## 3 Results and Discussion
### Microbiology
#### 3.9.1 Time-Kill (TK) Experiments
Results were reported both as the variation of Log\({}_{10}\) (CFU/mL) (dLLog\({}_{10}\) (CFU/mL) and as the percentage of killed bacteria when the mixture was exposed to R4 for 1, 2, and 4 h (Table 7, Figure 12a,b). The results unequivocally demonstrated that the optimal contact time for obtaining a higher reduction in bacterial contamination was 4 h, thus confirming what was previously observed with _E. coli_ 224. Even when it was used in a column simulating a sanitization system acting by filtration, R4 proved less efficient in reducing the bacterial contamination and lost the bactericidal \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Strains**} & \multicolumn{2}{c}{**1 h**} & \multicolumn{2}{c}{**2 h**} & \multicolumn{2}{c}{**4 h**} \\ \cline{2-7}  & **dLog\({}_{10}\)** & **Killed Cells** & **dLog\({}_{10}\)** & **Killed Cells** & **dLog\({}_{10}\)** & **Killed Cells** \\  & **(CFU/mL)** & **(\%)** & **(CFU/mL)** & **(\%)** & **(CFU/mL)** & **(\%)** \\ \hline Bacteria *,1 & \(-\)0.59 & 74.5 & \(-\)1.22 & 94.0 & \(-\)2.23 & 99.4 \\ Bacteria *2 & +0.55 & N.A. & +1.13 & N.A. & +1.25 & N.A. \\ \hline \hline \end{tabular}
* _E. facecium_ 503, _E. faecalis_ 365, _E. coli_ 462 (New Delhi metallo-\(\beta\)-lactamase producer), and _Salmonella_ 505; 1 in presence of R4; 2 control (CTR).  
\end{table} 
Table 7: Log\({}_{10}\) variation in the initial cell number of the tested bacteria expressed in CFU/mL and the percentage of killed bacteria when tested in mixture and exposed to R4 for 1, 2, and 4 h.  
Figure 12: Variation of Log\({}_{10}\) (CFU/mL) observed when a mixture of Group B _Salmonella_ species 505, _E. coli_ 462 (New Delhi metallo-\(\beta\)-lactamase producer), _E. facecium_ 503, and _E. faecalis_ 365 strains was exposed for 1, 2, and 4 h to R4 (red bars) vs. that of CTR (pink bars) (**a**); percentage of the bacterial cells killed by R4 after 1, 2, and 4 h of exposure when tested against the mixture (red bars) and against _E. coli_ 224 (yellow bars) (**b**). power demonstrated in the TK experiments against enterococci. It was unexpectedly more efficient in killing bacteria when used against the bacterial mixture than when it was used against a single species (_E. coli_ 224) (Figure 12b). Collectively, after 4 h of contact of the bacterial mixture with R4, followed by rapid filtration, the initial number of bacterial cells was reduced by 2.3 Log\({}_{10}\), corresponding to 99.4% of the bacteria killed, while an increase of 2.25 Log\({}_{10}\) was observed for the control. Recently, an iodine-doped pyrrolidone-based polymer poly-(N-vinyl-2-pyrrolidone-co-vinyl acetate) (P(VAc-NVP)-I) was used in a filtration system, made of three consecutive filtration columns containing it, for water purification experiments against water containing _E. coli_ as a model for bacteria-contaminated water [52]. According to the reported results, differently from the control, when the contaminated water was passed through the P(VAc-NVP)-I-packed columns, _E. coli_ was completely inactivated and the sterilization efficiency of P(VAc-NVP)-I in the columns was close to 100% [52], as in our case. Although the reported results are very promising and interesting, the bactericidal effects of R4 in the column were remarkably more potent. In fact, instead of filtration through three consecutive filtering columns containing unknown quantities of P(VAc-NVP)-I, three consecutive filtrations (after 1, 2, and 4 h) through the same minimum quantity of R4 in the column were sufficient to obtain the same sterilization efficiency. In addition, in this study, we extended the evaluation of the sterilization efficiency of R4 to other relevant bacteria of fecal origin that can be found in contaminated water. So, unlike the study by [PERSON] et al., where the sterilization efficiency of P(VAc-NVP)-I was demonstrated only in the presence of _E. coli_, R4 demonstrated a sterilization efficiency of over 99% even in the presence of a polymicrobial pollution by enterococci, _E. coli_, and group B _Salmonella_ species.