## 3 Results and Discussion
### Microbiology  
#### 3.9.1 Time-Kill (TK) Experiments  
Hypothesizing the possible use of R4 as a new material for the decontamination (by contact) of water infected by bacteria, we explored the possible bactericidal effects of R4 by carrying out TK assays in suspension. In particular, TK experiments were carried out for 4 h with R4 on selected fecal isolates, including MDR strains, potentially present in non-potable water as described in Section 2.9.2. The experiments were repeated until reproducibly was achieved. The Log10 (CFU/mL) of the surviving bacterial cells for a period of 4 h are reported in the dispersion graphs with respect to the time values (0, 1, 2, 4 h), thus obtaining the time-kill curves. The lines whose trend was observed most frequently are reported in Figures 10 and 11. Figure 10 shows the most representative curves obtained for each species studied singularly (Figure 10a-d) while Figure 11 shows the results obtained when R4 was tested on a mixture of the same isolates reported in Figure 11. In particular, Figure 10a-d show the results obtained with the Group B _Salmonella_ species strain 505, _E. coli_ 462 (a strain producing New Delhi metallo-\(\beta\)-lactamases), _E. faecium_ 503 (VRE), and _E. faecalis_ 365 (VRE), respectively. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Polymer** & **Weight** & **HCl 0.1 N** & **NH3+** & **MmolesNH3+/g** & **\(\mu\)molesNH3+/g** & **Error * (\%)** \\ \hline
**R4** & 36.8 & 6.00 \(\pm\) 0.02 & 0.6 \(\pm\) 0.002 & 16.30 \(\pm\) 0.05 & 16,300 \(\pm\) 50 & 1.15 \\ \hline \hline \end{tabular}  
* Respect to UV-Vis determination by the method of Gaur and Gupta.  
\end{table} 
Table 4: Content of NH3+ of R4 by potentiometric titration of an exactly weighed amount of resins. Figure 9: Potentiometric titration of R4 (**a**); first derivative (dpH/dV) of the curve in Figure 10a (**b**) (error bars knowingly omitted). In both cases, the results were expressed as the Log\({}_{10}\) reduction (RLog\({}_{10}\) (CFU/mL)), and the killing percentage efficiency (K (%)). The values reported in Tables 5 and 6 are those that were observed most frequently. Figure 11: Representative results of TK experiments obtained from treating a mixture of Group B _Salmonella_ species 505, _E. coli_ 462 (New Delhi metallo-\(\beta\)-lactamase producer), _E. faecium_ 503, and _E. faecalis_ 365, with R4 for 4 h.  
Figure 10: Representative results of the TK experiments obtained from treating each R4 species separately for 4 h. Group B _Salmonella_ species strain 505 (**a**), _E. coli_ 462 (a strain New Delhi metallo-\(\beta\)-lactamase producer (**b**), _E. faecium_ 503 (**c**), and _E. faecalis_ 365 (**d**). According to Figure 10a,b, R4 was bactericidal against Group B _Salmonella_ species 505 after 4 h of exposure while it cannot be considered bactericidal against _E. coli_ 462 since the Log\({}_{10}\) reduction was <3 (Table 5). When TK experiments were performed on a mixture of the same strains that were previously tested singularly, the observed bactericidal effects of R4 on _Salmonella_ 505 when tested alone were initially maintained, and a Log\({}_{10}\) reduction = 3 in the initial inoculum of both _E. coli_ 462 and _Salmonella_ 505 in mixture was detected after 2 h of exposure (Figure 10, Table 6). Up to 4 h, even if the simultaneous presence of different bacterial species favored a certain bacterial regrowth of both _E. coli_ and _Salmonella_ (Figure 11 and Table 6), the reduction in the killing percentage efficiency of R4 was trivial since it changed from 99.99% to 99.8%. Interestingly, against all strains of _Enterococcus_ considered, R4 showed rapid bactericidal effects after only 2 h of exposure, and a killing percentage of 100% was observed over all times of exposure, both when VRE _E. facecium_ 503 and _E. faecalis_ 365, chosen as representative strains, were singularly tested and when they were in a mixture in the presence of _E. coli_ 462 and Group B _Salmonella_ species 505.