**5. Antimicrobial Activity of AgNPs**

To evaluate the antibacterial activity of the AgNPs against gram-negative bacteria (*E. coli* ATCC 25922, *K. pneumoniae* ATCC 700603, *P. aeruginosa* ATCC 27853) and gram-positive ones (*S. aureus* ATCC 25923 and MRSA ATCC 43300), MIC and MBC values were obtained. Afterwards, we studied how the TS influenced the bacterial growth rate (at the initial moment and after 3 and 6 h of incubation).

For therapeutic consideration, only antimicrobial activity obtained below 1 mM concentration was considered. The MIC and MBC of nanoparticles against pathogenic bacterial strains are as mentioned in Table 2. Thus, it is found that almost all tested solutions exhibit inhibitory activity on the growth of tested bacteria (except TS1, where the MIC was determined only for MRSA, at the maximum analyzed concentration). It can be seen that AgNPs obtained at pH = 9 (TS2, TS4) favor the inhibitory activity for the tested bacterial strains. They have a lower MIC compared to those obtained at pH = 4 (TS1, TS3). TS2 (for *E. coli*, *K. pneumoniae*, and *P. aeruginosa*) and TS4 (for *S. aureus*, *E. coli*, *K. pneumoniae*, and *P. aeruginosa*) have lower MIC and MBC compared to those obtained for the control beech bark extract. For the most part, the MBC was registered for the TS where the MIC was determined, the results being presented in Table 2. The lowest MBC was recorded for TS4 (MRSA, *E. coli*, and *P. aeruginosa*) and for TS2 (*E. coli*).

As can be seen from Figure 7, TS4 had a bactericidal effect on *E. coli* and inhibited the growth of *S. aureus* and *P. aeruginosa* after 6 h of incubation.

**Figure 7.** Graphical and visual representation of the growth rate for *S. aureus* (**A**,**D**), *E. coli* (**B**,**E**), *P. aeruginosa* (**C**,**F**) in the presence of TS4 (beech extract + AgC2H3O2 pH = 9) and in the absence of TS4 (Control) at: initial time—H0, 3 h—H1, and 6 h—H2.



ATCC—American Type Culture Collection; MIC—minimal inhibitory concentration; MBC—minimum bactericidal concentration; MRSA—methicillin-resistant *S. aureus*, BBE—beech bark extract; AgNO3—silver nitrate; AgC2H3O2—silver acetate; TS1—beech bark extract, pH = 4, AgNO3; TS2—beech bark extract, pH = 9, AgNO3; TS3—beech bark extract, pH = 4, AgC2H3O2; TS4—beech bark extract, pH = 9, AgC2H3O2.

The antibacterial mechanism might beexplained by the interaction between silver nanoparticles and bacterial DNA. AgNPs can cause DNA damage, and ultimately bacterial cell death. This effect has been demonstrated on *S. aureus* and *E. coli* strains [32].

However, the antibacterial mechanism is different, depending on the type of bacteria to be inhibited. For example, silver nanoparticles have an inhibitory effect on *Helicobacter pylori*, stating two theories of action: small amounts of AgNPs enter the bacterium and inhibit the respiratory chain, while another theory considers that AgNPs inhibit urease from *H. pylori* [33]. Evidence of bacterial growth inhibition have also been demonstrated for strains of *S. aureus*, *Streptococcus pneumoniae*, *Proteus mirabilis*, and *E. coli* [34].
