Gold Nanoparticles and Laser As Antimicrobial Agents Against Some Types of Bacteria
Maysa S. M. Al-Shukri
(Received: 10 September 2017; accepted: 05 November 2017)
The goal of this studywas to assess the efficacy of gold nanoparticles (AuNPs) and AuNPs– Low level laser combined therapy as antibacterial agents against different types of bacterial isolates in vitro.Five concentrations of AuNPs (25,50,75, 100 and 200 μg/mL) were employed for assessingbacterial growth rate and the bacterial MIC. It was found that there wasslight reducein bacterial growth after treatment with gold nanopracticle.The influence of gold nanoparticles on Staphylococcus aureus biofilms was also studied.It was found that biofilm growth was decreased at different concentrations of AuNPs compared to absence of nanoparticles.Results of the cytotoxic act of AuNPs and low level laser effectin combination againstEscherichia coli and S.aureus referred that the low level laser light improved the antibacterialactionof AuNPs when it used in combination with low level laser
Gold nanoparticles, Laser, Antibacterial agent,Cytotoxicity, MIC.
Nanomaterials become a favorable and effective candidate that can replace standardsresources with maximumuses in all areas of technology and science, due totheir ultra-small size of that have higher ratio of surface area to volume (SA/V ratio) and at their outer surfaces, prolong the number of active atoms,like silver, gold and zinc, each with diversefeatures and wide range of activities1.
Among NPs, AuNPsare manythat applied as a catalyst for gene therapy, medical therapy and biological diagnosticmethods2.The importantbenefit of AuNPs is that they are simple to be made bythe chemicalreduction methods and they have a smalllevel of toxicity when compared withothers nanomaterials. Many element methods have been assumed for different dimensions of nanoparticles and to organizetheir surface to make the uses to beincreased3.
The development of multi-drug resistant (MDR) bacteria and their effect on human health inspiresinvestigators to create new methods to improveactiveantibacterial agents to conquer the bacterial drug resistance and diminish their cost4. One method being recently assessed is photodynamic therapy (PDT), which employsdyes that absorb light to make toxic oxygen radicals to destroy the bacterial cells. Nevertheless, this managementmay be not active for bacterial infections in hypoxic environments. An additionaltalentedmethod is to practice metal nanoparticles, and laser energy to kill the microbes through Photo Thermal Therapy (PTT)5.Low level laser have medical applications such as bio-stimulation, destructive and inhibitory effects6,Laser therapy is considered as a non-invasive treatment method with several special effects and applications in clinical work, including stages of tissue healing7.
Optical characteristics of conductive NPs, likethose make of gold have been connected with Surface Plasmon Resonance (SPR), which as soon asrestricted intiny colloids, is mentioned as localized surface Plasmon resonance (LSPR). This results in wavelength dependent photo thermal effect, in which the electrons oscillate cooperatively when irradiated with specific light energy resonated with its LSPR.
When gold nanoparticles absorb light energy, they emit heatin conformity with. The releasing of such heat makes gold nanoparticles suitable in different photo thermal therapy applications likethose directedagainst bacterial and cancer cells. The death of the bacterial cells is happened as a result to physical disruption of these cells induced by Laser photothermal phenomenon8
This studyaims todetect theeffectiveness of gold nanoparticles and AuNPs–laser induced therapeutic approach (at different energy densities of laser and different concentrations of AuNPs) as antibacterialagents against some pathogenicbacterial strains.
MATERIALS AND METHODS
Synthesis of the gold nanoparticles
Gold nanoparticles were produced as a solution by chemical procedure were 3mM HAuCl4 solution was reduced by the use of10 mM NaBH4 solution under continuous stirring. For additional and complete reduction of the mixture, the mixture was reduced again by 10 mg/ml solution of dextrose. The mixture was exposed to continuous stirring. After that, the mixture was washed numerous times with methanol using centrifugation at 65,000 rpm,AuNPssamples were characterized by using TEM. The size range of gold nanoparticles was 20-30 nm9.
The procedure for antibacterial effectof AuNPswasmade by using some types of bacterial isolates(E. coli and S.aureus)which were supplied by the Department of Microbiology, University of Babylon,collage of medicine, Iraq.
Detection of Antimicrobial Activities of AuNPs.
An aliquot of 20 mL from readily prepared culture medium (holding 105 CFU/mL of bacterial cells) was liquated and dispensed into test tubes,then a volume of 50 μL of these solutions was put into a 96-well plate and mixed with 50 μL of NP solutions in M9, making a final bacterial concentration of 5×105cfu/mL. NPs concentration differs ranging from (25, 50, 75,100, and 200 µg/mL). A growth control group without NPs and a sterile control group with only growth medium were carried out at the same time. For estimation of the inhibition zone and measuring the diameter of inhibitory zones in mm,the method of Shamailaet al.3was followed.
Effect of Nanoparticles on Bacterial Biofilms
Staphylococcus aureus and E. coliwere used for this test.Bacterial biofilm formed in polystyrene microtiter plate overnight cultured bacterial cell were suspended with medium containing AuNPs at different concentrations, ranging as (25, 50, 75,100, and 200 µg/mL).100µl of the suspension were add in to 96–well plates. The plate covered with lid andincubated at 37º Cfor 24 hr.After that the plate were wash with PBS and go to next steps which included the crystal violet staining10.
Analysis of Biofilm Formation with the Crystal Violet Staining
The method of crystal violetstaining11was done for detection of the effect of nanoparticles on the production of bacterial biofilm.
Experimental Design of Laser Irradiation
Bacterial cells were exposed to a diode laser, at a wavelength of about 532nm, outpower of 220mW in a continuous type of wave. The irradiation with 532nm light of laser was made according to standard methods12,13 at the doses of 3.9 ,7.8, 11.6 and 15.6 J/ cm² in term of deposit energy. The treated bacteria were separated into fivegroups: the group 1 as a control (not irradiated); group 2 (3.9 J/cm2); group 3 (7.8J/cm2); group 4 (11.6J/cm2), and group 5 (15.6J/cm2)6.
Antibacterial effect of Laser-induced AuNPs
The MIC for two treated bacteria groupswas carried out from a mixture of bacterial culture and AuNPs (25, 50, 75,100 and 200 µg/mL) and the culture tubes were incubated at a degree of 37°C inshaking incubator for 24 hours.
Earlierto incubation,the culture tubes were exposed to 532 nm laser light, 220 mW, at doses 15.6 J/ cm² for each group as consider the best dose,correspondingly. An aliquot of 50 µL from each culture tube wasspreaderd onto nutrient agar and incubated at a degree of 37°C for 24 h; the numbers of bacterial colonies grown on agar plates were counted. The measurements of optical density at 590 nm of treated bacteria groups were graphed forestimationof the bacterial growth curves14-15.
The Effects of gold nanoparticles on both Gram-negative and -positive bacteria are study, Bacterial strains were cultured with(25, 50, 75, 100 and 200 µg/mL)AuNPs and incubate at 37°C while untreated bacteria was used as a negative control,theOD590 values were monitored from each bacterial strain(E. coli andS. aureus) to each concentration of nanopracticle and the results showed that the influence of the AuNPs on different bacterial growth. The presence of AuNPs slightly inhibited bacterial growth(Figure 1).
Figure 1: Effect of Different Concentrations of AuNPs on the Growth of Bacterial Isolates
In vitro test results reveal significant inhibitory effect on bacterial biofilm production in the presence of the gold nanoparticles. The effect were depend the type of bacteria, nanoparticle, and concentrations of nanoparticle. At a concentration 75, 100 and 200 µg/mL, gold nanoparticles effect showed a reduction of about (13%) in S. aureus biofilm production, whereas in E. coli the nanoparticles showed no effect on biofilm production compared to control. Interestingly, at higher concentrations (75,100 and 200 µg/mL) a significant inhibition in biofilm production (S. aureus )was observed with the presence of gold nanoparticles when compared to its with low concentration (Figure 2).
Figure 2: Effect of Different Concentrations of AuNPs on the Staph aureus Biofilm Formation
The study also showed an inhibitory effect of 532nm diode laser on bacterial growth alone (Figure 3) and in combination with different concentration of gold nanoparticles for dose of 15.6 J/cm2. While the results revealed that S. aureus and E.coli showed no significant difference in growth when irradiated with the 532nm diode laser alone when exposed to different doses(3.9,7.8, and 11.6) J/cm² respectively
Figure 3: Effect of Different Doses ofDiode Laser on the Growth of Bacterial Isolates
The MIC of AuNPs-laser induced was estimated, and it revealed a relative study between applying of AuNPs alone as antibacterial agent and AuNPs-laser combined therapy. Laser enhancement it is a dose depended.The optimal dose was 15.6 J/cm2.It was obviously recorded in the equivalent growth curve (Figure 4).
Figure 4: Effect of Combinations Different Concentrations of AuNPs and LASER (for 20 min) on the Growth of Bacterial Isolates
Nano-methods hada considered worldwideconsiderationdue the fact thatthe nanoparticles have distinctiveand novel features from their bulk equivalents. The antimicrobialeffects of the different nanoparticles had been widely reported by several authors with various and important Gram positive and Gram negative pathogenic bacteria like E. coli and S. aureus10.
Chatterjeeet al.16found that when cultures of E. coli isolates were treated with several concentrations of AuNPs as (25 μg/mL, 50 μg/mL, 75 μg/mL and 100 μg/mL), there was no significant effect in the curve of growth. The experiment of bacterial growth under the influence of the gold nanoparticle, reveals the nontoxic nature of these nanoparticle in the bacterial organism (E. coli). Therefore, it can be appliedin the biological uses with the least probabilities of cytotoxicity.
The bactericidal activity of AuNPsagainst different pathogenic bacterial strains of Gram Gram negative bacteria like Salmonella typhi, Pseudomonasaeruginosa, E. coli, andKlebsiellapneumoniae, are vary, where MICs fluctuated from 20 to 40 µg/mL.Thismay be due to the fact that thicker cell wall of bacteriawilldiminishdiffusion rate of these nanoparticlethrough cell wall (at lower concentrations)and subsequentlyreducing the effective action of gold nanoparticleas antibacterial agents1.
AuNPs have been reported to aggregate on and react with both Gram- negative and Gram-positive bacterial cell membranes, with the inhibition of protein synthesis of bacteria and to preventsynthesis of the cell membranes17.
Shiet al.17found thatthe biofilm production of bacteria increased quicklyat the first 12 hours with the absence of the gold nanoparticles. This increment was inhibited by the nanoparticles atA 6 hours and more significant at 6 to 12 hours with the decrement in bacteria numbers in the biofilm. These results recommended that these nanoparticles are significantly effective in restriction of bacterial colonization in the biofilm.
Several author worldwide who studied the bactericidal effect of laser radiation reported that the radiation absorbed by chromophores may result inchanges in molecules conformation, creating free radicals and reactive oxygen which, subsequently, stimulatedisturbance in the structures of bacterial and fungalmembranes18.
It was reported that the laser light increasesthe antibacterial effect of AuNPsby as a minimum of one fold. This may be referred back to the fact that photo thermal effect causedby the exclusivecharacter of nano scaled gold colloidal liquid mixture that has robustboostedabsorption band at 524 nm equivalent to the AuNPs SPR oscillations. When subjecting to resonating laser emission line, the kinetic energy of AuNPsincreases19.
The createdphotothermalconsequence can be active for fast and effective bacterial cell destruction8. The laser enhancement of the antibacterial action of AuNPsrelysupon the exposure time at which the bacterial cells are treated.These results were corroborated by a previous study that investigated the antibacterial effect of AuNPs-laser,this effect was enhanced by photothermal degeneration in combined approach, AuNPs-laser, results inrapidloss of bacterial cell membrane integrity20.
AuNPs have been found to have a vital and effective revolution for drug delivery.Also, they perform as a non-dangerous and non-toxic antimicrobial agent refers to their functional effective nature when compared with antibiotics.This studycan conclude that AuNPsunited with laser exposure could be employed as confined effective antibacterial because that low level laser increases the antibacterial actionof AuNPsby as a minimum of one fold.
The authors are thankful to Department of Microbiology, College of Medicine, University of Babylon, Iraq, for the facilities provided in the completion of the work.
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