## 3 Results
### Microbial Re-Growth and Biofilm Persistence  
To assess the microbial regrowth under the different conditions, the 4 groups of OE, were incubated for further 24 h (up to time 48 h) in fresh medium and tested by CFU assay and MALDI-TOF MS/API analysis. Table 3 shows that the total CFU detected in all the groups were similar, ranging between 552 to 953 \(\times\) 10\({}^{4}\)/100 \(\mu\)L. Interestingly, _S. mitis/oralis_ was the most representative species both in saliva A/saline and saliva P/saline groups (91.01% and 85.3%, respectively), while, these same species were not detected in Tp-SUP treated groups, no matter whether A or P saliva groups were considered. In contrast, in the Tp-SUP-treated groups, most of the microbial population was identified as _Lactobacillus_ spp. (>90%); such bacteria were undetectable (<0.1 CFU \(\times\) 10\({}^{4}\)/100 \(\mu\)L) in the two groups that had not received the Tp-SUP treatment.  
Figure 2: Early biofilm formation onto saliva-contaminated OE exposed or not to Tp-SUP. The saliva-contaminated OE were incubated for 23 h at 37 \({}^{\circ}\)C + 5% CO\({}_{2}\) and then exposed to saline or Tp-SUP for additional 1 h, in the presence of CFDA, to allow fluorescence staining. Then, the OE were washed with warm saline and transferred in new wells. The plate was read by the Fluoroskan microplate reader and the fluorescence signal was recorded. The depicted values represent the mean \(\pm\) SEM (RFU/OE) of 13–16 replicates obtained by 3 independent experiments. ****\(p<\) 0.0001; ****\(p<\) 0.001.  
\begin{table}
\begin{tabular}{l c c c c c c c c} \hline \hline \multicolumn{1}{c}{**Species Identified at**} & \multicolumn{1}{c}{**Saliva A/Saline**} & \multicolumn{1}{c}{**Saliva P/saline**} & \multicolumn{1}{c}{**Saliva A/Tp-SUP**} & \multicolumn{1}{c}{**Saliva P/Tp-SUP**} \\ \hline  & **CFU** & **(\%)** & **CFU** & **(\%)** & **CFU** & **(\%)** & **CFU** & **(\%)** \\ \hline _Neisseria fatav/subflava_ & \textless{}0.1 & 0.018 & \textless{}0.1 & 0.01 & \textless{}0.1 & 0.01 & \textless{}0.1 & 0.01 \\ _Streptococcus thermophilus_ & 13 & 2.3 & 63 & 7.01 & 57 & 5.98 & 57 & 8.23 \\ _Streptococcus mitis/oralis_ & 503 & 91.0 & 763 & 85.3 & \textless{}0.1 & 0.01 & \textless{}0.1 & 0.01 \\ _Streptococcus salivarius_ & 36 & 6.5 & 68 & 7.61 & 6 & 0.63 & 5 & 0.72 \\ _Rothia aeria_ & \textless{}0.1 & 0.018 & \textless{}0.1 & 0.01 & \textless{}0.1 & 0.01 & \textless{}0.1 & 0.01 \\ _Lactobacillus_ spp. & \textless{}0.1 & 0.018 & \textless{}0.1 & 0.01 & 890 & 93.3 & 630 & 91.0 \\ \hline Total CFU/100 \(\mu\)L \(\times\) 10\({}^{4}\) & 552.3 & 100 & 894.3 & 100 & 953.3 & 100 & 692.3 & 100 \\ \hline \hline \end{tabular}  
* The OE, that had been contaminated with Saliva A and P (time 0 h), incubated for 23 h and exposed or not to the Tp-SUP (for 1 h), were further incubated at 37 \({}^{\circ}\)C + 5% CO\({}_{2}\) (up to 48 h) and then analyzed by CFU assay. The colonies isolated after incubation in selective media were counted, clustered and identify by MALDI-TOF MS or API gallery systems. The results are given as microbial load (mean of CFU \(\times\) 10\({}^{4}\)/100 \(\mu\)L) and as percentage of bacterial species.  
\end{table}
Table 3: Microbial re-growth onto saliva-contaminated and Tp-SUP treated OE.  
To evaluate biofilm persistence, at time 48 h, the OE were washed again, transferred to new wells and labelled with CFDA fluorescent reagent for 1 h.  
As shown in Figure 3, the RFU/OE were consistently lower in saliva P/saline than in saliva A/saline groups, although without reaching significance. The exposure to Tp-SUP significantly reduced the RFU/OE in both groups. When comparing saliva A/saline with saliva A/Tp-SUP or saliva P/saline with saliva P/Tp-SUP, statistically significant differences were observed.