Materials and methods

Clinical data collection

Twenty adult male participants from Sudan, who presented at the clinic at the Khartoum Centre for Research and Medical Training (Khartoum, Republic of the Sudan) were enrolled in the study after obtaining written informed consent. The study protocol was approved by the Ethics Committee of the El Razi College for Medical and Technological Sciences (Ref. KCRMT/Nov 2011). Among the 20 participants, ten were consumers of Sudanese smokeless tobacco, while the remaining ten did not use smokeless tobacco. Participants’ ages ranged from 19 to 70 years, with a median age of 29 (Q1: 23.75; Q3:40.5) and a mean age of 34. All participants were in good general health at the time of sample collection. Participants presenting with probing pocket depths ≥ 4 mm as well as individuals with a history of other tobacco use (e.g., cigarette smoking) were excluded during recruitment. Periodontal pockets probing was performed clinically at the study site during participant screening using a PCP-UNC-15 periodontal probe [35]. The inclusion criteria for the tobacco user group were written informed consent, an age above 18 years, male gender, regular daily long-term use of Sudanese smokeless tobacco of at least 5 years, no consumption of other kinds of tobacco (e.g., cigarettes), no antibiotic intake 3 months prior to the time of sample collection and no consumption of alcohol Participants with systemic conditions that could influence the oral microbiota or host immune response were excluded. These included diabetes mellitus, HIV infection, immunodeficiency disorders, autoimmune diseases, chronic inflammatory diseases, active malignancies and immunosuppressive therapy. The inclusion criteria for the control group were an age above 18 years, male gender, no history of Sudanese smokeless tobacco use, no use of mouthwash and no antibiotic intake 3 months prior to sample collection, no consumption of other kinds of tobacco (e.g., cigarettes), no consumption of alcohol and no systemic diseases. All participants provided written informed consent following verbal and written explanation at the Khartoum Centre for Research and Medical Training, where clinical examination and data acquisition were conducted by Dr. Fadil Elameen. The recorded data included name, date of birth, gender, ethnicity, medical history, medication usage, antibiotic intake, hospitalization history, documentation of remaining teeth and the sampling sites. Furthermore, the duration of tobacco use in years, the age at initiation of tobacco use, frequency of daily tobacco use, interruptions in tobacco use, intraoral sites of tobacco placement, the number of placement sites in the oral cavity and the approximate diameter of tobacco portions used were recorded for the group of tobacco users. Participant names were documented exclusively at the clinical site for the purpose of informed consent and administrative record-keeping. For all microbiological and statistical analyses, the data were pseudonymized, and no direct identifiers were transferred to or stored within the study database used for research evaluation.

All samples were collected at the Khartoum Centre for Research and Medical Training, Northern Sudan, using sterile cotton swabs, which were handled with sterile forceps throughout sample collection to avoid contamination. The mucosal site where the tobacco was habitually placed during consumption was swabbed and the swab was immediately transferred into 0.75 ml of Reduced Transport Fluid (RTF) [36]. For control participants, samples were obtained accordingly from the vestibular mucosa adjacent to the mandibular anterior teeth or the first quadrant, representing anatomically corresponding vestibular mucosal sites. Samples were stored at −80 °C and transported on dry ice by air to the Department of Operative Dentistry and Periodontology at the University Medical Center Freiburg, Germany, where they were subsequently stored at −80 °C to ensure uninterrupted cold chain maintenance.

Following thawing in a 36 °C water bath, samples were vortexed for 30 s (Vortex Genie 2, Scientific Industries, New York, USA) to homogenize. Dilutions were prepared in Peptone-Yeast broth (PY) and an aliquot of 100 µL thereof were plated on Columbia blood agar (CoBl) plates, yeast-cysteine blood agar (YCB) plates and Trypticase-Soy-Bacitracin-Vancomycin-Agar (TSBV) plates, whereby the latter were used for detection of Aggregatibacter actinomycetemcomitans. YCB plates were sealed in anaerobic jars using chemical gas generators (GENbox anaer, bioMerieux, Marcy-l’Étoile, France) and an indicator strip verifying anaerobic conditions (Merck KGaA, Darmstadt, Germany). Plates were incubated at 36 °C for 10 days. All other plates were incubated in a CO2 incubator (Heraeus Holding GmbH, Hanau, Germany) at 36 °C with 5–10% CO2 for 5 days. CoBl plates were used for aerobic bacteria cultivation, and YCB plates for anaerobic bacteria. Quantification of the total bacterial count and the different bacterial isolates was conducted by determination of the colony forming units (CFU).

Microbiological analysis of clinical samples

For the identification of cultured microorganisms, a standardized approach was applied to each subculture, consisting of macroscopic and microscopic differentiation as well as MALDI-TOF mass spectrometry. If these methods did not yield a conclusive result, Vitek2 System (bioMérieux, Marcy-l’Étoile, France), 16S rRNA PCR with subsequent Sequencing, Catalase Test, Nitrate Reductase Test, Spot Indole Test, Cytochrome Oxidase Test, Urease Test, API 20 Strep System (bioMérieux, Marcy-l’Étoile, France) and Optochin and Oxacillin Sensitivity Tests were employed depending on the bacterial species under investigation.

Microbial profile of toombak

To determine the bacterial content of chewing tobacco, a dilution series of a typical Sudanese chewing tobacco application was prepared. One gram of chewing tobacco was suspended in 9 ml phosphate-buffered saline (PBS), vortexed, and incubated for 2 h at 36 °C. The suspension was centrifuged at 845 g and 20 °C for 10 min. The supernatant was subjected to a tenfold dilution series in PBS and plated on CoBl plates and YCB plates. All plates were incubated for 5 days at 36 °C in a CO₂ incubator. Subsequent quantification, isolation of subcultures, freezing of pure cultures, and microbial identification followed procedures described above.

Testing of antimicrobial activity of chewing tobacco

To evaluate the antimicrobial activity of chewing tobacco, three different dilutions (Initial, 1:10, 1:102) of typical Sudanese chewing tobacco were prepared: 3 g of chewing tobacco were added to 10 ml of 0.9% NaCl and incubated for 24 h at an incubation temperature of 36 °C. The solution was then centrifuged at 845 g for 10 min at 20 °C and the supernatant was used as initial dilution. 100 µl of the initial dilution was diluted with 900 µl of 0.9% NaCl resulting in a 1:10 dilution. Then, 100 µl of this dilution was again diluted with 900 µl of 0.9% NaCl resulting in a 1:102 dilution. Simultaneously, test plates were prepared with the following bacterial strains: Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Streptococcus sanguinis (DSM 20068), Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (DSM 6633).

Bacteria were inoculated into separate tubes containing 0.9 ml PY-medium and homogenized using a vortex mixer. Subsequently, 40 µl aliquots were transferred to tubes containing 4 ml 0.9% NaCl to achieve a 1:100 dilution. These dilutions were plated on Mueller-Hinton agar plates (for E. faecalis,_ E. coli_,_ S. aureus_ and B. subtilis) or CoBl plates (for S. sanguinis) and evenly spread by swirling. Excess liquid was removed using a disinfected aspirator and plates were pre-incubated for 15 min with open lids. Afterwards, three wells were punched per plate, and 100 µl of each chewing tobacco dilution was added. After incubation for 24 h at 36 °C, zones of inhibition surrounding the wells were examined under 1.7-fold magnification (Xylem Analytics, Weilheim, Germany), and their diameters were measured. Larger inhibition zone diameters were interpreted as indicating greater antimicrobial activity.

Statistical analysis

Data visualization was performed GraphPad Prism 10 (GraphPad Software, Boston, USA). The present investigation was conceived as an exploratory pilot study intended to generate initial microbiological data in a population and exposure context that has received little prior scientific attention. Accordingly, no a priori power calculation was performed. Analyses were conducted in a descriptive and hypothesis-generating manner and no formal adjustment for multiple comparisons was applied. Differences between chewing tobacco users and the control group were analyzed using the Wilcoxon rank-sum test. The significance level α was set to 5%. All statistical analyses were performed using STATA 14.2 software (StataCorp LLC, College Station, USA).