## 3 Results  
The subjects were 26-56 years old (median 38; interquartile range 34-54), and 42/66 (64%) were females. A total of 30 subjects (50.8%) had periodontal disease. All subjects had periodontitis stage III and grade B except smokers, who had stage III and grade C. There were seven dropouts and, so, 59 microbiological samples were analyzed. Periodontitis prevalence increased in the order of IQOS (41%) \(<\) non-smokers (44%) \(<\) cigarette smokers (68%), without statistically significant differences between groups (Table 1). FMPS was similar between the three groups (non-smokers 58.5, cigarette smokers 58.3, IQOS 57.1). The number of perio-pathogenic species (scale 0-9 species) was similar in all three groups: non-smokers (median 6 species) \(=\) smokers (6) \(<\) IQOS (6.5); and it was similar between smokers and non-smokers (both median 6). It was significantly higher in those with periodontitis than without the disease, with moderate effect size (7 vs. 6; \(p=0.005\); \(\mathrm{r}=-0.368\); Figure 1). When combining exposure and periodontal status, each exposure group had more perio-pathogenic species than non-smokers: non-smokers without periodontitis (median 5 species) \(<\) smokers without periodontitis (5.5) \(<\) IQOS without periodontitis (6); non-smokers with periodontitis (6.5) \(<\) IQOS with periodontitis (7) \(=\) smokers with periodontitis (7); however, the differences did not reach the level of statistical significance. The number of perio-pathogenic species was not related to age, sex, or FMPS. In multiple linear regression, periodontitis was the only predictor of the number of perio-pathogenic species (dependent variable), when controlling for the effect of consumption of burning and heated tobacco, sex, age, and oral hygiene (adjusted \(\mathrm{R}^{2}=0.163\); \(p=0.012\)). Periodontitis, burning and heated tobacco, age, sex, and FMPS were considered as independent variables. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & & **Presence of Periodontitis** & \\ \hline
**Exposure to Tobacco** & **Non-Periodontitis** & **Periodontitis** & **Total** \\ \hline CTRL (N (\%)) & 10 (55.6\%) & 8 (44.4\%) & 18 (100\%) \\ IQOS (N (\%)) & 13 (59.1\%) & 9 (40.9\%) & 22 (100\%) \\ S (N (\%)) & 6 (31.6\%) & 13 (68.4\%) & 19 (100\%) \\ Total (N (\%)) & 29 (49.2\%) & 30 (50.8\%) & 59 (100\%) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Distribution of periodontal disease between control group (non-smokers, CTRL), IQOS users, and classic smokers (S). The median ratio of perio-pathogenic species in biofilm in the whole sample was \(<\)7%, with _F. nucleation_ being the highest (4%), followed by _T. forysthia_ (3%), _P. gingivalis_ (0.3%), _E. nodatum_ (0.03%), and _T. denticola_ (0.02%), while the ratio of other species was less than 0.01%. A significant difference between smokers, non-smokers, and IQOS in terms of the ratio of perio-pathogenic species was only present for _A. actinomycetemomitans_ (\(p=0.013\)), where non-smokers had the highest and IQOS the lowest ratio. When comparing all types of smokers with non-smokers, they differed in terms of the ratios of _A. actinomycetemontans_ and _C. rectus_ (\(p\leq 0.047\)), with _A. actinomycetemontans_ having a higher ratio in non-smokers and _C. rectus_ in smokers. Subjects with periodontitis had higher ratios of _P. gingivalis_, _P. intermedia_, _T. forysthia_, and _E. nodatum_ than those without periodontitis (\(p\leq 0.039\)), with the highest ratio in the case of _T. forysthia_ (2 vs. 0.1%; \(p=0.009\)). According to tobacco exposure, the highest levels of _C. rectus_ and _F. nucleatum_ were observed in the smoker group, while _F. nucleatum_ was the most dominant bacterium in the IQOS group. _A. actinomycetemontans_ was the most abundant in the CTRL group. When groups with tobacco exposure and the presence of periodontitis were compared, _P. intermedia_ showed the highest abundance in the group of non-smokers with periodontitis (CTRL peri), while _P. gingivalis_ and _T.