# ZnO/Ag Nanocomposites with Enhanced Antimicrobial Activity
## 3 Results and Discussion
### Antimicrobial Activity of ZnO/Ag Nanocomposites  
The effect of ZnO/Ag nanocomposites at different concentrations of Ag was analyzed in vitro against _S. aureus_ and _E. coli_. The results showed a reduction in the growth of both bacteria in the presence of ZnO and ZnO/Ag nanocomposites when compared to controls. The antimicrobial effect of ZnO nanoparticles has been previously demonstrated, with an increase in the action of some antibiotics being observed in the presence of NP [33; 34]. In the same way, there is clear evidence that the use of Ag nanoparticles increases the antimicrobial effect of certain antibiotics or defense-inducing molecules such as chitosan, which makes it difficult to analyze the effect of the nanoparticles [13; 23; 35; 36; 37]. Other authors have tested in vitro the effects of Ag NPs against _E. coli_ and _S. aureus_, noting that Ag NPs were effective against _E. coli_ but not against _S. aureus_. However, the mechanism of action of the Ag NPs is still unknown, since it depends on the type of nanoparticle, the size, the arrangement of the ions, and their concentration [14; 34; 38; 39] as well as the morphology of the bacteria (type of cell wall, type of metabolism, virulence mechanism, etc.) [40]. Previous studies have suggested that the action of Ag NPs on bacteria may be due to their ability to penetrate the cell [41], the production of reactive oxygen species  
Figure 5: **(A)** Raman scattering spectra of nanocomposite samples (Zn1, Zn2, Zn3, Zn4, Zn5, and Zn6) were measured under 325 nm excitation wavelength in different points of the samples and (**B**) averaged per sample. Figure 6: Relative integrated intensity of 2 LO peak from the spectra measured under 325 nm excitation wavelength. (ROS) [42], or the formation of free radicals, as well as the inactivation of proteins in the cell by silver ions [15]. The variability of these results led us to test the effect of ZnO combined with different concentrations of silver on _E. coli_ and _S. aureus_. The novelty of our study lies in the synthesis method of the AgNPs, which is a novel synthesis method based on sol-gel methodology, and in the fact that AgNPs synthized in this study were able to inhibit high concentrations of both bacteria, which rarely occurs in real-life systems. The results obtained are shown in Figure 7.  
Under our conditions, a reduction in bacterial growth was observed from 3 h of inoculation in both bacteria and all treatments when compared to the control. In _S. aureus_, a similar effect in growth inhibition was observed in ZnO with or without Ag NPs; in _E. coli_, bacterial growth was greater in the absence of Ag NPs at all sampling times, although no significant differences were observed (Figure 8). These results are in concordance with previous works observing that Gram-negative bacteria are more susceptible to silver nanoparticles. This may be due to the fact that the cellular wall of Gram-negative bacteria is narrower than that of Gram-positive strains, which may reduce the penetration of nanoparticles into the bacterial cells [43]. When analyzing the effect of the different concentrations of Ag against both bacteria, it can be seen that an increase in the concentration of Ag does not correlate with an increase in its effect against the bacteria. Furthermore, it seems that lower concentrations of Ag have greater efficacy against _E. coli_. In order to check whether the effect of the Zn/Ag nanocomposites was bacteriostatic or bactericidal, the samples were stained with the LIVE/DEAD BacLight kit and further analyzed through flow cytometry. All treatments showed a bactericidal effect when compared to controls, as seen in Figure 8. The treatments reduced the percentage of living cells by more than 90% in most cases. Table 1 shows the results of the percentages of live bacteria concerning the total number of cells counted, which were obtained using the flow cytometer after being stained with the live/dead kit. Figure 7: Results of the effect of the treatments on _S. aureus_ and _E.