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.