## 3 Results
### Microbial Adhesion onto OE, Growth and Biofilm Formation  
The ability of microorganisms occurring in the saliva A and P to mediate adhesion onto OE was evaluated. Accordingly, two sets of pre-sterilized OE were exposed to each pool of saliva in the presence of CFDA to allow fluorescence staining of cells. After 1 h at 37 \({}^{\circ}\)C, a condition commonly used to allow microbial adhesion onto abiotic surfaces [37], the OE were washed with warm saline, transferred to new wells containing fresh medium and analyzed for fluorescence emission. As shown in Figure 1, the fluorescence signal, indicating the amounts of viable and metabolically active cells, was significantly lower in saliva P, as compared to saliva A contaminated OE (about 50% difference). Subsequently, the contaminated OE were incubated for further 23 h, under standard culture conditions, to allow microbial growth. Then, each set of OE (saliva A and saliva P contaminated groups) was split in two sub-groups and exposed to saline or Tp-SUP (in both cases, 100 \(\upmu\)L/well were added) for additional 1 h at 37 \({}^{\circ}\)C. Subsequently, each sample was assessed for microbial growth by CFU assay, followed by MALDI-TOF MS/API colonies identification. As depicted in Table 2, when comparing the total CFU from saliva A- and saliva P-contaminated OE maintained in saline, about 1 log lower values were found in the latter group. Moreover, the treatment with Tp-SUP strongly reduced the CFU, in both saliva A- and saliva P-contaminated OE; particularly, the reduction was approximately \begin{table}
\begin{tabular}{l c c c c} \hline \hline
**Species Identified at Time 0 h** & \multicolumn{2}{c}{**Saliva A**} & \multicolumn{2}{c}{**Saliva P**} \\ \hline  & **CFU** & **(\%)** & **CFU** & **(\%)** \\ \hline _Neisseria flavus_subflava_ & 27 & 7.1 & 55 & 10.9 \\ _Streptococcus thermophilus_ & 34 & 8.7 & 24 & 4 \\ _Streptococcus mitis/paralis_ & 254 & 67.0 & 406 & 81 \\ _Streptococcus salivarius_ & 64 & 16.8 & 15 & 2.9 \\ _Rothia aerial_ & \textless{}0.1 & 0.02 & 1 & 0.1 \\ _Candidida_ spp. & \textless{}0.1 & - & \textless{}0.1 & - \\ _Lactobacillus_ spp. & \textless{}0.1 & - & \textless{}0.1 & - \\ \hline Total CFU/100 \(\upmu\)L \(\times\) 10\({}^{4}\) & 379.1 & 100 & 501 & 100 \\ \hline \hline \end{tabular} The two pools of saliva A and P, collected from six healthy volunteers, during mastication of gum A and P, were analyzed to assess the total microbial load, at time 0 h, by CFU assay. The colonies isolated after incubation in selective media were counted and identified by MALDI-TOF MS or API gallery system. The microbial load, expressed as mean of CFU/100 \(\upmu\)L \(\times\) 10\({}^{4}\) saliva, and the percentage of bacterial species detected are shown.  
\end{table} 
Table 1: Initial microbial load and evaluation of the main microbial species in Saliva A and P.  
Figure 1: Microbial adhesion onto saliva-contaminated orthodontic elastics (OE). Two sets of OE were exposed to saliva A and P for 1 h at 37 \({}^{\circ}\)C. During that time, fluorescence staining of viable cells was allowed by Carboxyl-fluorescein Diacetate, Acetoxymethyl Ester (CFDA) addition. Then, the OE were washed with warm saline and transferred to new wells. The plate was read by the Fluoroskan reader and the fluorescence signal was recorded. The depicted values represent the mean (RFU/OE) \(\pm\) SEM of 48 replicates obtained by 3 independent experiments. \({}^{***}\)\(p\)\(<\) 0.001.  
of 2 logs when comparing saliva A/saline vs. saliva A/Tp-SUP and of about 1 log considering saliva P/saline vs. saliva P/Tp-SUP. In parallel, species identification revealed that the most representative one, both in saliva A and P/saline groups, was _S. mitis/oralis_ (97.1% and 99.1%, respectively). Tp-SUP treatment did not substantially change this percentage. Furthermore, when considering the crude numbers, the _S.