## 3 Results and Discussion
### Microbiology
#### 3.9.1 Time-Kill (TK) Experiments
9.2 Determination of the Optimal Contact Time of a Highly Contaminated Water Model with a Column Containing R4  
In this part of the study on R4, we evaluated its potential environmental application as a new material for sanitation systems capable of reducing the fecal bacterial contamination of polluted water. In particular, a small amount of R4 was inserted in a glass column (\(\mathcal{O}\) = 2.5 cm), up to a thickness of 3 cm, to imitate a hypothetical disinfection system working by contact followed by rapid filtration. Then, the best contact time between the aqueous models of contaminated water (artificially prepared) and R4, before filtration, for obtaining the best decontaminant activity was determined. Figure S2 in the Supplementary Materials shows the column and the appearance of R4 in contact with the aqueous model of contaminated water (Figure S2a) and just after filtration (Figure S2b). In an early investigation, an aqueous model of water containing _E. coli_ 224, a strain fully susceptible to antibiotics, was used to explore whether, after 4 h of exposure, bacterial regrowth could occur. According to the obtained results, over 4 h, regrowth occurred, and a decrease in the killing efficiency percentage from 97% (4 h) to 31% (24 h) was evidenced (Figures S3 and S4 \begin{table}
\begin{tabular}{c c c c} \hline  & **1 h** & **2 h** & **4 h** \\
**Strains** & **RLog\({}_{10}\) (CFU/mL) (k\%)** & **RLog\({}_{10}\) (CFU/mL) (k\%)** & **RLog\({}_{10}\) (CFU/mL) (k\%)** \\ \hline _E. faecalis_ 365 * & \(-\)3.1, 99.9 & \(-\)5.3, 100 & \(-\)5.3, 100 \\ _E. facecium_ 503 * & \(-\)3.1, 99.9 & \(-\)5.3, 100 & \(-\)5.3, 100 \\ _E. coli_ 462 ** & \(-\)1.41, 96.1 & \(-\)2.89, 99.9 & \(-\)2.75, 99.8 \\ _Salmonella_ 505 & \(-\)1.41, 06.1 & \(-\)2.89, 99.9 & \(-\)2.75, 99.8 \\ \hline \end{tabular}
* denotes vancomycin-resistant isolates (VRE); ** New Delhi metallo \(\beta\)-lactamase producer.  
\end{table} 
Table 6: Log \({}_{10}\) reduction in the initial cell number of the tested bacteria expressed in CFU/mL and the killing percentages of bacteria when tested in a mixture exposed to 25 \(\mu\)g/mL R4 for 1, 2, and 4 h. \begin{table}
\begin{tabular}{c c c c} \hline  & **1 h** & **2 h** & **4 h** \\
**Strains** & **RLog\({}_{10}\) (CFU/mL) (k\%)** & **RLog\({}_{10}\) (CFU/mL) (k\%)** & **RLog\({}_{10}\) (CFU/mL) (k\%)** \\ \hline \multicolumn{4}{c}{_Gram-positive species of genus Enterococcus_} \\ \hline _E. faecalis_ 365 * & \(-\)2.9, 99.9 & \(-\)5.3, 100 & \(-\)5.3, 100 \\ _E. facecium_ 503 * & \(-\)3.1, 99.9 & \(-\)5.2, 100 & \(-\)5.2, 100 \\ \hline \multicolumn{4}{c}{_Gram-negative species of Enterobacteriaceae family_} \\ \hline _E. coli_ 462 ** & \(-\)1.2, 93 & \(-\)1.8, 99 & \(-\)2.6, 99.8 \\ _Salmonella_ 505 & \(-\)0.8, 83 & \(-\)1.7, 98 & \(-\)3.0, 99.9 \\ \hline \end{tabular}
* denotes vancomycin-resistant isolates (VRE); ** New Delhi metallo \(\beta\)-lactamase producer.  
\end{table} 
Table 5: Log \({}_{10}\) reduction in the initial cell number of the tested bacteria expressed in CFU/mL and killing percentages of bacteria tested singularly when exposed to R4 for 1, 2, and 4 h.  
in Supplementary Materials). After establishing this, in order to obtain the best efficient decontamination effects, it is not necessary to exceed contact times between R4 and bacteria of four hours. A more realistic aqueous model of infected water was prepared using MDR strains of _E. facecium_ 503, _E. faecalis_ 365, _E. coli_ 462, and strain 505 of a _Salmonella_ species of Group B. Therefore, experiments of the treatment of the contaminated aqueous model were carried out in the column containing R4, as previously described, and determinations of the Log\({}_{10}\) (CFU/mL) after contact times of 1, 2, and 4 h before rapid filtrations were made. Determinations of Log\({}_{10}\) (CFU/mL) were made in parallel on the same mixture of bacteria not treated with R4 at the same times of exposure (control (CTR).