Fig. 4 Bacteriostasis rate ofE. coli (A) and S. aureus (B) incubated with different concentrations of C-AgNPs, R12-AgNPs and R12-AgNPs+H2O2 (concentration of H2O2 = 10-6 M); Bacteriostasis rate of E. coli (C) and S. aureus (D) incubated with different concentrations of H2O2(10-7-10-3M) after treatment with or without R12-AgNPs (1 μg/mL); Bacterial colonies (E) treated with C-AgNPs, R12-AgNPs and R12-AgNPs+H2O2(concentration of H2O2 = 10-6 M, concentration of AgNPs = 1 μg/mL).
To study the synergistic antimicrobial activity of R12-AgNPs+H2O2, the antimicrobial activity of H2O2, R12-AgNPs and R12-AgNPs+H2O2 were qualitatively evaluated by measuring the bacterial inhibition rate (colony count). While R12-AgNPs demonstrated excellent bacteriostatic activity forE. coli and S. aureus , a significantly stronger synergistic antibacterial activity was observed when R12-AgNPs were combined with H2O2 (Fig. 4). For example, at a concentration of 1 µg/mL, R12-AgNPs exhibited a bacterial inhibition rate of 41.34±3.12% and 33.97±2.59% for E. coli andS. aureus , respectively (Fig. 4A, B). In comparison, H2O2 at a concentration of 10-6 M was reported to have a notably low inhibitory effect [46]. Notably, the bacterial inhibition rate increased by over 54.45% and 27.82% for E. coli (63.85±2.70%) and S. aureus (43.42±1.83%), respectively, when R12-AgNPs (1 µg/mL) were combined with H2O2(10-6 M). Moreover, as the concentration of H2O2 was increased from 10-6 M to 10-5 M, the inhibition rates achieved 87.93±2.59% and 47.80±1.19% for E. coli andS. aureus (Fig. 4C, D), respectively. As seen in Fig.4, this phenomenon can be attributed to the dual role of R12-AgNPs, which not only act as an antimicrobial agent but also as a nano catalyst to enhance the activity of H2O2 by converting it into hydroxyl radicals. These generated hydroxyl radicals possess high oxidative activity against nucleic acids, proteins and polysaccharides within the biofilm matrix [17].
Mechanism analysis of synergistic antibacterial activity
While the antibacterial properties of R12-AgNPs have been well documented, the precise mechanism of their action remains to be fully elucidated, necessitating further investigation. To determine the antibacterial mechanism of R12-AgNPs, we conducted experiments to measure the levels of reactive oxygen species (ROS) generated by the nanoparticles and analyzed the morphological properties of the bacteria [47]. The generation of ROS, leading to oxidative stress, has been proposed as a potential mechanism explaining the antibacterial effects of AgNPs [48]. In this study, we exposed bacterial cells to different concentrations of AgNPs (1, 5 μg/mL), and then detected intracellular ROS production was detected by microplate reader. As seen in Fig. 5, both types of AgNPs induced a dose-dependent increase in ROS levels within the bacteria. Notably, R12-AgNPs induced higher ROS production in bacteria compared to C-AgNPs, which is consistent with the stronger antibacterial effect of R12-AgNPs (Fig. 5A, C). In the presence of H2O2, R12-AgNPs induced even greater ROS production in bacteria. These results indicated that in the presence of H2O2, the peroxidase activity of R12-AgNPs stimulated the cells to produce more ROS and this synergistic effect killed more bacteria.
In order to further insights into the antibacterial mechanism, we examined the morphological changes of E. coli and S. aureus using SEM [49]. As shown in Fig. 5 (B, D), untreatedE. coli and S. aureus displayed intact cells with smooth membranes, exhibiting their typical rod and round shapes. Meanwhile, the bacteria treated with H2O2 showed no discernible differences compared to the untreated control group, indicating the preserved structural integrity of the bacteria. Nonetheless, when E. coli and S. aureus were subjected to different antibacterial agents, they underwent distinct changes in morphology. The SEM images revealed that both bacteria treated with C-AgNPs, R12-AgNPs and R12-AgNPs+H2O2exhibited disrupted cell morphology with wrinkled cell surfaces (Fig. 6B). Notably, in the group of R12-AgNPs+H2O2, the fine vesicle walls of the bacteria were more severely disrupted. Similar observations regarding the effects of AgNPs on bacteria have been reported in earlier studies [50, 51]. However, S. aureus treated with C-AgNPs did not show obvious change in the cell surfaces, which may be due to the thicker cell wall of S. aureus .