odontolytica was detected exclusively in four samples from the control group (p = 0.0306). Aerobic Gram-positive rods were more frequently observed in the control group. Analysis of different detected bacterial species for Gram-positive rods and specifically aerobic Gram-positive rods revealed statistically significant differences, with p-values of 0.0137 and 0.0157, respectively.

 | Chewing Tobacco Group | Control Group |  | P-Value Wilcoxon Rank Sum Test | 
Isolates Gram-positive aerobic cocci |  |  |  |  | 
Streptococcus gordonii | 1 | 2 |  | 0.5432 | 
S. mitis/oralis | 10 | 10 |  | 0.3643 | 
S. salivarius | 6 | 5 |  | 0.3833 | 
S. anginosus group | 2 | 1 |  | 0.6267 | 
S. sanguinis | 3 | 4 |  | 0.4780 | 
S. parasanguinis | 2 | 1 |  | 0.5432 | 
S. constellatus | 1 | 0 |  | 0.3173 | 
S. intermedius | 1 | 0 |  | 0.3173 | 
S. vestibularis | 1 | 0 |  | 0.3173 | 
Gemella hämolysans | 3 | 5 |  | 0.2494 | 
G. sanguinis | 0 | 1 |  | 0.3173 | 
Granulicatella adiacens | 1 | 1 |  | 0.9422 | 
G. elegans | 0 | 1 |  | 0.3173 | 
Isolates Gram-negative aerobic cocci | 
Neisseria flavescens | 3 | 3 |  | 0.6749 | 
N. macacae/mucosa | 6 | 4 |  | 0.2582 | 
N. elongata | 1 | 0 |  | 0.3173 | 
N. perflava | 1 | 0 |  | 0.3173 | 
N. cinerea | 0 | 2 |  | 0.1468 | 
Isolates Gram-positive aerobic rods | 
Actinomyces oris/viscosus | 4 | 7 |  | 0.2048 | 
A. naeslundii | 2 | 2 |  | 0.9139 | 
(A) denticolens | 1 | 0 |  | 0.3173 | 
Schaalia odontolytica | 0 | 4 |  | 0.0306 | 
Rothia mucilaginosa | 5 | 7 |  | 0.2261 | 
R. dentocariosa | 5 | 4 |  | 0.3627 | 
R. aeria | 5 | 9 |  | 0.2351 | 
Corynebacterium durum | 1 | 0 |  | 0.3173 | 
Bacillus licheniformis | 2 | 0 |  | 0.1468 | 
(B) subtilis | 3 | 3 |  | 0.6749 | 
B. thermoamylovorans | 1 | 0 |  | 0.3173 | 
Isolates Gram-negative aerobic rods | 
Kingella oralis | 1 | 0 |  | 0.3173 | 
Eikenella corrodens | 0 | 1 |  | 0.3173 | 
Capnocytophaga granulosa | 1 | 0 |  | 0.3173 | 
(C) gingivalis | 4 | 2 |  | 0.2634 | 
C. sputigena | 1 | 2 |  | 0.6267 | 
C. ochracea | 0 | 2 |  | 0.1468 | 
Cardiobacterium hominis | 0 | 1 |  | 0.3173 | 
Haemophilus parainfluenzae | 1 | 0 |  | 0.3173 | 
Isolates Gram-positive anaerobic cocci | 
Parvimonas micra | 0 | 2 |  | 0.1468 | 
Isolates Gram-negative anaerobic cocci | 
Veillonella parvula | 9 | 7 |  | 0.5189 | 
V. dispar | 1 | 0 |  | 0.3173 | 
Veillonella sp. oral taxon | 1 | 1 |  | 0.9422 | 
Megasphaera micronuciformis | 0 | 2 |  | 0.1468 | 
Isolates Gram-positive anaerobic rods | 
Lancefieldella parvulum | 1 | 0 |  | 0.3173 | 
L. rimae | 1 | 1 |  | 0.9422 | 
Bifidobacterium longum | 0 | 1 |  | 0.3173 | 
Isolates Gram-negative anaerobic rods | 
Campylobacter showae | 1 | 0 |  | 0.3173 | 
C. concisus | 1 | 0 |  | 0.3173 | 
Prevotelle nigrescens | 1 | 0 |  | 0.3173 | 
P. histicola | 2 | 1 |  | 0.5432 | 
Non culturable | 0 | 1 |  | 0.3173 | 
Fusobacterium nucleatum | 4 | 1 |  | 0.1362 | 
Leptotrichia sp. oral taxon | 1 | 0 |  | 0.3173 | 
Selenomonas sp. oral taxon | 0 | 1 |  | 0.3173 | 
Selenomonas noxia clone | 0 | 2 |  | 0.1468 | 
CFU | 
CFU Total | 1.83E + 08 | 3.31E + 09 |  | 0.8089 | 
CFU aerobic bacteria | 1.76E + 08 | 2.82E + 09 |  | 0.9397 | 
CFU anaerobic bacteria | 7.29E + 06 | 4.93E + 08 |  | 0.9397 | 
CFU Gram-positive bacteria | 1.69E + 08 | 2.88E + 09 |  | 0.2568 | 
CFU Gram-negative bacteria | 1.48E + 07 | 4.34E + 08 |  | 0.2568 | 
Species Level Statistics | 
Different detected Species Total | 102 | 104 |  | 1 | 
Number dif. det. aerobic Species | 79 | 84 |  | 0.3064 | 
Number dif. det. anaerobic Species | 23 | 20 |  | 0.3064 | 
Number dif. det. gram-positive Species | 62 | 71 |  | 0.0523 | 
Number dif. det. gram-negative Species | 40 | 33 |  | 0.0523 | 
Isolate Level Statistics | 
Gram-positive cocci | 31 | 33 |  | 0.8197 | 
Gram-positive rods | 31 | 38 |  | 0.0137 | 
Gram-negative cocci | 22 | 19 |  | 0.1694 | 
Gram-negative rods | 18 | 14 |  | 0.4943 | 
Gram-positive aerobic cocci | 31 | 31 |  | 0.9698 | 
Gram-positive aerobic rods | 29 | 36 |  | 0.0157 | 
Gram-negative aerobic cocci | 11 | 9 |  | 0.3802 | 
Gram-negative aerobic rods | 8 | 8 |  | 0.6705 | 
Gram-positive anaerobic cocci | 0 | 2 |  | 0.1468 | 
Gram-positive anaerobic rods | 2 | 2 |  | 0.8285 | 
Gram-negative anaerobic cocci | 11 | 10 |  | 0.5644 | 
Gram-negative anaerobic rods | 10 | 6 |  | 0.4995 | 
CFU Level Statistics | 
CFU Gr+ aerobic cocci | 1.55E + 08 | 2.23E + 08 |  | 0.6501 | 
CFU Gr- aerobic cocci | 2.45E + 06 | 1.56E + 06 |  | 0.0756 | 
CFU Gr+ aerobic rods | 1.34E + 07 | 5.34E + 07 |  | 0.6501 | 
CFU Gr- aerobic rods | 5.07E + 06 | 3.29E + 06 |  | 0.2835 | 
CFU Gr+ anaerobic cocci | 0.00E + 00 | 1.60E + 06 |  | 0.1468 | 
CFU Gr- anaerobic cocci | 6.11E + 06 | 2.51E + 07 |  | 0.4961 | 
CFU Gr+ anaerobic rods | 3.00E + 04 | 9.10E + 06 |  | 0.9139 | 
CFU Gr- anaerobic rods | 1.15E + 06 | 1.35E + 07 |  | 0.9368 | 
CFU Gr+ cocci | 1.55E + 08 | 2.25E + 08 |  | 0.4963 | 
CFU Gr- cocci | 8.56E + 06 | 2.67E + 07 |  | 0.4963 | 
CFU Gr+ rods | 1.34E + 07 | 6.25E + 07 |  | 0.7055 | 
CFU Gr- rods | 6.22E + 06 | 1.67E + 07 |  | 0.5447 | 
Statistical Analysis of Bacillus spp. | 
Number of different Bacillus spp. | 3 | 1 |  | 0.6749 | 
Participants with Bacillus spp. | 3 | 3 |  | 1 | 
Mean CFU Bacillus spp. | 6.80E + 04 | 1.70E + 03 |  | 0.6749 | 

Bacterial cultivation from chewing tobacco

Table 3 lists the bacterial species that were successfully cultivated from Sudanese chewing tobacco. Notably, only aerobic endospore-forming bacteria comprising B. subtilis, Bacillus licheniformis and Bacillus pumilus were culturable. Among these bacteria directly detected in the Sudanese chewing tobacco, B. subtilis was identified in samples from both chewing tobacco users and the control group. However, B. licheniformis was detected only in two samples from chewing tobacco users, while B. pumilus could not be found in any sample.

Cultivated Species | CFU/ml
Bacillus subtilis | 3,00E + 07
Bacillus licheniformis | 1,00E + 07
Bacillus pumilus | 1,50E + 07
CFU | 5,50E + 07

Antimicrobial activity of sudanese chewing tobacco

No inhibitory effects of commonly used Sudanese chewing tobacco, measured as described above as zones of inhibition (0 mm), were observed against Enterococcus faecalis,_ Escherichia coli_,_ Streptococcus sanguinis_,_ Staphylococcus aureus_ or Bacillus subtilis.