## 3 Results and Discussion
### Antibacterial Activity of Biosynthesisized AgNPs  
The antibacterial activity of biosynthesized AgNPs was carried out using the agar well diffusion method (Figure 7a) against Gram-positive (_S. aureus_ and _S. epidermidis_) and Gram-negative (_E. coli_ and _Salmonella_ sp.) pathogens employing various concentrations of AgNPs (12.5, 25, 50, 100, and 200 ug/mL). Figure 7b depicts the increased inhibition zone associated with the increase of AgNPs concentration, which may be due to more Ag\({}^{+}\) being released at a higher concentration. It was found that the inhibitory effects of AgNPs against all tested pathogens were similar. Previous studies reported that the inhibitory effect of AgNPs was more pronounced on Gram-negative bacteria compared to Gram-positive bacteria due to the thickness of the cell wall and cell composition [50; 51].  
Although the efficacy of AgNPs as an antibacterial agent has been demonstrated, the exact mechanisms behind the operation have not been completely deciphered. The most common mechanism involved is the release of Ag\({}^{+}\) from the AgNPs. AgNPs act in the same way as silver salt; however, the bactericidal activity of AgNPs and silver salt are at nanomolar and micromolar concentrations, respectively [52]. It is well documented that the Ag\({}^{+}\) released from AgNPs may interact with the surface of the bacterial cell, particularly with functional groups of protein resulting in their deactivation [6]. Furthermore, the potential antibacterial activity of AgNPs may be attributed to the physical contact of NPs via electrostatic forces with the bacterial cell wall and leads to the penetration of NPs into the cells, which could be toxic to biomolecules of the cell [53]. A recent study has revealed that the contact  
Figure 7: **(a)** Antibacterial activity of biosynthesized AgNPs using agar well diffusion method against (i) _S. aureus_, (ii) _S. epidermidis_, (iii) _E. coli_, and (iv) _Salmonella_ sp. (**b**) Zone of inhibition of biosynthesized AgNPs at different concentrations. Each value represents the mean \(\pm\) SD of three replicates.  
between AgNPs and _E. coli_ cell membrane for a few minutes led to the disruption of cells, as visualized by TEM [54]. Moreover, it was also suggested that AgNPs exert their antibacterial activity through the inactivation of the respiratory membrane chain of bacteria, which resulted in the generation of excess Reactive oxygen species (ROS) and accumulation in the cell, leading to cell death [55]. It should be noted that the ROS are generated intracellularly during the mitochondrial oxidative phosphorylation of bacterial cell.  
Researchers also suggested that the most prominent antibacterial mechanisms are related to the size of NPs. [PERSON] et al. [56] reported that the smaller size of AgNPs has a greater bactericidal effect than the larger ones due to the large surface area of NPs, which increases the surface reactivity. For instance, the smallest size of NPs releases a high concentration of Ag\({}^{+}\), while the biggest NPs release the lowest concentration of Ag\({}^{+}\)[57]. [PERSON] et al. [58] demonstrated that different sizes of AgNPs (5, 15, and 55 nm) exhibited different inhibition rates against oral pathogenic bacteria. In their study, the AgNPs of 5 nm size presented greater antibacterial activity compared to the AgNPs of 15 and 55 nm sizes due to their greater surface area. Similarly, AgNPs with the size of 10 nm completely inhibited pathogenic bacteria at the lowest minimum inhibitory concentration (MIC) of 1.0 \(\mu\)g/mL, whereas at the size of 90 nm exhibited higher MIC of 11.5 \(\mu\)g/mL [59]. In our study, it can be postulated that the significant inhibitory effect of biosynthesized AgNPs against tested pathogens is due to its smaller particle size.