Enhancement of the Antibacterial Efficiency of Silver Nanoparticles against Gram-Positive and Gram-Negative Bacteria Using Blue Laser Light
Silver nanoparticles (Ag-NPs) possess excellent antibacterial properties and are considered to be an alternative material for treating antibiotic-resistant bacteria. The present study was aimed at enhancing the antibacterial efficiency of Ag-NPs using visible laser light against Escherichia coli and Staphylococcus aureus in vitro. Four concentrations of Ag-NPs (12.5, 25, 50, and 100 μg/ml), synthesized by the chemical reduction method, were utilized to conduct the antibacterial activity of prepared Ag-NPs. The antibacterial efficiencies of photoactivated Ag-NPs against both bacteria were determined by survival assay after exposure to laser irradiation. The mechanism of interactions between Ag-NPs and the bacterial cell membranes was then evaluated via scanning electron microscopy (SEM) and reactive oxygen species analysis to study the cytotoxic action of photoactivated Ag-NPs against both bacterial species. Results showed that the laser-activated Ag-NP treatment reduced the surviving population to 14% of the control in the E. coli population, while the survival in the S. aureus population was reduced to 28% of the control upon 10 min exposure time at the concentration of 50 μg/ml. However, S. aureus showed lower sensitivity after photoactivation compared to E. coli. Moreover, the effects depended on the concentration of Ag-NPs and exposure time to laser light. SEM images of treated bacterial cells indicated that substantial morphological changes occurred in cell membranes after treatment. The results suggested that Ag-NPs in the presence of visible light exhibit strong antibacterial activity which could be used to inactivate harmful and pathogenic microorganisms.
Silver nanoparticles (Ag-NPs) have been used as antibacterial, antifungal, antiviral, anti-inflammatory, and anti-antiangiogenic due to their unique properties such as physical, chemical, and biological properties . These previous studies demonstrated that the antibacterial activity of Ag-NPs is dependent on their size and specific surface area, with smaller particles showing the better antibacterial activities [2, 3]. This difference in efficacy based on sizes is related to their large surface area to volume ratio, which allows efficient binding with the bacterial surface. Recently, researchers have discovered how to activate the bactericidal effect of Ag-NPs using laser irradiation [4, 5], thus offering a promising development in the fight against antibacterial resistance. The size of metal particles (NPs) also correlates with their optical properties, which have been extensively studied and found to depend on their size, shape, composition, and dielectric surrounding medium . These unique optical properties of metal NPs are closely associated with the localized surface plasmon resonance (LSPR) effect, in which free electrons oscillate collectively on the metal surface when irradiated with certain light energies, causing wavelength-dependent absorption and scattering .
Ag-NPs can potentially be engineered as drug delivery tools due to their strong interaction with light. Hence, Ag-NPs have attracted considerable attention for their possible use in diagnostics and therapeutics . Ag-NPs can strongly absorb light in the visible spectrum region by inducing collective oscillation of conduction bands in strong resonance with specific light frequencies. This phenomenon is known as surface plasmon resonance (SPR) and is one of the most frequently studied characteristics of Ag-NPs .
Photoactivation has been suggested to influence the antibacterial activity of NPs. Therapy with photoactive NPs has been proposed as an alternative method for eradicating pathogenic bacteria . The light source used to activate Ag-NPs should have a wavelength within the absorption band. In recent years, a number of works have attempted to explore the toxicity of Ag-NPs after photoactivation using UV and visible laser light. These studies have shown that the antimicrobial effects of Ag-NPs can be enhanced through light activation . Moreover, exposure of cells to Ag-NPs irradiated by low-power visible light can cause oxidative stress owing to the production of reactive oxygen species (ROS) such as, ⋅OH, O2⋅-, and ROS, produced by photoactive NPs which can damage cellular components, e.g., proteins and DNA of treated cells [11, 12].
Recently, the use of blue light has attracted increasing attention for the development of a novel phototherapy-based antimicrobial agent with significant antimicrobial activity against a broad range of bacterial and fungal pathogens but with less chance of resistance compared with antibiotics . Blue light with a wavelength of 400-500 nm has been shown to be highly effective against both Gram-negative and Gram-positive bacteria . Despite the broad range of Ag-NPs synthesized and biologically evaluated in the literature, there have been few studies of the interaction between Ag-NPs and laser light on cells. Therefore, this study was conducted to investigate the antibacterial activity of Ag-NPs on the growth inhibition, cytotoxicity, and membrane cell integrity of Escherichia coli and Staphylococcus aureus after photoactivation by visible laser light.
2. Materials and Methods
2.1. Bacteria and Culture Conditions
Two bacterial representatives were used in this study, namely, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). The bacterial cultures were cultivated on nutrient agar (Oxoid) at 37°C for 24 h. A single colony was transferred to nutrient broth and incubated aerobically under shaking at 37°C for 16 h. Cells were then harvested by centrifugation and resuspended in phosphate-buffered saline (PBS) to an optical density of 0.03 at 600 nm corresponding to approximately 106 colony-forming units per ml (CFU/ml).
2.2. Synthesis of Ag-NPs
For preparation of Ag-NPs, two reducing and stabilizing agents were used, i.e., sodium borohydride (NaBH4) as the primary reductant and trisodium citrate (TSC) as the secondary reductant with silver nitrate solution as a metal salt precursor, according to the method published in . To freshly prepare the solution containing 1 mM sodium borohydride and 3 mM TSC solution, 1 mM AgNO3 solution was added drop-wise accompanied by a color change from colorless solution to brown-yellow indicating the formation of Ag-NPs. The Ag-NPs were purified by centrifugation and washing three times with deionized ultrapure distal water.
2.3. Characterization of Ag-NPs
2.3.1. UV-Visible Spectroscopy
An ultraviolet-visible (UV-Vis) spectrophotometer (JASCO, V-630) (Japan) was used to observe the absorbance mode in the range of 300–500 nm with appropriate dilutions of the silver colloid in a quartz cuvette.
2.3.2. Transmission Electron Microscopy (TEM)
To study the size and morphology of the Ag-NPs, transmission electron microscopy (TEM) was used. The histogram of the size distribution was calculated from TEM images by measuring the diameters of at least 100 particles. Samples for TEM studies were prepared by placing one drop of Ag-NP solution on a carbon-coated copper grid.
2.4. Antibacterial Activity of Ag-NPs
The minimum inhibitory concentration (MIC) of Ag-NPs was determined using the well agar diffusion method . Briefly, the bacterial cell cultures were adjusted to 105 CFU/ml, and 100 μl of the microbial suspension was spread on Mueller-Hinton agar plates. 50 μl of 12.5, 25, 50, or 100 μg/ml Ag-NPs was added to each well of plates under normal light condition of the laminar flow cabinet. The plates were then incubated at 37°C for 24 h, and the diameter of the zone of inhibition of bacterial growth surrounding the wells was measured in mm.
2.5. Laser Light Source
The laser irradiation source used in this study was a diode laser (Dragon Lasers Ltd, China) emitting light at a wavelength of 410 nm and with an actual power output of 100 mW. The distance of the laser probe to the plate surface was adjusted to give a power density of 2.040 W/cm2.
2.6. Photoactivation of Ag-NPs against Bacteria
To evaluate the effect of irradiation on the antibacterial activity of Ag-NPs, a survival assay based on the method of Nakonieczna et al.  was used. Briefly, 100 μl of bacterial cell suspensions from overnight cultures containing approximately 106 CFU/ml in sterile nutrient broth was transferred into a 96-well plate, and 100 μl aliquots of Ag-NPs were added to each well to reach a final concentration of 12.5, 25, or 50 μg/ml. After addition of the Ag-NPs, the wells were left in the incubator for 1 h preirradiation time and then exposed to intense laser light at 2.040 W/cm2 with exposure times of 1, 3, 5, or 10 min. Each experimental condition was tested in triplicate. Three experimental groups were considered as follows: (i)The control group comprising bacterial cells cultured in wells without treatment of either Ag-NPs or irradiation (L- Ag-NPs-)(ii)Bacterial cells exposed to irradiation in the absence of Ag-NPs (L+ Ag-NPs-)(i)Bacterial cells irradiated in the presence of Ag-NPs (L+ Ag-NPs+)
To enumerate the survival of the bacteria, 100 μl from each well was spread in triplicate on nutrient agar petri dishes and then incubated for 24 to 48 h at 37°C. Next, the bacterial colonies were counted and the values converted into colony-forming units (CFU/ml). The survival of the bacterial cells () was calculated as follows: where is the number of bacterial cells (CFU/ml) before treatment and is the number of bacterial cells (CFU/ml) after treatment.
2.7. Measurement of Temperature during Irradiation (Thermography)
Per thermography analysis, the irradiation did not generate sufficient heat to cause damage to the living bacteria. The temperature of bacterial suspensions was measured during irradiation for all exposure times using a noncontact thermometer (Microlife, Germany).
2.8. Reactive Oxygen Species (ROS) Assay
ROS generation from the surface of postirradiation Ag-NPs was determined using an intracellular ROS assay kit (Cell Biolabs, USA). Generation of ROS from Ag-NPs was measured using 2,7-dichlorofluorescein diacetate (DCFH2-DA) as a fluorescent probe based on the method in . Bacterial cells were inoculated in fresh nutrient broth and incubated at 37°C and 150 rpm for 24 h. Afterwards, the bacterial cell suspensions were adjusted to 106 CFU/ml, 100 μl was transferred to each well of a 96-well plate, 50 or 25 μg/ml of Ag-NPs was added to the wells, and the plates were incubated for 2 h. After incubation, the wells were irradiated with laser light for 5 or 10 min. Cells were collected by centrifugation at 5,000 g for 10 min and then washed and resuspended in PBS. The supernatant was labelled with 10 μM DCFH2-DA and incubated for 30 min at 37°C in dark conditions. A positive control obtained by treating the cells with 0.8 mM H2O2 and a negative control comprising cells, without treatment, were also labeled with 10 μM DCFH2-DA. The fluorescence intensity was measured using a microplate reader (AccuReader, Taiwan) with excitation at 485 nm and emission at 530 nm.
2.9. Preparation of Cells for SEM
Scanning electron microscopy (SEM) was performed on a midexponential growth-phase bacterial culture diluted in nutrient broth medium to a cell density of 106 CFU/ml and treated with 50, 25, or 12.5 μg/ml of Ag-NPs and incubated at 37°C for 2 h. The cultures were then irradiated with laser light for 1, 3, 5, or 10 min. Untreated controls were maintained in nutrient medium. The bacterial cells were washed and resuspended in PBS. Samples were placed on membrane filters and fixed for 3 h with 2.5% glutaraldehyde, then washed twice in PBS. The samples were dehydrated with a graded ethanol series (30%, 50%, 70%, 80%, 95%, and 100%) and dried with a critical point drier using liquid CO2, then coated with a gold coater. The coated samples were observed under scanning electron microscopy (JEOL, Japan) .
2.10. Statistical Analysis
All mean values were obtained from triplicate independent experiments. The results were expressed as and analyzed using SPSS (version 22) software. A one-way analysis of variance (ANOVA) test was used to test the significance among the different treatment groups, and value ≤ 0.05 was considered to indicate a statistically significant difference.
3.1. Synthesis and Characterization of Ag-NPs
Figure 1 shows the UV-Vis spectrum of the Ag-NPs and characteristic SPR. An absorption peak was observed centered at 410 nm, indicating formation of Ag-NPs. However, the spectrum was either red or blue shifted depending on the surface, size, and shape of the NPs. The average particle size and shape of the synthesized Ag-NPs were measured using TEM. TEM results showed that Ag-NPs were spherical in shape (Figure 2(a)), while the size of the nanoparticles were (Figure 2(b)).
3.2. Antibacterial Activity of Ag-NPs
The MICs (minimum inhibitory concentrations) of Ag-NPs required to inhibit the growth of 90% of the isolates for E. coli and S. aureus were 50 and 100 μg/ml, respectively, as shown by the inhibition zones in Figure 3.
3.3. Antibacterial Activity of Ag-NPs after Photoactivation against Bacteria
The antibacterial effect of photoactivated Ag-NPs was demonstrated by testing the bactericidal effect against the Gram-negative E. coli and Gram-positive S. aureus bacteria. This study examined the effect of photoactivation utilizing laser light (at a different exposure time) on the Ag-NPs on the survival of the two test bacteria. Growth of E. coli in the presence of the Ag-NPs is shown in Figure 4(a). The inhibitory effect of Ag-NPs for different concentrations was investigated (under normal light condition) over a period of 24 h, which allows for the different phases of bacterial growth to take place. Cells not treated with Ag-NPs were used as controls. After 24 h of treatment with 12.5 μg/ml Ag-NPs, bacterial growth was observed to be , with no growth inhibition observed in the controls (only bacteria and medium). A significant difference was observed at the higher concentration, 100 μg/ml, when compared with the concentration not exposed to Ag-NPs, and the microbial number decreased significantly, by up to ().
The bacterial cells were exposed to Ag-NPs at concentrations of 12.5, 25, and 50 μg/ml and a light wavelength of 410 nm for 1, 3, 5, and 10 min. The effect of exposure to blue laser light without Ag-NPs on the survival of E. coli (L+ Ag-NPs-) is shown in Figure 4(b). In comparison with the control group (L- Ag-NPs-, control), no significant reduction was observed in the viable number of E. coli cells exposed to blue laser light for 1, 3, or 5 min. However, the microbial number decreased significantly, by up to 85%, when the bacteria were exposed for 10 min, as compared with that of the control group (L- Ag-NPs-).
The effect of photoactivated Ag-NPs on E. coli cells using concentrations of 12.5, 25, and 50 μg/ml (L+ Ag-NPs+) was also determined. The lowest significant reduction in the number of bacteria cells was observed when cultures were exposed to Ag-NPs at concentrations of 12.5 μg/ml followed by light irradiation for 1, 3, 5, and 10 min reductions to 83%, 58%, 54%, and 37% (), respectively, compared to that of the control group. However, there was a noticeable decrease in the number of viable cells for all exposure times and concentrations of Ag-NPs compared with the control group (L- Ag-NPs-).
Using 25 μg/ml Ag-NPs, exposure to blue laser light for 5 and 10 min resulted in a significant reduction in the survival of E. coli to 31% and 22%, respectively (). The highest reduction in survival of the bacterial cells was observed when treating with 50 μg/ml of photoactivated Ag-NPs at 10 min exposure time and a power density of 2.040 W/cm2 against E. coli cells. A highly significant reduction was seen at 10 min for all concentrations of Ag-NPs, whereas much smaller reductions were observed for 1 min exposure. The results indicated that the antibacterial activity of photoactivated Ag-NPs increases with an increasing NP concentration and irradiation exposure time. Furthermore, at all concentrations of photoactivated Ag-NPs (L+ Ag-NPs+), there was a decrease in the number of viable bacterial cells compared to that with irradiation with laser light alone (L+ Ag-NPs-) (Figure 4(b)).
The antibacterial activity of Ag-NPs on the survival of S. aureus is shown in Figure 5(a). As is clear from the results, no significant reduction was observed in the viable number of S. aureus cells () exposed to 12.5 μg/ml when compared with that of the control group of cells. However, a significant inhibition of bacterial growth was observed for bacteria treated with the higher concentration of Ag-NPs (100 μg/ml), and the microbial number decreased significantly, by up to (), when compared with that of the control group ().
The effects of blue laser light on the survival of S. aureus cells (L+ Ag-NPs-) are shown in Figure 5(b). The highest sensitivity to laser irradiation alone occurred for the 10 min exposure (82%) compared with the control group (L- Ag-NPs-). Thus, similar to E. coli, inactivation of the bacterial growth was dependent on the exposure time of laser light illumination. However, for S. aureus, no significant inactivation was observed when cells were illuminated for 1, 3, or 5 min. The smallest reduction in survival in the presence of photoactivated Ag-NPs was for a concentration of 12.5 μg/ml when cells were illuminated for 1, 3, 5, or 10 min.
Irradiation of 50 μg/ml Ag-NPs exhibited the highest significant decrease in survival bacterial cells for all exposure times to 81%, 68%, 53%, and 28% (Figure 5(b)).
These results demonstrate that for all concentrations of Ag-NPs, E. coli was more sensitive to the photoactivated Ag-NPs than S. aureus; i.e., a lower concentration of Ag-NPs caused higher inactivation of E. coli compared to S. aureus cells. In addition, the results show that, by using laser light with Ag-NPs, the sensitivity of the microorganisms to radiation increased as cells exposed to laser light in the absence of Ag-NPs showed no significant decrease in viability. Therefore, the results demonstrated that the use of Ag-NPs together with laser irradiation enhances the antibacterial activity.
3.4. Measurement of Temperature during Irradiation
During laser light irradiation, changes in the temperature of the bacterial suspensions were monitored using a digital noncontact thermometer. The temperature of the bacterial suspensions increased slightly during the irradiation experiments, as shown in Table 1. In particular, the temperature of the bacterial suspensions increased by 1°C after laser irradiation for 10 min compared with that of the nonirradiated samples. Under the same irradiation conditions, the temperature of bacterial suspensions containing Ag-NPs increased by 3°C for S. aureus and 2°C for E. coli.
3.5. ROS Assay
ROS generation is a marker of oxidative stress and causes oxidative damage to cellular components. Therefore, ROS generation is monitored in E. coli and S. aureus by using the fluorescent probe DCFH-DA. DCFH-DA can be used to detect a wide spectrum of different ROS species, including singlet oxygen, super oxide, and hydrogen peroxide, in addition to hydroxyl radicals. Figure 6 shows ROS formation after labeling E. coli with 10 μM DCFH-DA and subjecting them to laser light, Ag-NPs, or photoactivated of Ag-NPs. After 2 h incubation with DCFH-DA, the negative control samples (untreated cells, control-) did not show any significant increase in the fluorescence intensity, indicating a lack of H2O2 formation in the bacterial suspensions. Therefore, the background fluorescence of the control- group was assumed to reflect the endogenous ROS. In contrast, the positive control (control+) obtained by treating the cells with 0.8 mM H2O2 showed much higher fluorescence. Treatment with Ag-NPs alone did not induce significant ROS formation at concentrations of either 25 or 50 μg/ml. The level of ROS was slightly increased at 5 and 10 min exposure times in cells irradiated with only laser light in comparison with the control samples.
In contrast, treatment with photoactivated Ag-NP concentrations at concentrations of 25 or 50 μg/ml and 5 or 10 min exposure time resulted in a detectable increase in fluorescence intensity, indicating formation of ROS. Laser activation of Ag-NPs at a concentration of 50 μg/ml for the 10 min exposure time induced highly significant ROS generation, approximately twofold higher than that of the negative control sample, suggesting the presence of significant oxidative stress. In comparison, ROS generation was significantly lower at 50 μg/ml when cells were treated with Ag-NPs alone. These results demonstrate that ROS formation increased with increasing exposure time to laser light and concentration of Ag-NPs.
Figure 7 shows ROS formation levels in S. aureus cells treated with laser light or photoactivated Ag-NPs. After 2 h incubation with DCFH-DA, the negative control samples (untreated bacterial cells, control-) again did not display any increase in fluorescence. However, when the cells were treated with Ag-NPs (25 and 50 μg/ml) or laser light (5 and 10 min) alone, ROS production slowly increased within the first 2 h. Bacterial cells of S. aureus treated with both laser light and Ag-NPs at 25 and 50 μg/ml also showed higher levels of ROS formation compared with the control cells. The maximum level of ROS formation was observed with 50 μg/ml of Ag-NPs and 10 min exposure to laser light. The results demonstrated that the use of photoactivated Ag-NPs increased ROS production.
The level of ROS formation in E. coli cells after treatment was higher than that of S. aureus cells. In other words, E. coli cells were more sensitive to Ag-NPs after photoactivation by laser light. In E. coli, treatment with photoactivated Ag-NPs induced ROS concentrations that were two times higher than that of the negative control (). ROS production appeared to be dependent on the laser exposure time and Ag-NP concentration as observed previously in the growth inhibition assay. The photoactivated Ag-NPs enhanced a remarkable increase in ROS formation against S. aureus cells after treatment.
3.6. Observation of Morphology by SEM
SEM images were used to examine the membrane surface integrity of bacterial cells before and after exposure to the photoactivated Ag-NPs, as well as to determine any changes or damage to cell membranes caused by the treatment for different exposure times. Figure 8 presents SEM images of E. coli cells. The images show that untreated cells were typically rod shaped with smooth and intact cell walls. Immediately after exposure to photoactivated Ag-NPs, Ag-NPs were observed to adhere to the surface of the bacteria accompanied by simultaneous shrinkage of the cells and leakage of intracellular components. Regarding morphological alterations, no differences were detected between the control group and cells irradiated for 1 or 3 min when treated with 12.5 μg/ml Ag-NPs. For cells treated with 25 μg/ml Ag-NPs and exposed to laser light for 3, 5, or 10 min, damage to the cell wall was significant. Increasing the concentration of Ag-NPs to 50 μg/ml always resulted in damage after laser light irradiation, regardless of the exposure time.
The SEM images in Figure 8 show that the morphology of the S. aureus cell was round shaped, with an intact membrane and smooth cell wall before treatment. Some structural damage was visible after the cells had been treated with 12.5 μg/ml Ag-NPs for 2 h and exposed to laser light. No damage was evident after treatment for 1 and 3 min irradiation, but cell ruptures were prominent following 5 and 10 min exposure. Moreover, cells exposed to 25 and 50 μg/ml showed severe damage to the bacterial structure when exposed to laser light for 10 min. The S. aureus cell walls became wrinkled and damaged. The shape and size of the cells also dramatically changed, and a number of particles attached to the bacterial surface. Treatment with 50 μg/ml of Ag-NPs caused more structural changes to the bacterial cells than with 25 and 12.5 μg/ml Ag-NPs, as can be seen in the SEM images after 10 min exposure.
Based on these results, the photoactivated Ag-NPs seemed to affect E. coli and S. aureus cells differently. The results provide evidence of damage to the membrane in both bacteria, i.e., rupture of the cytoplasmic membrane and internal organization, leading to leakage of the cytoplasmic contents. The results of SEM analysis supported the findings on ROS formation and bacterial growth inhibition after treatment with different concentrations of Ag-NPs and laser exposure times.
This study set out to assess the impact of blue light (410 nm) on the antibacterial efficiency of Ag-NPs against E. coli and S. aureus cells. The results showed that Ag-NPs exposed to laser light exhibited a stronger antibacterial effect compared to unexposed Ag-NPs. Laser irradiation alone had only a slight effect in both strains. It is interesting to compare the observations of this research with other studies reporting phototherapeutic applications based on metal NPs. Akram et al.  studied the antimicrobial effects of combining Ag-NPs, visible blue light, and antibiotics against methicillin-resistant S. aureus (MRSA). Their findings indicated that the antimicrobial activity against MRSA isolates was enhanced by using a combination of Ag-NPs, laser light, and antibiotics. The combined treatment had positive synergistic antibacterial effects, when compared with antibiotic treatment combined with either laser light or Ag-NPs alone. Nourbakhsh and Ashjaran  studied the effect of laser light on cotton fabrics coated with Ag-NPs against E. coli and S. aureus and concluded that the presence of Ag-NPs prevented the growth of bacteria on cotton fabrics after laser irradiation.
Panas et al.  reported that excitation of Ag/TiO2 NPs by blue laser light at 455 nm for 5 min inhibited the proliferation of Streptococcus salivarius due to high photocatalytic activity that irreversibly damages cell walls and membranes. In another study, Fuertes et al.  analyzed the antibacterial activity of Ag-NPs coated with porous silica and irradiated at their SPR frequency of 387 nm, which induced production of toxic ROS and subsequent cell death in E. coli. They suggested that a possible mechanism for the synergistic effect of Ag-NPs with laser light is the production of cytotoxic ROS by the visible blue light resulting in destruction of the bacterial cell membrane.
In the present study, exposure of the Ag-NPs to laser irradiation was mediated by the SPR originating at the Ag-NP surface. The surface of the metal was expected to absorb the light and convert this energy into a photochemical effect, forming cytotoxic ROS and enhancing the antibacterial effect [21, 22]. Our results demonstrated that a significant activation energy is generated in Ag-based materials upon exposure to laser irradiation, which in turn causes a notable antibacterial effect. During the irradiation experiments, temperature changes were monitored in the bacteria suspensions. Only slight increases in temperatures were observed during irradiation. Therefore, heat effects were considered negligible.
Hence, the observed effects were most likely related to the plasmon induced at the surface of the Ag-NPs, which may initiate photochemical reactions and affect the survival viability of bacterial cells. A number of possible mechanisms for the action of photoactivated Ag-NPs on bacterial cells can be proposed based on the observations during the experiments. It is important to note that the amount of ROS produced in the illuminated bacteria correlated with the phototoxic effect. ROS production by two bacterial strains was measured using a DCFH-DA probe. Once inside cells, DCFH-DA is hydrolyzed to DCFH by cellular esterases, which becomes trapped inside the cells. Upon oxidation by ROS, the nonfluorescent DCFH is converted to fluorescent DCF . After irradiation with blue laser light in the presence of Ag-NPs, the fluorescence results showed an increase in hydrogen peroxide (H2O2) formation in both the Gram-negative and Gram-positive bacteria. A possible mechanism for the observed synergy effect could be production of highly cytotoxic ROS in a similar manner to photodynamic therapy (PDT).
PDT involves the use of exogenous photosensitizers, such as hematoporphyrin derivatives, which are introduced to the cells and then irradiated with an appropriate wavelength of visible or near infrared (NIR) light [13, 23]. The activated photosensitizer molecules pass their energy to the surrounding molecular oxygen, resulting in the formation of ROS .
Our study suggests that exposure to high-intensity visible light for 10 min can generate sufficient ROS to kill bacteria.
Ratti et al.  suggested that the mechanism of photoactivated Ag-NPs irradiated with laser light involves the release of Ag+ ions due to an increase in thermal energy and such ions can nucleate to form small Ag-NPs.
It is well known that the toxicity of visible light irradiation is due to the photochemical production of ROS. Toxic ROS can be formed from oxygen bound to or in the neighborhood of bacterial endogenous chromophores, such as porphyrins and flavins, when they are excited by laser light in the region of 320 to 450 nm. In turn, ROS react with vital molecular components of cells, including DNA, membrane components, lipids, and proteins, leading to growth delay and eventual cell death [25–27]. Thus, for an improved understanding of the antibacterial mechanism, it is necessary to elucidate which cellular components are directly affected by ROS. In this study, the cell membrane was expected to be one of the main targets of ROS generation under oxidative stress conditions induced by irradiation. Therefore, the morphology of the bacterial cell wall upon irradiation of Ag-NPs was analyzed. When E. coli and S. aureus were treated with Ag-NPs exposed to laser irradiation, visible changes and damage to the cell membrane morphology occurred, resulting in cell death.
Several mechanisms may be involved in the loss of membrane integrity and physical function, such as disruption of the permeability barrier, enzyme activity, membrane potential, and pump activity associated with the membrane [28, 29]. The present results strongly suggest that alteration of the bacterial membrane permeability via photoactivation of Ag-NPs with laser light may be the main reason for the loss of membrane integrity due to ROS formation. The cellular membrane damage resulting from the photoactivated Ag-NPs may be due to alteration of the membrane lipids, which are one of the major targets of ROS under oxidative stress conditions. ROS generated by photoactivated Ag-NPs may interact directly with unsaturated fatty acids in bacterial membranes and initiate lipid peroxidation, decreasing membrane fluidity, altering membrane components, and disrupting membrane-bound proteins [26, 30]. Comparable results were obtained by Kumar et al.  who reported that cytoplasmic membrane damage occurred in Bacillus cereus and S. aureus strains under LED illumination at 405 nm, due to oxidative damage probably triggered by generation of ROS or free radicals. Based on the results of this study, the effect of photoactivated Ag-NPs was more prominent in E. coli than in S. aureus.
This difference in antimicrobial activity between Gram-positive and Gram-negative microorganisms is often attributed to the structure of their different cell walls. S. aureus may have a stronger defence system against Ag-NPs due to the presence of a thicker cell wall that prevents the action of the Ag-NPs, rendering the bacterium comparatively more resistance to the antimicrobial activity of Ag-NPs . Moreover, the cell wall of Gram-negative bacteria possesses a stronger negative charge than Gram-positive bacteria due to the presence of lipopolysaccharides (LPS), which promotes adhesion of Ag-NPs, causing the bacteria to be more susceptible to Ag-NPs’ antimicrobial action . Hence, electrostatic attraction between negatively charged bacterial cells and positively charged NPs is crucial for the activity of NPs as bactericidal materials .
In this study, irradiation of Ag-NPs was proposed to cause oxidative stress and the generation of ROS leading to destruction of bacterial components. This mechanism for the bactericidal activity of photoactivated Ag-NPs can be explained by the photochemical toxic effect enhanced by visible light, resulting in the production of highly toxic ROS that can kill cells. When Ag-NPs absorb laser light of a wavelength corresponding to the Ag-NP absorption peak in the visible region, energy is transferred to form the short-lived first-excited singlet state. Intersystem crossing can then convert the excited singlet state to form a much longer lived excited triplet state, which can survive long enough to complete chemical reactions as shown in Figure 9.
In the presence of ambient molecular oxygen, the triplet Ag-NPs can react in photochemical reactions involving hydrogen or electron transfer reactions between the excited state PS and other molecules in the environment (usually oxygen). These electron transfer reactions produce (directly or indirectly) ROS that are harmful to cells, such as superoxide (O2⋅-), hydrogen peroxide (H2O2), hydroxyl radicals (HO⋅), and hydroperoxyl radicals (HOO⋅) [32, 35].
In this work, the results showed that the antibacterial activity of Ag-NPs can be significantly enhanced by illumination with blue laser light of 20 nm Ag-NPs with different concentrations to produce a phototoxic effect in both Gram-negative and Gram-positive bacteria. Our findings suggested that Gram-positive S. aureus are more resistant to photoactivated Ag-NPs than Gram-negative E. coli. ROS, such as ⋅OH, O2⋅-, and H2O2 generated when Ag-NP surfaces are irradiated, attack cell components leading to a loss of membrane permeability and ultimately reduced viability. It is important to note that laser light irradiation alone also reduced bacterial viability, as it is absorbed by cellular endogenous PS leading to generation of significant numbers of ROS. The phototoxic effect of photoactivated Ag-NPs was found to be dependent on irradiation time and concentration of NPs. Increasing the exposure time to laser light and concentration of Ag-NPs enhanced bacterial growth reduction.
Combined treatment of Ag-NPs and visible light irradiation for killing bacteria may offer several advantages, such as the need for lower concentration of Ag-NPs and shorter incubation time of cells, which are less toxic to the host tissue. In addition, the use of metal Ag-NPs combined with visible light irradiation opens new possibilities for surface decontamination. Based on these findings, irradiated Ag-NPs are expected to be more efficient nanomaterials for preventing bacterial regrowth than conventional antibacterial agents.
Data generated in this study are included in this article. The raw data generated using statistical analysis is available from the corresponding author on reasonable request.
Highlights. (i) Ag-NP was synthesized by the reduction method and the morphology was uniform with the spherical shape, and its average particle size was . (ii) Photoactivation of Ag-NPs demonstrated that a concentration of 50 μg/ml and 10 min exposure time reduced E. coli and S. aureus populations by 14% and 28%, respectively. (iii) SEM images of treated bacteria showed that substantial morphological changes occurred in cell membranes after treatment. (iv) Gram-positive S. aureus is more resistant to photoactivated Ag-NPs than Gram-negative E. coli. (v) Irradiation of Ag-NPs caused oxidative stress that induced the generation of ROS leading to the destruction of the bacterial cell membrane.
Conflicts of Interest
The authors declare no conflict of interest.
The authors thank Mr. Shafri Bin Semawi for his help in SEM image observation. This study was financially supported by the Iraqi Government Program, Ministry of Higher Education, through a scholarship to Anes Al-Sharqi.
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