## 4 Discussion
It seems that, although IQOS is presented as a less-harmful tobacco product with less severe consequences for human health, it still has a detrimental effect on the periodontal tissues in terms of the prevalence and abundance of certain periodontal pathogens. There were some limitations and concerns associated with our investigation. The study was observational and, so, all data and samples were collected before eventual periodontal therapy. It would be interesting if resampling was carried out after periodontal treatment in a longitudinal study. In this way, detailed insights, on a per-bacteria basis, into how initial periodontal therapy affects the numbers and prevalence of micro-organisms when different tobacco products are used would be enabled. Our study showed that increased bacterial load is related mainly to the presence of the disease, rather than the smoking habit. However, there was no evidence of a potential stratification according to the severity and the extent of the disease, which could clarify the results a bit more. The sample size was small, but metagenomic studies such as the present one are mostly small due to the high cost of analyses, complexity of the procedure itself, and interpretation of the large amount of received data. NGS technology has revolutionized genomics research with the speed and extent of the received results, but it still has some disadvantages beyond those already mentioned above. It is prone to errors and biases in sequencing data, which can affect the accuracy and completeness of the results [40; 41; 42]. However, its advantages in terms of enabling the sequencing of microbial communities provide invaluable contributions to science. NGS technology can enable and upgrade the possibility of personalized dental medicine through the identification of microbial communities found in different lesions caused by bacteria in the oral cavity [42; 43]. Personalized treatment protocols targeting, for example, specific periodontitis, is one of possible applications. This research is the first of its kind that used 16s rRNA NGS analysis to determine the exact composition of perio-pathogenic polymicrobial communities in subgingival dental plaque in IQOS users, classic smokers, and non-smokers with or without periodontal disease. The data obtained in this study have the potential to help in better understanding the effect of IQOS aerosols on the periodontal microbiome. No tobacco products can be completely safe. The consequences for human health can be minimized but, for now, not nullified. In the case of IQOS usage, there are concerns regarding its short- and long-term health effects due to its novelty on the world market. Studies have confirmed that IQOS users are exposed to lower levels of some toxicants, when compared to cigarette smoke; however, their exposure to higher levels of some (not known and not sufficiently researched) other toxicants and health consequences are yet to be revealed in the future [17]. Smoking does not have a stronger influence on the oral subgingival microbiome than periodontal disease, but it also cannot be claimed that smoking is harmless and without any consequences no matter which tobacco product we use, as our research demonstrated. Guidelines for future research would include a longitudinal study concept, ideally before and after periodontal therapy was conducted. It would be interesting to carry out resampling every few months through one year, in order to monitor the reduction (stagnation or growth?) in the periodontal pathogens after periodontal therapy and recall visits. The additional inclusion of different tobacco products (e.g., e-cigarettes, cigars, hookah, and so on) and comparing their effects on the diversity and abundance of the periodontal microbiome using 16S rRNA sequencing would certainly clarify the effects of different kinds of tobacco products on the periodontal microbiome.