## 4 Discussion  
None of the groups except group F (positive control) presented bacterial infiltration during the time of our experiment. The use of Eppendorf tubes as experimental model of cavity access was considered to exclude possible communications present in natural teeth (exposed dentinal tubules, lateral canals, enamel/cement cracks) that could determine false positive bacterial infiltrations. The complete immersion of Eppendorf tubes in a bacterial suspension of _S. mutans_ was a condition not naturally observable in in vivo studies, considered to test the sealing ability of filling material and temporary restorations in simulated extreme experimental conditions. Agar culture medium was inserted in Eppendorf tubes to have a solid base to support spacer and filling materials and to facilitate the extraction of cells and genetic material for PCR and microbiological tests. Positive control demonstrated the accuracy of microbiological analysis and negative control excluded external contaminations during collection procedures of samples. Moreover, absence of _S. mutans_ genome was confirmed through RT-PCR, in order to further support isolation results. In other words, we have planned to carry out also the real-time PCR assay which, as is well known, is able to detect the nucleic acids of pathogens. Therefore, even the presence of dead pathogens eventually passed through the restoration may be detected. Accordingly, the lack of isolation of _S. mutans_ from the Agar samples of Eppendorf tubes was confirmed as the absence of microorganisms that passed through the filling material. _Streptococcus mutans_ is generally considered to be a major pathogen of human caries and is also involved in periodontal diseases. Moreover, _S. mutans_ can migrate from periodontal pocket into endodontic system without clinically coronal leakages such as caries, traumatic tooth fractures, or old restorations. _S. mutans_ strains are classified into serotypes c, e, f, and k on the basis of the biochemical composition of serotype-specific polysaccharides. From the literature, it is known that approximately 70-80% of strains found in the human oral cavity are classified as serotype c, followed by the serotype e (-20%), and the serotypes f and k (less than 5% each). The virulence of _S. mutans_ as a dental pathogen mostly resides in its ability to adhere and form biofilms on tooth surfaces, produce large quantities of organic acids, and tolerate low pH and oxidative stress. The oral host tissue attachment \begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**Groups**} & \multicolumn{2}{c}{**T5**} & \multicolumn{2}{c}{**T10**} \\ \cline{2-5}  & **I** & **RT** & **I** & **RT** \\ \hline A & - & - & - & - \\ B & - & - & - & - \\ C & - & - & - & - \\ D & - & - & - & - \\ E & - & - & - & - \\ F & + & + (22.50) * & + & + (19.00) * \\ G & - & - & - & - \\ \hline \end{tabular} Legend: +: positive sample; -: negative sample. * In brackets the Ct values of RT.  
\end{table} 
Table 1: Detection of _S. mutans_ by isolation (I) and real-time PCR (RT) in agar samples from Eppendorfs vials after 5 days (T5) and 10 days (T10) of incubation. could be due to a group of glycotransferases, which act as a cell surface protein antigen, recognized to have a collagen-binding activity. The most described collagen-binding proteins (CBPs), described only in the _S. mutans_ strains, are the Cnm and Cbm proteins. These act as adhesins, i.e., they have the ability to adhere to type I collagen, thus promoting the biofilm formation, the development, and the duration of the periodontal pathologies. In addition, _S. mutans_ strains can became invasive, and the cnm gene is required for _S. mutans_ adherence to endothelial and epithelial cells and intracellular invasion, also being an important virulence factor for systemic disease secondary to bacteremia such as endocarditis. It is well-known that _S.