Highlights:
AgNPs biosynthesized from the
supernatant of Deinococcus wulumuqiensis R12 have low toxicity
excellent, bacteriostatic activity and peroxidase-like properties
without additional modifications.
Upon combination of R12-AgNPs and
H2O2, significant synergistic
inhibitory activity was observed against both E. coli andS. aureus , due to peroxidase-like activity of R12-AgNPs.
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 MoS2 [5], Fe3C/N-C
[6], Au/g-C3N4 [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.
H2O2 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 H2O2 (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
H2O2 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 H2O2. Several
nanocatalysts, including Fe3O4,
C3N4, MoS2 and
carbon-based nanomaterials, have demonstrated peroxidase-like activity
[7, 17, 18]. These nanocatalysts can synergistically inhibit
bacterial growth when combined with AgNPs and
H2O2. 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
H2O2 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
H2O2 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 Deinococcus wulumuqiensis R12 (D.
wulumuqiensis R12) could successfully produce AgNPs without addition of
stabilizer at room temperature. D. wulumuqiensis R12, a member of
the Deinococcus 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 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 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 H2O2. 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.
Materials andmethods
Materials and chemical reagents
D. wulumuqiensis R12 was kindly offered by Xinjiang Academy of
Agricultural Sciences (Xinjiang, China). Sodium borohydride
(NaBH4) and silver nitrate (AgNO3) 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 D.
wulumuqiensis R12, and they were labeled as R12-AgNPs. For comparison,
another type of AgNPs were produced by utilizing sodium borohydride
(NaBH4) 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 μL of 80 mM TMB was introduced into a mixture of 0.89 mL of
PBS buffer solution (pH=4.0) containing AgNPs and
H2O2, 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 Escherichia
coli (E. coli ) and Gram-positive bacteria Staphylococcus
aureus (S. aureus) [29,
30] were selected for experiments. The bacterial fluids were diluted
to 108 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
H2O2 and R12-AgNPs, E. coli andS. aureus were mixed with different concentrations (0, 1, 5, 10
μg/mL) of R12-AgNPs and hydrogen peroxide
(H2O2) at a final concentration of
10-6 M. Meanwhile, the effect of
H2O2 concentration on combination
therapy was investigated by mixing bacteria with varying concentrations
of H2O2(10-7-10-3 M) and a final
concentration of 1 μg/mL of R12-AgNPs. All experiments were performed as
follows: after incubating with antibacterial agents for 30 minutes at
37°C, 100 µL of diluted bacterial suspension was removed and dispersed
evenly on blood agar plates and left at 37°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 E.
coli and S. aureus after incubation with AgNPs and
H2O2.
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 1×104CFU/mL using culture medium. After incubation in enzyme labelled plates
for 24 h at 37 ℃, the original culture medium was aspirated out and 100
µL 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 µL of MTT solution (5 mg/mL) was added to each
well, and the supernatant was removed after 4 h of incubation in a
CO2 incubator, and 150 µL 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.
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 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-1, The peaks at 2971 cm−1 and
2921 cm−1 referred to C–H bonds; the band at 1066
cm−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−1 assigned to –C = CH– was attributed to aromatic
groups [40]. In our case, the
cell-free supernatant of 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 D. wulumuqiensisR12 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 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.