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 .