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\begin{document}

\title{Deinococcus wulumuqiensis R12 synthesized silver nanoparticles with
peroxidase-like activity for synergistic antibacterial application}



\author[1]{Jingjia Liu}%
\author[1]{Nan Zhang}%
\author[1]{Bowen Shen}%
\author[1]{Liling Zhang}%
\author[1]{Zhidong Zhang}%
\author[1]{Liying Zhu}%
\author[1]{Ling Jiang}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The use of a combination of several antibacterial agents for therapy
holds great promise in reducing the dosage and side effects of these
agents, improving their efficiency, and inducing potential synergistic
therapeutic effects. Herein, R12-AgNPs were produced with the
supernatant of an ionizing radiation-tolerant bacterium Deinococcus
wulumuqiensis R12 by one-step under room temperature. The biosynthesized
AgNPs presented fascinating antibacterial activity and peroxidase-like
properties, which endowed it with the capability to catalyse the
decomposition of H2O2 to generate hydroxyl radical. After combination of
R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was
observed for both E. coli and S. aureus, especially at low
concentrations. In addition, in vitro cytotoxicity tests showed
R12-AgNPs had good biocompatibility. Thus, this work presents a novel
antibacterial agent that exhibits favorable synergistic antibacterial
activity and low toxicity, without the use of antibiotics or a
complicated synthesis process.%
\end{abstract}%



\sloppy


\emph{Deinococcus wulumuqiensis}R12 synthesized silver nanoparticles
with peroxidase-like activity for synergistic antibacterial application

Jingjia Liu\textsuperscript{1}, Nan Zhang\textsuperscript{1}, Bowen
Shen\textsuperscript{2}, Liling Zhang\textsuperscript{3}, Zhidong
Zhang\textsuperscript{4}, Liying Zhu\textsuperscript{1, *}, Ling
Jiang\textsuperscript{2,3}

\textsuperscript{1} School of Chemistry and Molecular Engineering,
Nanjing Tech University, Nanjing 210009, PR China.

\textsuperscript{2}College of Biotechnology and Pharmaceutical
Engineering, State Key Laboratory of Materials-Oriented Chemical
Engineering, Nanjing 211816, China.

\textsuperscript{3}College of Food Science and Light Industry, Nanjing
Tech University, Nanjing 210009, PR China.

\textsuperscript{4}Xinjiang Key Laboratory of Special Environmental
Microbiology, Institute of Microbiology, Xinjiang Academy

of Agricultural Sciences, Urumqi 830091, China.

\textsuperscript{*}Corresponding author: Liying Zhu, Email:
zlyhappy@njtech.edu.cn, Tel: +86-25-58139430

\textbf{Abstract}

The use of a combination of several antibacterial agents for therapy
holds great promise in reducing the dosage and side effects of these
agents, improving their efficiency, and inducing potential synergistic
therapeutic effects. Herein, R12-AgNPs were produced with the
supernatant of an ionizing radiation-tolerant bacterium
\emph{Deinococcus wulumuqiensis} R12 by one-step under room temperature.
The biosynthesized AgNPs presented fascinating antibacterial activity
and peroxidase-like properties, which endowed it with the capability to
catalyze the decomposition of H\textsubscript{2}O\textsubscript{2} to
generate hydroxyl radical. After combination of R12-AgNPs and
H\textsubscript{2}O\textsubscript{2}, an excellent synergistic
bacteriostatic activity was observed for both\emph{E. coli} and \emph{S.
aureus} , especially at low concentrations. In addition, in vitro
cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus,
this work presents a novel antibacterial agent that exhibits favorable
synergistic antibacterial activity and low toxicity, without the use of
antibiotics or a complicated synthesis process.

\textbf{Keyword:} AgNPs; \emph{Deinococcus wulumuqiensis} ;
Peroxidase-like activity; Antibacterial activity; Combination therapy;
Synergistic antibacterial activity

\textbf{Graphical Abstract}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Scheme 1. Schematic of preparation R12-AgNPs and designing of synergetic
antibacterial system.

\textbf{Highlights:}

AgNPs biosynthesized from the supernatant of \emph{Deinococcus
wulumuqiensis} R12 have low toxicity excellent, bacteriostatic activity
and peroxidase-like properties without additional modifications.

Upon combination of R12-AgNPs and H\textsubscript{2}O\textsubscript{2},
significant synergistic inhibitory activity was observed against both
\emph{E. coli} and\emph{S. aureus} , due to peroxidase-like activity of
R12-AgNPs.

\textbf{Introduction}

Bacterial infections have consistently posed a significant challenge in
healthcare and have remained a primary cause of pathological disease
{[}1{]}. While the application of antibiotics has significantly reduced
mortality rates and shortened treatment durations for both humans and
animals afflicted with bacterial infections in recent years {[}2, 3{]},
the inappropriate use of antibiotics has led to the emergence of
antibiotic-resistant bacteria {[}4{]}. Consequently, there is an urgent
need for the development of antimicrobial drugs that are not only safe
but also exhibit potent antibacterial properties while being incapable
of inducing bacterial resistance. Various nanomaterials have been
explored for their potential in combatting drug-resistant bacteria,
including MoS\textsubscript{2} {[}5{]}, Fe\textsubscript{3}C/N-C
{[}6{]}, Au/g-C\textsubscript{3}N\textsubscript{4} {[}7{]}, AgNPs
{[}8{]},AuNPs {[}9{]}, Ag-CuNPs {[}10{]}. However, the utility of most
nanomaterials as antimicrobial agents is constrained due to their
inherent toxicity to humans and animals.

Fortunately, silver nanoparticles (AgNPs) have emerged an excellent
antibacterial agent due to their remakable bacteriostatic capabilities
against both Gram-positive and Gram-negative bacteria, along with their
high biocompatibility {[}11, 12{]}. So far, various methods for the
synthesizing of different types of AgNPs have been developed, comprising
physical, chemical, biological and hybrid approaches {[}13, 14{]}. In
recent years, reports of AgNPs synthesis with biological methods have
come into bloom for their advantages of being eco-friendly and not using
additional stabilizers {[}8, 12, 15{]}. However, it is worth noting that
antimicrobial agents based on these noble materials can be costly. The
creation of cost-effective antimicrobial agents with potent
bacteriostatic properties remains a significant concern.
H\textsubscript{2}O\textsubscript{2} has been widely used as a
therapeutic agent for wound disinfection due to its affordability
{[}16{]}. However, it is important to acknowledge that high
concentrations of H\textsubscript{2}O\textsubscript{2} (0.5-3\%) can be
harmful to healthy tissue {[}16{]}, even though it is commonly employed
in healthcare as a disinfectant.

Combination therapy involving AgNPs and
H\textsubscript{2}O\textsubscript{2} offers a potential solution to the
previously mentioned challenges. Through peroxidase catalysis, hydroxyl
radicals with enhanced antimicrobial activity can be generated from low
concentrations of H\textsubscript{2}O\textsubscript{2}. Several
nanocatalysts, including Fe\textsubscript{3}O\textsubscript{4},
C\textsubscript{3}N\textsubscript{4}, MoS\textsubscript{2} and
carbon-based nanomaterials, have demonstrated peroxidase-like activity
{[}7, 17, 18{]}. These nanocatalysts can synergistically inhibit
bacterial growth when combined with AgNPs and
H\textsubscript{2}O\textsubscript{2}. However, overly complex
composition can introduce instability into the disinfection treatment
process. Fortunately, some biosynthesized AgNPs were also exhibited
peroxidase-like properties, enabling them to catalyze the oxidation of
various horseradish peroxidase (HRP) substrates by
H\textsubscript{2}O\textsubscript{2} without modifying functional groups
on their surfaces {[}19, 20{]}. These properties made AgNPs widely
applicable in biosensors {[}21{]}, antibiosis {[}22{]}, and therapeutic
interventions {[}23{]}. Therefore, the straightforward combination of
H\textsubscript{2}O\textsubscript{2} and AgNPs with peroxidase-like
activity can achieve a synergistic bacterial inhibition effect.

In our previous report {[}24{]}, we demonstrated that the cell-free
supernatant of \emph{Deinococcus wulumuqiensis} R12 (\emph{D.
wulumuqiensis} R12) could successfully produce AgNPs without addition of
stabilizer at room temperature. \emph{D. wulumuqiensis} R12, a member of
the \emph{Deinococcus} \emph{radiodurans genus} , was well-known for its
ionizing radiation-tolerant ability and contains a large number of
natural product synthesis genes and oxidoreductase genes {[}25-27{]}. It
is possible that certain components within \emph{D. wulumuqiensis} R12
contribute to the remarkable properties exhibited by AgNPs. Therefore,
the present study is focused on evaluating the bacteriostatic properties
and peroxidase-like activity of AgNPs synthesized by \emph{D.
wulumuqiensis} R12 in comparison to chemically synthesized AgNPs using
sodium borohydride. Additionally, the study explored the synergistic
bacteriostatic effects of combination therapy involving AgNPs and low
concentrations of H\textsubscript{2}O\textsubscript{2}. This work not
only presents a highly effective antibacterial agent produced through an
eco-friendly synthesis process but also provides a promising strategy
for the treatment of bacterial infections.

\textbf{Materials andmethods}

Materials and chemical reagents

\emph{D. wulumuqiensis} R12 was kindly offered by Xinjiang Academy of
Agricultural Sciences (Xinjiang, China). Sodium borohydride
(NaBH\textsubscript{4}) and silver nitrate (AgNO\textsubscript{3}) were
supplied from Sigma (St. Louis, Missouri, USA). Tryptone, glucose and
yeast extract (TGY) medium were obtained from OXOID Ltd. (Basingstoke,
Hampshire, England). 3, 3', 5, 5'-tetramethylbenzidine (TMB) was
supplied by Aladdin Reagent Company (Shanghai, China).

Preparation and characterization of AgNPs

AgNPs were synthesized by using the cell-free supernatant of \emph{D.
wulumuqiensis} R12, and they were labeled as R12-AgNPs. For comparison,
another type of AgNPs were produced by utilizing sodium borohydride
(NaBH\textsubscript{4}) as the reducing and stabilizing agent, and they
were labeled as C-AgNPs. UV-vis spectroscopy, SEM, FT-IR and XRD were
used to confirm the successful synthesis of AgNPs. In vitro release of
silver ions from AgNPs was analyzed by Inductively Coupled Plasma
Optical Emission Spectrometer (ICP-OES: Therm Fisher iCAP PRO).

Performance of AgNPs

Peroxidase-like activity of AgNPs

In the presence of AgNPs, the intrinsic peroxidase mimetic activity was
investigated by monitoring the conversion of TMB to an oxidized form,
which would display a blue color with absorption bands at 652 nm
{[}28{]}. To measure the peroxidase-like activity, 10 \selectlanguage{greek}μ\selectlanguage{english}L of 80 mM TMB
was introduced into a mixture of 0.89 mL of PBS buffer solution (pH=4.0)
containing AgNPs and H\textsubscript{2}O\textsubscript{2}, and the
resulting mixture was incubated for 10 minutes. To obtain the optimum
result, various influencing factors including pH of the solution, the
concentration of the AgNPs, reaction temperature and time were studied
accordingly.

Antibacterial tests

In this study, model bacteria Gram-negative bacteria \emph{Escherichia
coli} (\emph{E. coli} ) and Gram-positive bacteria \emph{Staphylococcus
aureus} (\emph{S. aureus)} {[}29, 30{]} were selected for experiments.
The bacterial fluids were diluted to 10\textsuperscript{8} cfu/mL and
each material was divided into 3 groups: (I) Bacterial control, (II)
Bacteria + R12-AgNPs, (III) Bacteria + C-AgNPs. Parallel tests were
conducted in each of the three groups.

To further evaluate the synergistic antimicrobial activity of
H\textsubscript{2}O\textsubscript{2} and R12-AgNPs, \emph{E. coli}
and\emph{S. aureus} were mixed with different concentrations (0, 1, 5,
10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) of R12-AgNPs and hydrogen peroxide
(H\textsubscript{2}O\textsubscript{2}) at a final concentration of
10\textsuperscript{-6} M. Meanwhile, the effect of
H\textsubscript{2}O\textsubscript{2} concentration on combination
therapy was investigated by mixing bacteria with varying concentrations
of
H\textsubscript{2}O\textsubscript{2}(10\textsuperscript{-7}-10\textsuperscript{-3}
M) and a final concentration of 1 \selectlanguage{greek}μ\selectlanguage{english}g/mL of R12-AgNPs. All experiments
were performed as follows: after incubating with antibacterial agents
for 30 minutes at 37\selectlanguage{ngerman}°C, 100 \selectlanguage{greek}µ\selectlanguage{english}L of diluted bacterial suspension was
removed and dispersed evenly on blood agar plates and left at 37\selectlanguage{ngerman}°C for
12-24 hours.

Antibacterial mechanism analysis

Intracellular ROS content was determined by detecting highly fluorescent
2,7-dichlorofluorescin diacetate (DCF) resulting from the oxidation of
the nonfluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA)
{[}31, 32{]}. Based on the method of Tian et al. {[}33{]}, SEM (S-4800)
was employed to observe the changes in surface morphology of \emph{E.
coli} and \emph{S. aureus} after incubation with AgNPs and
H\textsubscript{2}O\textsubscript{2}.

Biocompatibility evaluation of R12-AgNPs

Based on the method suggested by Lu et al. {[}34{]}, mouse fibroblasts
(L929) were used to evaluate the biocompatibility of R12-AgNPs. Cells
were firstly cultivated in high glucose medium until the cell fusion
rate reaches 80\%. Then the cells were obtained by centrifugation and
the cell density was adjusted to a suspension of
1×10\textsuperscript{4}CFU/mL using culture medium. After incubation in
enzyme labelled plates for 24 h at 37 \selectlanguage{english}, the original culture medium was
aspirated out and 100 \selectlanguage{greek}µ\selectlanguage{english}L of different concentrations of R12-AgNPs and
blank control solution were added to the culture, with a total of 5
parallel samples in each group. 24 hours later, 20 \selectlanguage{greek}µ\selectlanguage{english}L of MTT solution (5
mg/mL) was added to each well, and the supernatant was removed after 4 h
of incubation in a CO\textsubscript{2} incubator, and 150 \selectlanguage{greek}µ\selectlanguage{english}L of DMSO was
added to each well plate to dissolve the crystals and mixing and shaking
for 10 min. The OD values of the individual supernatant solutions were
measured at 490 nm on an enzyme marker.

\textbf{Results and discussion}

Characterization of AgNPs

As seen in Fig. 1, the synthesis and morphology of AgNPs were observed
by both UV--vis adsorption spectroscopy and SEM (A). The UV-vis
spectroscopy is a handy method for confirming the formation of metal
nanoparticles with characteristic surface plasmon band. In the case of
UV-visible analysis, C-AgNPs and R12-AgNPs showed distinct adsorption
peaks at 400 nm and 450 nm, respectively, which corresponded to the
specific surface plasmon resonance (SPR) of AgNPs {[}35{]}. Spherical
shaped nanoparticles were observed in the SEM images (Fig. 1 A) of both
C-AgNPs and R12-AgNPs. From Fig. 1, it can also be noted that the
absorbance peak width of R12-AgNPs is significantly larger than that of
C-AgNPs. The wavelength and half-peak width of the UV absorption peak
were mainly decided by the size of the nanoparticles {[}36{]}. This
result, therefore, may be attributed to the larger droplet size and
wider size range of R12-AgNPs, which might be a result of the prolonged
synthesis time and complex composition present in the cell-free
supernatant of \emph{D. wulumuqiensis} R12. This observation is
consistent with the SEM results where the size of R12-AgNPs exhibited a
wider range.

The crystalline nature of the obtained nanoparticles was confirmed by
XRD spectra for both C-AgNPs and R12-AgNPs. As shown in Fig. 1B, there
are 5 sharp diffraction peaks in both XRD patterns, corresponding to the
(111), (200), (220), (311) and (222) crystal planes. These peaks are in
agreement with the typical face-centered cubic structure of silver
reported from the standard atlas JCPDS card 04-0783 {[}37{]}. The XRD
pattern therefore illustrates that AgNPs formed with both synthesis
methods are crystalline in nature {[}38{]}.

The FTIR analysis was performed to evaluate the coating compounds of
AgNPs. Fig. 1C showed that the FTIR spectrum of C-AgNPs, was in
consistent with the FTIR results reported by Fadaka et al., where prime
bands were associated with the C--H stretch and alkenyl C=C stretch
{[}39{]}. Compared to C-AgNPs, the FTIR spectrum of R12-AgNPs showed
more adsorption bands, including 2971, 2921, 1066 and 880
cm\textsuperscript{-1}, The peaks at 2971 cm\textsuperscript{-1} and
2921 cm\textsuperscript{-1} referred to C--H bonds; the band at 1066
cm\textsuperscript{-1} can be assigned to --C--O--C--stretching and
--C = C-- of the amide group, corresponding to stretching from
carboxylic acid, ether, alcohol and ester; the peak at 880
cm\textsuperscript{-1} assigned to --C = CH-- was attributed to aromatic
groups {[}40{]}. In our case, the cell-free supernatant of \emph{D.
wulumuqiensis} R12 used for the synthesis of AgNPs was found to contain
rich components, including NADPH-dependent oxidoreductase {[}24{]},
deinoxanthin {[}25{]}, DNA-binding protein {[}26{]}, reactive oxygen
species (ROS) scavenging enzymes (e.g., catalase and superoxide
dismutase) and non-enzyme antioxidants (e.g., carotenoids and manganese
species) {[}41{]}. Since the FTIR peaks of the cell-free supernatant of
\emph{D. wulumuqiensis}R12 were similar to those of R12-AgNPs, it can be
inferred that some biomolecules and functional groups involved in
reduction, capping and stabilization of R12-AgNPs and may also confer
some attractive properties to R12-AgNPs. The slight differences in the
intensity and position of the peaks may be attributed to the interaction
of chemical bonds between the supernatant and the nanoparticles. The
findings indicate that R12-AgNPs exhibited a higher number of surface
functional groups than C-AgNPs. Additionally, the results demonstrated
that the cell-free supernatant of \emph{D. wulumuqiensis} R12 served as
both a reducing agent and a stabilizer during the synthesis of R12-AgNPs
{[}24{]}.

The Ag release rate was known as one of the most important parameter for
evaluating silver-based antibacterial materials {[}34{]}. Fig. 1D showed
the release concentration of silver ions of the R12-AgNPs and C-AgNPs
after 7 days. As is shown in Fig. 1D, the silver ion release from
R12-AgNPs (5.07 mg/L) was much greater than that from C-AgNPs (0.46
mg/L), thus validating the direct correlation between the quantity of
silver ions emitted from the AgNPs and their ability to inhibit
microbial growth.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 1 UV-Vis absorption spectra and SEM (A), X-ray diffraction (B),
FTIR spectra (C) and silver ion release concentration (concentration of
AgNPs is 10 mg/L) (D) of AgNPs.

Peroxidase-like activity of AgNPs

The peroxidase-like activity of the AgNPs nanozyme was assessed by
monitoring the oxidation of TMB by H\textsubscript{2}O\textsubscript{2}.
As shown in Fig. 2, several solutions containing different components
were subjected to using UV/visible absorption spectroscopy measurements.
The solutions containing TMB (a), TMB+ R12-AgNPs (b) and TMB+ C-AgNPs
(c) showed no peak at 652 nm associated with ox-TMB absorption. In the
case of both groups of TMB + C-AgNPs +
H\textsubscript{2}O\textsubscript{2} (d) and TMB +
H\textsubscript{2}O\textsubscript{2} (e), the TMB oxidation products
appeared slightly blue with weak adsorption bonds at 652 nm, signifying
the limited catalytic potential for C-AgNPs and a slow oxidation of TMB
in the presence of H\textsubscript{2}O\textsubscript{2}, respectively.
In contrast, the combinations of R12-AgNPs + TMB +
H\textsubscript{2}O\textsubscript{2} (f) exhibited a strong blue color
with the highest absorbance at 652 nm (Fig. 2), which indicated the
peroxidase-like catalytic activity of R12-AgNPs (Eq. 3). However, the
catalytic mechanism remains unclear. Sun et al. deactivated the hydroxy
groups, carboxylic groups, or ketonic carbonyl groups on purpose and
assessed the catalytic activities of these derivatives of graphene
quantum dots (GQD). Their findings suggested that the --C=O groups
served as the catalytically active sites, while the O=C--O-- groups
acted as substrate-binding sites {[}42{]}. It can therefore be inferred
that the peroxidase-like activity of R12-AgNPs may be attributed to the
presence of the additional functional groups on their surface (Fig. 1C).

H\textsubscript{2}O\textsubscript{2}+TMBH\textsubscript{2}O+ox-TMB (3)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig. 2 UV-vis spectra of different combinations after 10 min of reaction
at room temperature.

The impact of various factors, including pH, temperature, catalyst
concentration and reaction time. On the catalytic abilities of R12-AgNPs
was further explored using UV-Vis absorption spectra. To determine the
optimal pH value, absorption spectra were recorded across a pH range
from 3 to 8 (Fig. 3A). It was observed that the absorption peak at 652
nm was absent at pH 8, whereas the highest absorbance at this peak was
attained at pH 4. Therefore, the optimum reaction pH of R12-AgNPs was
determined to be pH=4, consistent with the previous report {[}43{]}. In
addition, the absorbance values increased with rising temperature and
reached peaked at 50  (Fig. 3B). However, a decline in the
concentration of ox-TMB was noticed at higher temperature (60 ), which
may be attributed to the reduced thermal stability of
H\textsubscript{2}O\textsubscript{2} in an acidic environment {[}20{]}.
The effect of catalyst concentration was analyzed by adding AgNPs in the
range of 0-100 \selectlanguage{greek}μ\selectlanguage{english}L to the solution. The results in Fig. 3C indicated that
the catalytic activity of TMB oxidation increased with a higher catalyst
concentration, with the highest absorbance at the 652 nm peak occurred
at 100 \selectlanguage{greek}μ\selectlanguage{english}L of R12-AgNPs solution was used. Finally, the changes in
catalytic activity over to the course of the reaction time were
demonstrated in Fig. 3D, revealing the maximum catalytic activity was
achieved within 10 min. Consequently, the optimal conditions for maximum
catalytic activity in the oxidation of TMB were discovered to be at a pH
of 4, a temperature of 50 , 100 \selectlanguage{greek}μ\selectlanguage{english}L of AgNPs, and a reaction time of 10
min.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig. 3 Optimization parameters for efficient sensing with variation in
pH (A), temperature (B), R12-AgNPs concentration (C) and time (D).

Determination of immediate antimicrobial effect

Standard Plate Count (SPC) is the most widely accepted and frequently
utilized approach for estimating the number of viable bacteria. In this
study, the antibacterial activity of both R12-AgNPs and C-AgNPs (0-10
\selectlanguage{greek}μ\selectlanguage{english}g/mL) was evaluated against Gram-positive and Gram-negative bacteria
(\emph{S. aureus} and \emph{E. coli} ) with a microdilution method (Fig.
4). The results indicated that the inhibitory effect of AgNPs on both
types of bacteria was enhanced with the increase of AgNPs, signifying a
positive correlation between the concentration of AgNPs and their
antibacterial activity. According to Fig. 4 (A, B), almost 100\% the
inhibition of \emph{E. coli} and \emph{S. aureus} were achieved when the
concentration of R12-AgNPs was 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL and 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL, respectively.
However, the antibacterial effect of C-AgNPs was notably weaker at all
concentrations of AgNPs (Fig. 4). Especially at lower concentrations,
C-AgNPs exhibited negligible antibacterial activity. This difference may
be attributed to the fact that C-AgNPs have a lower
Ag\textsuperscript{+} release, which was confirmed by the
Ag\textsuperscript{+} release test (Fig. 1D). Overall, R12-AgNPs
demonstrated higher inhibitory effects against all tested bacteria, both
low and high concentrations, in compared to C-AgNPs. This finding was
consistent with previous results, highlighting that biosynthesized
AgNPs, stabilized by biomolecules, possess a greater tendency to exhibit
enhanced antibacterial activity when compared to chemically synthesized
AgNPs {[}44, 45{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig. 4 Bacteriostasis rate of\emph{E. coli} (A) and \emph{S. aureus} (B)
incubated with different concentrations of C-AgNPs, R12-AgNPs and
R12-AgNPs+H\textsubscript{2}O\textsubscript{2} (concentration of
H\textsubscript{2}O\textsubscript{2} = 10\textsuperscript{-6} M);
Bacteriostasis rate of \emph{E. coli} (C) and \emph{S. aureus} (D)
incubated with different concentrations of
H\textsubscript{2}O\textsubscript{2}(10\textsuperscript{-7}-10\textsuperscript{-3}M)
after treatment with or without R12-AgNPs (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL); Bacterial colonies
(E) treated with C-AgNPs, R12-AgNPs and
R12-AgNPs+H\textsubscript{2}O\textsubscript{2}(concentration of
H\textsubscript{2}O\textsubscript{2} = 10\textsuperscript{-6} M,
concentration of AgNPs = 1 \selectlanguage{greek}μ\selectlanguage{english}g/mL).

To study the synergistic antimicrobial activity of
R12-AgNPs+H\textsubscript{2}O\textsubscript{2}, the antimicrobial
activity of H\textsubscript{2}O\textsubscript{2}, R12-AgNPs and
R12-AgNPs+H\textsubscript{2}O\textsubscript{2} were qualitatively
evaluated by measuring the bacterial inhibition rate (colony count).
While R12-AgNPs demonstrated excellent bacteriostatic activity
for\emph{E. coli} and \emph{S. aureus} , a significantly stronger
synergistic antibacterial activity was observed when R12-AgNPs were
combined with H\textsubscript{2}O\textsubscript{2} (Fig. 4). For
example, at a concentration of 1 \selectlanguage{greek}µ\selectlanguage{english}g/mL, R12-AgNPs exhibited a bacterial
inhibition rate of 41.34\selectlanguage{ngerman}±3.12\% and 33.97±2.59\% for \emph{E. coli}
and\emph{S. aureus} , respectively (Fig. 4A, B). In comparison,
H\textsubscript{2}O\textsubscript{2} at a concentration of
10\textsuperscript{-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 \emph{E. coli} (63.85±2.70\%) and \emph{S.
aureus} (43.42±1.83\%), respectively, when R12-AgNPs (1 \selectlanguage{greek}µ\selectlanguage{english}g/mL) were
combined with
H\textsubscript{2}O\textsubscript{2}(10\textsuperscript{-6} M).
Moreover, as the concentration of H\textsubscript{2}O\textsubscript{2}
was increased from 10\textsuperscript{-6} M to 10\textsuperscript{-5} M,
the inhibition rates achieved 87.93\selectlanguage{ngerman}±2.59\% and 47.80±1.19\% for \emph{E.
coli} and\emph{S. 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 H\textsubscript{2}O\textsubscript{2} 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 \selectlanguage{greek}μ\selectlanguage{english}g/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 H\textsubscript{2}O\textsubscript{2}, R12-AgNPs induced even greater
ROS production in bacteria. These results indicated that in the presence
of H\textsubscript{2}O\textsubscript{2}, 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 \emph{E. coli} and \emph{S.
aureus} using SEM {[}49{]}. As shown in Fig. 5 (B, D), untreated\emph{E.
coli} and \emph{S. aureus} displayed intact cells with smooth membranes,
exhibiting their typical rod and round shapes. Meanwhile, the bacteria
treated with H\textsubscript{2}O\textsubscript{2} showed no discernible
differences compared to the untreated control group, indicating the
preserved structural integrity of the bacteria. Nonetheless, when
\emph{E. coli} and \emph{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+H\textsubscript{2}O\textsubscript{2}exhibited disrupted
cell morphology with wrinkled cell surfaces (Fig. 6B). Notably, in the
group of R12-AgNPs+H\textsubscript{2}O\textsubscript{2}, 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, \emph{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 \emph{S. aureus} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig. 5 ROS generation (A, B) and SEM pictures (C) of bacteria after
treatments with bactericidal agents.

Based on the comprehensive results discussed earlier, it is evident that
R12-AgNPs exhibit a synergistic antibacterial effect when combined with
H\textsubscript{2}O\textsubscript{2}. The proposed mechanism for
bacterial inhibition may involve the following steps (Fig. 6): (1)
R12-AgNPs capture and adhere to the negatively charged bacterial
surfaces through electrostatic attraction; (2) R12-AgNPs facilitate the
sustained release of Ag\textsuperscript{+} ions in the vicinity of the
bacteria. These released Ag\textsuperscript{+} ions can interact with
thiol groups found in critical proteins within the bacterial cells. As a
result, many essential enzyme proteins may become inactivated {[}52{]};
(3) R12-AgNPs possess peroxidase-like activity, which allows them to
catalyze the decomposition of H\textsubscript{2}O\textsubscript{2},
yielding a substantial quantity of hydroxyl radicals. These hydroxyl
radicals act on biofilms, lipids, proteins, and DNA, leading to the
degradation and disruption of these critical structures within the
bacterial cells. Ultimately, this process results in bacterial death
{[}53{]}. The antibacterial mechanism of R12-AgNPs in combination with
H\textsubscript{2}O\textsubscript{2} involves multiple synergistic
steps, which collectively lead to the effective inhibition and
destruction of bacterial cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Fig. 6 Schematic representation of the synergistic antimicrobial
mechanism of R12-AgNPs with H\textsubscript{2}O\textsubscript{2}.

Biocompatibility evaluation of R12-AgNPs

The biocompatibility of nanoparticles is a critical factor in
determining their potential biomedical applications. Therefore, it is
essential to evaluate the biocompatibility of R12-AgNPs. The
cytotoxicity of R12-AgNPs was assessed in vitro using mouse fibroblast
(L929). As shown in Fig. 7, the cell viability remained at 92.39\% when
the concentration of R12-AgNPs was below 10 \selectlanguage{greek}µ\selectlanguage{english}g/mL. Even a higher
concentration of 75 \selectlanguage{greek}µ\selectlanguage{english}g/mL, a cell viability of 80\% was achieved. The
results indicate that R12-AgNPs had low cytotoxicity and their potential
suitability as a material for biomedical applications. The favorable
biocompatibility of R12-AgNPs is an important finding, as it opens the
door to various biomedical applications where these nanoparticles can be
utilized without causing significant harm to living cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Fig.7 Cell viability of R12-AgNPs after 24 h incubation at different
concentrations.

\textbf{Conclusion}

In conclusion, the AgNPs synthesized using the cell-free supernatant
of\emph{D. wulumuqiensis} R12 exhibit several advantageous properties,
including peroxidase-like activity, strong bacteriostatic capabilities,
and excellent biocompatibility. These AgNPs effectively deactivate
bacteria by generating toxic ROS that disrupt bacterial membranes.
Moreover, the intrinsic peroxidase-like activity of R12-AgNPs enables
the catalysis of H\textsubscript{2}O\textsubscript{2} decomposition,
producing hydroxyl radicals at low concentrations and enhancing
synergistic antimicrobial effects while maintaining biosafety. Through
the combination with H\textsubscript{2}O\textsubscript{2}, R12-AgNPs
have demonstrated exceptional effectiveness in antimicrobial therapy.
This approach holds promise as a candidate for antibiotic-free treatment
of bacterial infections. The combination of R12-AgNPs and
H\textsubscript{2}O\textsubscript{2} presents a novel strategy that
could address the growing concern of antibiotic resistance and offer a
safer and effective alternative for combatting bacterial infections.

\textbf{CRediT authorship contribution statement}

Conceived and designed the experiments: Liying Zhu. Performed the
experiments and wrote the paper: Jingjia Liu. Analyzed the data: Ling
Jiang, Zhidong Zhang. Contributed reagents/materials/analysis tools:
Bowen Shen, Nan Zhang. All authors read and approved the final
manuscript.

\textbf{Declaration of Competing Interest}

The authors report no declarations of interest.

\textbf{Data availability}

Data will be made available on request.

\textbf{Acknowledgements}

This work was supported by National Key Research and Development Program
(2022YFB3805001), Mining of Actinobacteria resource from extremely arid
and hot area in China by culturomics and screening of quorum sensing
inhibitors (32060004), the Jiangsu Synergetic Innovation Center for
Advanced Bio-Manufacture (XTC2205).

\textbf{References}

{[}1{]} G. Hughes, M.A. Webber, Novel approaches to the treatment of
bacterial biofilm infections, British Journal of Pharmacology 174(14)
(2017) 2237-2246.

{[}2{]} H.K. Kang, H.Y. Kim, J.D. Cha, Synergistic effects between
silibinin and antibiotics on methicillin-resistant \emph{Staphylococcus
aureus} isolated from clinical specimens, Biotechnology Journal 6(11)
(2011) 1397-1408.

{[}3{]} R.A. Aleinein, H. Sch\selectlanguage{ngerman}äfer, M. Wink, Secretory ranalexin produced
in recombinant \emph{Pichia pastoris} exhibits additive or synergistic
bactericidal activity when used in combination with polymyxin B or
linezolid against multi\selectlanguage{english}-drug resistant bacteria, Biotechnology Journal
9(1) (2013) 110-119.

{[}4{]} C. Ferriol-Gonzalez, P. Domingo-Calap, Phage Therapy in
Livestock and Companion Animals, Antibiotics 10(5) (2021) 559.

{[}5{]} Y. Sang, W. Li, H. Liu, L. Zhang, H. Wang, Z. Liu, J. Ren, X.
Qu, Construction of Nanozyme\selectlanguage{english}-Hydrogel for Enhanced Capture and
Elimination of Bacteria, Advanced Functional Materials 29(22) (2019)
1900518.

{[}6{]} Y. Li, W. Ma, J. Sun, M. Lin, Y. Niu, X. Yang, Y. Xu,
Electrochemical generation of Fe\textsubscript{3}C/N-doped graphitic
carbon nanozyme for efficient wound healing in vivo, Carbon 159 (2020)
149-160.

{[}7{]} Z. Wang, K. Dong, Z. Liu, Y. Zhang, Z. Chen, H. Sun, J. Ren, X.
Qu, Activation of biologically relevant levels of reactive oxygen
species by Au/g-C\textsubscript{3}N\textsubscript{4} hybrid nanozyme for
bacteria killing and wound disinfection, Biomaterials 113 (2017)
145-157.

{[}8{]} S. Lethongkam, J. Glaser, A.V. Ammanath, S.P. Voravuthikunchai,
F. Gotz, In vitro and in vivo comparative analysis of antibacterial
activity of green\selectlanguage{english}-synthesized silver nanoparticles, Biotechnology
Journal (2023) .

{[}9{]} S. Paosen, S. Bilhman, S. Wunnoo, S. Ramanathan, A.W. Septama,
S. Lethongkam, S.P. Voravuthikunchai, Control of biomaterial\selectlanguage{english}-associated
infections through biofabrication of gold nanoparticles using \emph{Musa
sapientum} extract, Biotechnology Journal 18(10) (2023).

{[}10{]} A.M. Khalil, A.H. Hashem, S. Kamel, Bimetallic hydrogels based
on chitosan and carrageenan as promising materials for biological
applications, Biotechnology Journal 18(10) (2023).

{[}11{]} Y. Qin, Y. Liu, L. Yuan, H. Yong, J. Liu, Preparation and
characterization of antioxidant, antimicrobial and pH-sensitive films
based on chitosan, silver nanoparticles and purple corn extract, Food
Hydrocolloids 96 (2019) 102-111.

{[}12{]} S. Paosen, S. Lethongkam, S. Wunnoo, N. Lehman, E.
Kalkornsurapranee, A.W. Septama, S.P. Voravuthikunchai, Prevention of
nosocomial transmission and biofilm formation on novel biocompatible
antimicrobial gloves impregnated with biosynthesized silver
nanoparticles synthesized using \emph{Eucalyptus citriodora} leaf
extract, Biotechnology Journal 16(9) (2021).

{[}13{]} P. Nie, Y. Zhao, H. Xu, Synthesis, applications, toxicity and
toxicity mechanisms of silver nanoparticles: A review, Ecotoxicol
Environ Saf 253 (2023) 114636.

{[}14{]} K. Vijayaraghavan, S.P.K. Nalini, Biotemplates in the green
synthesis of silver nanoparticles, Biotechnology Journal 5(10) (2010)
1098-1110.

{[}15{]} G. Gahlawat, A.R. Choudhury, A review on the biosynthesis of
metal and metal salt nanoparticles by microbes, RSC Advances 9(23)
(2019) 12944-12967.

{[}16{]} J. Xi, G. Wei, Q. Wu, Z. Xu, Y. Liu, J. Han, L. Fan, L. Gao,
Light-enhanced sponge-like carbon nanozyme used for synergetic
antibacterial therapy, Biomaterials Science 7(10) (2019) 4131-4141.

{[}17{]} L. Gao, K.M. Giglio, J.L. Nelson, H. Sondermann, A.J. Travis,
Ferromagnetic nanoparticles with peroxidase-like activity enhance the
cleavage of biological macromolecules for biofilm elimination, Nanoscale
6(5) (2014) 2588-2593.

{[}18{]} H. Chen, X. Zhao, B. Cui, H. Cui, M. Zhao, J. Shi, J. Li, Z.
Zhou, Peroxidase-like MoS\textsubscript{2}/Ag nanosheets with
synergistically enhanced NIR-responsive antibacterial activities,
Frontiers in Chemistry 11 (2023).

{[}19{]} A. Shafiq, A.R. Deshmukh, K. AbouAitah, B.-S. Kim, Green
Synthesis of Controlled Shape Silver Nanostructures and Their
Peroxidase, Catalytic Degradation, and Antibacterial Activity, Journal
of Functional Biomaterials 14(6) (2023) 325.

{[}20{]} V.-D. Doan, V.-C. Nguyen, T.-L.-H. Nguyen, A.-T. Nguyen, T.-D.
Nguyen, Highly sensitive and low-cost colourimetric detection of glucose
and ascorbic acid based on silver nanozyme biosynthesized
by\emph{Gleditsia australis} fruit, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy 268 (2022) 120709.

{[}21{]} Y. Wu, J.-Y. Chen, W.-M. He, Surface-enhanced Raman
spectroscopy biosensor based on silver nanoparticles@metal-organic
frameworks with peroxidase-mimicking activities for ultrasensitive
monitoring of blood cholesterol, Sensors and Actuators B: Chemical 365
(2022) 154125.

{[}22{]} X. Tan, S. Liu, X. Hu, R. Zhang, X. Su, R. Qian, Y. Mai, Z. Xu,
W. Jing, W. Tian, L. Xie, Near-Infrared-Enhanced Dual Enzyme-Mimicking
Ag-TiO\textsubscript{(2-x)}@Alginate Microspheres with Antibactericidal
and Oxygeneration Abilities to Treat Periodontitis, ACS Appl Mater
Interfaces 15(1) (2023) 391-406.

{[}23{]} A. Mohammadzadeh, P. Pashazadeh-Panahi, M. Hasanzadeh, Visual
monitoring and optical recognition of digoxin by functionalized AuNPs
and triangular AgNPs as efficient optical nano-probes, J Mol Recognit
34(10) (2021) e2917.

{[}24{]} A. Xiao, B. Wang, L. Zhu, L. Jiang, Production of extracellular
silver nanoparticles by radiation-resistant \emph{Deinococcus
wulumuqiensis} R12 and its mechanism perspective, Process Biochemistry
100 (2021) 217-223.

{[}25{]} X. Xu, L. Jiang, Z. Zhang, Y. Shi, H. Huang, Genome Sequence of
a Gamma- and UV-Ray-Resistant Strain, \emph{Deinococcus
wulumuqiensis}R12, Genome Announc 1(3) (2013) e00206-13.

{[}26{]} Y. Chen, Z. Yang, X. Zhou, M. Jin, Z. Dai, D. Ming, Z. Zhang,
L. Zhu, L. Jiang, Sequence, structure, and function of the Dps
DNA-binding protein from \emph{Deinococcus wulumuqiensis} R12, Microb
Cell Fact 21(1) (2022) 132.

{[}27{]} S. Sun, B. Shen, L. Jiang, L. Zhu, Potential for efficient
microbial remediation of Cr(VI) in wastewater using \emph{Deinococcus
wulumuqiensis} R12, Journal of Applied Microbiology 134(6) (2023).

{[}28{]} Y. Wu, J.-M. Zhou, Y.-S. Jiang, W. Li, M.-J. He, Y. Xiao, J.-Y.
Chen, Silver nanoparticles@metal-organic framework as peroxidase mimics
for colorimetric determination of hydrogen peroxide and blood glucose,
Chinese Journal of Analytical Chemistry 50(12) (2022).

{[}29{]} E. Jamroz, P. Kopel, L. Juszczak, A. Kawecka, Z. Bytesnikova,
V. Milosavljevi\selectlanguage{polish}ć, \selectlanguage{english}M. Kucharek, M. Makarewicz, V. Adam, Development and
characterisation of furcellaran-gelatin films containing SeNPs and AgNPs
that have antimicrobial activity, Food Hydrocolloids 83 (2018) 9-16.

{[}30{]} D. Feng, R. Zhang, M. Zhang, A. Fang, F. Shi, Synthesis of
Eco-Friendly Silver Nanoparticles Using Glycyrrhizin and Evaluation of
Their Antibacterial Ability, Nanomaterials 12(15) (2022) 2636.

{[}31{]} S.-P. Deng, J.-Y. Zhang, Z.-W. Ma, S. Wen, S. Tan, J.-Y. Cai,
Facile Synthesis of Long-Term Stable Silver Nanoparticles by Kaempferol
and Their Enhanced Antibacterial Activity Against \emph{Escherichia
coli} and \emph{Staphylococcus aureus} , Journal of Inorganic and
Organometallic Polymers and Materials 31(7) (2021) 2766-2778.

{[}32{]} Y. Wang, Y. Liu, N. Zhao, J. Wang, Y. Yang, D. Cui, M. Zhao,
Fe\textsubscript{3}O\textsubscript{4} nanozyme coating enhances
light-driven biohydrogen production in
self-photosensitized\emph{Shewanella oneidensis} -CdS hybrid systems,
Biotechnology Journal (2023).

{[}33{]} X. Tian, X. Jiang, C. Welch, T.R. Croley, T.Y. Wong, C. Chen,
S. Fan, Y. Chong, R. Li, C. Ge, C. Chen, J.J. Yin, Bactericidal Effects
of Silver Nanoparticles on \emph{Lactobacilli} and the Underlying
Mechanism, ACS Appl Mater Interfaces 10(10) (2018) 8443-8450.

{[}34{]} B. Lu, F. Lu, Y. Zou, J. Liu, B. Rong, Z. Li, F. Dai, D. Wu, G.
Lan, In situ reduction of silver nanoparticles by chitosan-l-glutamic
acid/hyaluronic acid: Enhancing antimicrobial and wound-healing
activity, Carbohydrate Polymers 173 (2017) 556-565.

{[}35{]} M.S. Mechouche, F. Merouane, C.E.H. Messaad, N. Golzadeh, Y.
Vasseghian, M. Berkani, Biosynthesis, characterization, and evaluation
of antibacterial and photocatalytic methylene blue dye degradation
activities of silver nanoparticles from \emph{Streptomyces
tuirus}strain, Environmental Research 204 (2022) 112360-112373.

{[}36{]} Y. Zaman, M.Z. Ishaque, S. Ajmal, M. Shahzad, A.B. Siddique,
M.U. Hameed, H. Kanwal, R.J. Ramalingam, M. Selvaraj, G. Yasin, Tamed
synthesis of AgNPs for photodegradation and anti-bacterial activity:
Effect of size and morphology, Inorganic Chemistry Communications 150
(2023) 110523-110529.

{[}37{]} G. Rajkumar, R. Sundar, Biogenic one-step synthesis of silver
nanoparticles (AgNPs) using an aqueous extract of \emph{Persea
americana} seed: Characterization, phytochemical screening,
antibacterial, antifungal and antioxidant activities, Inorganic
Chemistry Communications 143 (2022) 109817.

{[}38{]} X. Li, M. Liu, H. Cheng, Q. Wang, C. Miao, S. Ju, F. Liu,
Development of ionic liquid assisted-synthesized nano-silver combined
with vascular endothelial growth factor as wound healing in the care of
femoral fracture in the children after surgery, J Photochem Photobiol B
183 (2018) 385-390.

{[}39{]} A.O. Fadaka, S. Meyer, O. Ahmed, G. Geerts, M.A. Madiehe, M.
Meyer, N.R.S. Sibuyi, Broad Spectrum Anti-Bacterial Activity and
Non-Selective Toxicity of Gum Arabic Silver Nanoparticles, Int J Mol Sci
23(3) (2022) 1799.

{[}40{]} S. Palithya, S.A. Gaddam, V.S. Kotakadi, J. Penchalaneni, V.N.
Challagundla, Biosynthesis of silver nanoparticles using leaf extract
of\emph{Decaschistia crotonifolia} and its antibacterial, antioxidant,
and catalytic applications, Green Chemistry Letters and Reviews 14(1)
(2021) 137-152.

{[}41{]} H.-z. Qi, W.-z. Wang, J.-y. He, Y. Ma, F.-z. Xiao, S.-y. He,
Antioxidative system of \emph{Deinococcus radiodurans} , Research in
Microbiology 171(2) (2020) 45-54.

{[}42{]} H. Sun, A. Zhao, N. Gao, K. Li, J. Ren, X. Qu, Deciphering a
nanocarbon-based artificial peroxidase: chemical identification of the
catalytically active and substrate-binding sites on graphene quantum
dots, Angew Chem Int Ed Engl 54(24) (2015) 7176-80.

{[}43{]} X. Shu, Y. Chang, H. Wen, X. Yao, Y. Wang, Colorimetric
determination of ascorbic acid based on carbon quantum dots as
peroxidase mimetic enzyme, RSC Advances 10(25) (2020) 14953-14957.

{[}44{]} K. Gudikandula, S. Charya Maringanti, Synthesis of silver
nanoparticles by chemical and biological methods and their antimicrobial
properties, Journal of Experimental Nanoscience 11(9) (2016) 714-721.

{[}45{]} M. Mousavi-Khattat, M. Keyhanfar, A. Razmjou, A comparative
study of stability, antioxidant, DNA cleavage and antibacterial
activities of green and chemically synthesized silver nanoparticles,
Artificial Cells, Nanomedicine, and Biotechnology 46(sup3) (2018)
1022-1031.

{[}46{]} N. Yu, T. Cai, Y. Sun, C. Jiang, H. Xiong, Y. Li, H. Peng, A
novel antibacterial agent based on AgNPs and
Fe\textsubscript{3}O\textsubscript{4} loaded chitin microspheres with
peroxidase-like activity for synergistic antibacterial activity and
wound-healing, International Journal of Pharmaceutics 552(1-2) (2018)
277-287.

{[}47{]} A.A. Dayem, B. Kim, S. Gurunathan, H.Y. Choi, G. Yang, S.K.
Saha, D. Han, J. Han, K. Kim, J.H. Kim, S.G. Cho, Biologically
synthesized silver nanoparticles induce neuronal differentiation of
SH-SY5Y cells via modulation of reactive oxygen species, phosphatases,
and kinase signaling pathways, Biotechnology Journal 9(7) (2014)
934-943.

{[}48{]} M.A. Quinteros, C.A. Viviana, R. Onnainty, V.S. Mary, M.G.
Theumer, G.E. Granero, M.G. Paraje, P.L. P\selectlanguage{ngerman}áez, Biosynthesized silver
nanoparticles: Decoding their mechanism of action in\emph{Staphylococcus
aureus} and \emph{Escherichia coli} , The International Journal of
Biochemistry \& Cell Biology 104 (2018) 87-93.

{[}49{]} H. Wang, Y. Jiang, Y. Zhang, Z. Zhang, X. Yang, M.A. Ali, E.M.
Fox, K.S. Gobius, C. Man, Silver nanoparticles: A novel antibacterial
agent for control of Cronobacter sakazakii, Journal of Dairy Science
101(12) (2018) 10775-10791.

{[}50{]} S. Liao, Y. Zhang, X. Pan, F. Zhu, C. Jiang, Q. Liu, Z. Cheng,
G. Dai, G. Wu, L. Wang, L. Chen, Antibacterial activity and mechanism of
silver nanoparticles against multidrug-resistant \emph{Pseudomonas
aeruginosa} , Int J Nanomedicine 14 (2019) 1469-1487.

{[}51{]} J.C. Ontong, S. Paosen, S. Shankar, S.P. Voravuthikunchai,
Eco-friendly synthesis of silver nanoparticles using \emph{Senna
alata}bark extract and its antimicrobial mechanism through enhancement
of bacterial membrane degradation, J Microbiol Methods 165 (2019)
105692.

{[}52{]} H.S. Jiang, Y. Zhang, Z.W. Lu, R. Lebrun, B. Gontero, W. Li,
Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of
Thiol Groups, Small 15(27) (2019) 1900860.

{[}53{]} M.A. Quinteros, V. Cano Aristizábal, P.R. Dalmasso, M.G.
Paraje, P.L. Páez, Oxidative stress generation of silver nanoparticles
in three bacterial genera and its relationship with the antimicrobial
activity, Toxicology in Vitro 36 (2016) 216-223.

\textbf{Figure legends}

\textbf{Fig. 1} UV-Vis absorption spectra and SEM \textbf{(A)} , X-ray
diffraction \textbf{(B)} , FTIR spectra \textbf{(C)} and silver ion
release concentration (concentration of AgNPs is 10 mg/L) \textbf{(D)}of
AgNPs.

\textbf{Fig. 2} UV-vis spectra of different combinations after 10 min of
reaction at room temperature.

\textbf{Fig. 3} Optimization parameters for efficient sensing with
variation in pH \textbf{(A)} , temperature \textbf{(B)} , R12-AgNPs
concentration \textbf{(C)} and time \textbf{(D)} .

\textbf{Fig. 4} Bacteriostasis rate of \emph{E. coli} \textbf{(A)}
and\emph{S. aureus} \textbf{(B)} incubated with different concentrations
of C-AgNPs, R12-AgNPs and
R12-AgNPs+H\textsubscript{2}O\textsubscript{2}(concentration of
H\textsubscript{2}O\textsubscript{2} = 10 \selectlanguage{greek}μ\selectlanguage{english}M); Bacteriostasis rate of
\emph{E. coli} \textbf{(C)} and \emph{S. aureus}\textbf{(D)} incubated
with different concentrations of
H\textsubscript{2}O\textsubscript{2}(10\textsuperscript{-7}-10\textsuperscript{-3}M)
after treatment with or without R12-AgNPs (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL); Bacterial colonies
\textbf{(E)} treated with C-AgNPs, R12-AgNPs and
R12-AgNPs+H\textsubscript{2}O\textsubscript{2} (concentration of
H\textsubscript{2}O\textsubscript{2} = 10 \selectlanguage{greek}μ\selectlanguage{english}M, concentration of AgNPs = 1
\selectlanguage{greek}μ\selectlanguage{english}g/mL).

\textbf{Fig. 5} ROS generation \textbf{(A, B)} and SEM
pictures\textbf{(C)} of bacteria after treatments with bactericidal
agents.

\textbf{Fig. 6} Schematic representation of the synergistic
antimicrobial mechanism of R12-AgNPs with
H\textsubscript{2}O\textsubscript{2}.

\textbf{Fig.7} Cell viability of R12-AgNPs after 24 h incubation at
different concentrations.

\textbf{Hosted file}

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