Dental caries is a multifactorial disease primarily caused by supragingival dental biofilm. Its progression is influenced by many environmental factors, which include smoking. Tobacco heating systems (THS) are a novel tobacco product whose effect on the microbiome of the supragingival dental biofilm has not yet been investigated.

The aim of the proposed research is to determine and compare the composition of the microbiomes of the supragingival dental biofilm of THS smokers, cigarette smokers, and nonsmokers using the Next Generation Sequencing method and to assess and compare the risk of new caries lesion formation between the test groups using the Cariogram method.

The results of this research will provide insight into changes in the microbiome of the supragingival dental biofilm resulting from exposure to aerosols from tobacco combustion and tobacco heating.

Dental caries is a dynamic disease caused by the presence of dental biofilm which leads to the demineralization and loss of hard dental tissues. Historically, the main microbes which were considered responsible for developing caries lesions were Streptococcus mutans and Lactobacilli, but results of recent studies have shown tha a far greater number of microorganisms and microbial interactions are present. Furthermore the progress of the disease is also affected by many environmental factors like tobacco smoking.

A wide number of studies have already confirmed a positive correlation between tobacco smoking and the incidence/presence of caries lesions in different populations of all ages. Also, invitro studies have confirmed that bacterial proliferation and metabolic activity is affected by the components, and concentrations of components of cigarette smoke, especially nicotine.

Tobacco heating systems (THS) are a relatively novel nicotine delivery product that when consumed creates an aerosol that has the same contents as cigarette smoke, but the concentration of it's components is greatly reduced. The effect of THS smoking on the bacterial microbiome of supragingival dental biofilm has not yet been researched.

In the present study a total of 60 subject have been enrolled: 20 THS smokers, 20 cigarette smokers and 20 non-smokers. Subject from each group are age and gender matched.

The goal of the study is to compare the bacterial microbiomes of supragingival dental biofilm of the 3 examined groups. This will be done by collecting supragingival dental biofilm samples, isolating bacterial DNA from them and conducting 16s RNA metagenomics through next generation sequencing.

Furthermore, caries risk assessments will be conducted through the Cariogram method . Necessary data for the Cariogram analysis is obtained through routine anamnesis, intraoral clinical examination and tests.

The results of the study will give more insight into the effects of THS and cigarette smoking on the microbiome and microbial interactions in supragingival dental biofilm.

Inclusion Criteria:

• smokers of tobacco heating systems that have smoked at least 100 heating sticks during their lifetime and have smoked exclusively tobacco heating systems for at least 6 months
• cigarette smokers that have smoked at least 100 cigarettes during their lifetime and have smoked exclusively factory made cigarettes for at least 6 months
• non-smokers that have never smoked during their lifetime
• signed informed consent

Exclusion Criteria:

• use of antibiotics 3 month prior to examination and sampling
• active orthodontic treatment
• patients suffering from autoimmune diseases
• patients suffering from type II diabetes
• constant use of oral antiseptics
• use of corticosteroids of other immunosuppressive drugs/treatments
• patients who have less than 10 teeth

In the present study a total of 60 subject are enrolled. The subjects are divided into 3 groups: tobacco heating system smokers, cigarette smokers and non-smokers. In each group there are 20 subjects which are age and gender matched.