Antibacterial activity and characteristics of silver ...
Antibacterial activity and characteristics of silver ...
UV'Vis spectra analysis and color change
The visual color change from pale yellow to dark brown in response to time can be seen as evidence of silver ion reduction to AgNP. The change in color of biosynthesized AgNP is due to the excitation of surface plasmon resonance (SPR). Several studies done on the synthesis of AgNP via medicinal plant suggest the absorption peak around 412'470 nm with the duration of synthesis from 4 h till 24 h, these include medicinal plants, such as Abutilon indicum, Aegle marmelos, Azadirachta indica, Calliandra haematocephala, Calotropis procera, Carica papaya, Helicteres isora, Lawsonia inermi, Leptadenia reticulate, Rheum palmatum, Tecomella undulata, Tagetes erecta, Urtica dioica. The rate of color change from light yellow to dark brown varied in these studies, the earliest color change began within 1 h till 4 h4,20,23,24,30,31,32,33,34,35,36,37,38. Alternatively, different studies utilizing non-medicinal plants for the AgNP synthesis, such as Allium cepa, Chenopodiastrum murale, Cyperus rotundus, Eleusin indica, Euphorbia hirta, Melastoma malabathricum, Musa acuminate, Pachyrhizus erosus, Rubus glaucus exhibited absorption peak from 401'780 nm and was synthesized for 72 h till 14 days. The color change of AgNP synthesized via C. murale turned to brown color after incubating overnight39,40,41,42,43. The difference in color change rate might be due to the different properties of the plant, specifically, the medicinal plant contains a wide range of phytochemicals, such as flavonoids, polyphenols, terpenoids, etc.44 that assist in the formation of silver nanoparticles. Iravani et al.5 reported in their studies that flavonoids, polyphenols, terpenoids, alkaloids and proteins are the main constituents responsible for the reduction and stabilization of silver nanoparticles. Figure 1 shows the result of color change of the synthesized silver nanoparticle with different organs of Carduus crispus, such as stem, flower and the whole plant. It can be seen that different plant organs affected differently on silver nanoparticle synthesis, and particularly whole plant extract facilitated better silver nanoparticle formation compared to the stem and flower extract. The synthesis of silver nanoparticles with whole plant extract exhibited a darker color change. The variation in color change might be due to the different phytochemical content in the plant organs. Following the visual color change study, the formation and stability of silver nanoparticles synthesized with flower, stem, and whole plant of Carduus crispus were characterized using a UV'Vis spectrophotometer (Fig. 2). The results revealed that silver nanoparticles synthesized with whole plant (AgNP-W) exhibited higher absorption compared to silver nanoparticles synthesized using plant organs such as flower (AgNP-F) and stem (AgNP-S). The higher absorption is directly proportional to the higher yield of silver nanoparticles in colloidal solution45. Additionally, the size of the synthesized silver nanoparticle was studied by observing the shift of the absorption peak towards a longer or shorter wavelength8,46. In Fig. 2 b-d, silver nanoparticles were measured at various times, and according to our results, the AgNP-W exhibited blueshift in contrast to AgNP-F and AgNP-S, which can be interpreted as the formation of smaller-sized silver nanoparticles.
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Figure 1Color changes in biosynthesized silver nanoparticle with different parts of Carduus crispus. S-stem, F-flower and W-whole plant.
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Figure 2UV'Vis spectra for the reaction mixture containing of silver nanoparticles synthesized from Carduus crispus flower (AgNP-F), stem (AgNP-S) and whole plant (AgNP-W). Shown are the UV'Vis absorption spectra from 370 to 700 nm of all plant organs and synthesized (A) AgNPs, (B) AgNP-W, (C) AgNP-S, and (D) AgNP-F.
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Zeta potential analysis
Zeta potential explains the stability, dispersion and surface charge of the nanoparticles. The zeta potential greater than'+'30 mV or less than ''30 mV indicates high stability of nanoparticles in dry powder form31. The high negative value produces repulsion between similarly charged particles in suspension, therefore resisting aggregation47. Several studies were done on silver nanoparticle synthesis with a medicinal plant such as Potentilla fulgens, Alpinia calcarata, Pestalotiopsis micospora, Urtica dioica, Jatropha curcas which resulted inzeta potential of ''18 mV, ''19.4 mV, ''35.7 mV, ''24.1 mV, and ''23.4 mV respectively4,6,12,47,48. Our results showed that zeta potential of the synthesized AgNP-W, AgNP-S, AgNP-F had an average zeta potential of ''46.0 2'±'4.17 (AgNP-W), ''54.29'±'4.96 (AgNP-S) and ''42.64'±'3.762 (AgNP-F) (Table 1). The zeta potential of AgNP-S exhibited a higher average value compared to the AgNP-W and AgNP-F, this may be due to the presence of different phytochemicals in each sample that reduces and cap silver nanoparticles. The results of the zeta potential analysis suggest that silver nanoparticles synthesized with Carduus crispus exhibit high stability and resist agglomeration. Figure 3 showed that zeta potential values of AgNP-W, AgNP-S, and AgNP-F fall within the normal distribution curve, which indicates that synthesized silver nanoparticles are fairly monodisperse.
Table 1 Average zeta potential and mobility of AgNP-W, AgNP-S and AgNP-F.Full size table
Figure 3Zeta potential analysis of (A) AgNP-W, (B) AgNP-F and (C) AgNP-S.
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FTIR spectral analysis of synthesized AgNP by Carduus crispus
The presence of the functional groups capping AgNP synthesized using Carduus crispus is analyzed by FTIR and shown in Fig. 4. The presence of various organic compounds in the plant reveals multiple peaks compared to the chemical method where only a few and strong peaks are displayed49,50. The results of our FTIR analysis showed the presence of several functional groups in AgNP-W, AgNP-S, AgNP-F. Additionally, the functional groups in AgNP-F and AgNP-S were present in AgNP-W samples as well, this may be attributed to the various phytochemicals capping the silver nanoparticles that are found both in flower and stem of Carduus crispus. The strong characteristic bands at'~' cm'1 to cm'1 and cm'1 in all samples AgNP-S, AgNP-F, AgNP-W are assigned to the O'H stretching/N'H stretching of amides and cm'1 to the C'C stretching. Additionally, the weak band at'~' cm'1 to cm'1 and'~'828 cm'1 assigned to carbohydrates and 'C'='O bending were found in all samples AgNP-S, AgNP-F, and AgNP-W. C'O stretching is present in AgNP-F which was observed from the very strong band at cm'1. The weak bands at cm'1 and cm'1 of CH3 stretch of alkane/carboxylic acids present in AgNP-F and were absent in AgNP-S. The band detected at'~' cm'1 to cm'1 and .35 cm'1 correspond to the presence of phenolic compounds and flavonoids, and the band found on .35 cm'1 indicates carboxylic acid, ester, and ether groups of proteins and metabolites that may be involved in the synthesis of nanoparticles33. Our result show that the strong band detected at cm'1 and cm'1 from AgNP-F correspond to the presence of flavonoids and proteins. On the other hand, weak bands detected at'~' cm'1 to cm'1 correspond to alcohol, carboxylic acids, alkyl halides/carboxylic acids/ester, alkenes/alkyl halides/aromatics, alkynes/alkyl halides stretch that peaks found from AgNP-S. According to Baumberger27 the major compounds detected in Carduus crispus are flavonoids and coumarins, in addition, alkaloids, saccharides, essential oil, rubber and lipids contained in small quantities which is in line with the presence of flavonoids and phenolic compounds in our synthesized AgNP. The AgNP-F and AgNP-S contained different functional groups that correspond to various compounds, and AgNP-F revealed that it has a strong correlation with flavonoids from Carduus crispus. The results of FTIR and UV'Vis spectra analysis confirm that these functional groups are the capping and reducing agents responsible for the synthesis of AgNPs.
Figure 4Fourier transform infrared spectra of (a) AgNP-W, (b) AgNP-S, and (c) AgNP-F.
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XRD, PCCS, SEM/EDX and AFM analysis
The crystalline nature of the synthesized AgNP was confirmed by X-ray crystallography. The XRD pattern of the nanoparticles was analyzed with an XRD instrument and shown in Fig. 5. Bragg reflection of the 2θ peaks was observed at 32.25˚ to 81.62˚ and corresponded to (111), (200), (220), (311), (222) plane lattice which can be indexed to the face-centered cubic crystal nature of the silver. The average crystallite size was calculated using the Scherrer equation. The average crystallite sizes were 13 nm (AgNP-F), 14 nm (AgNP-W) and 36 nm (AgNP-S). The results of our study are in line with other published literature, the crystal nature of silver nanoparticles synthesized with Tagetes erecta 31, Urtica dioica4, Aegle marmelos was face-centered cubic with diffraction peaks of (111), (200), (220), (311) respectively 34. PCCS is a technique based on the Brownian motion that measures the average nanoparticle size (grain size). In Fig. 6, the average particle size of AgNP-W, AgNP-F and AgNP-S was 99.6 nm, 22.5 nm and 145.1 nm respectively. The difference between PCCS and XRD analysis lies in the measurement method of the particle. Application of the Scherrer equation on XRD data gives the average crystallite size, specifically the size of a single crystal inside the particle or grain. The morphological and elemental analysis was done on Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX). The elemental composition of the synthesized silver nanoparticle was assessed using EDX spectroscopy (Table 2). The results in Fig. 7 showed that AgNP-W, AgNP-S, and AgNP-F contained silver and potassium elements together with several other elements that differed in AgNP-F and AgNP-S samples, i.e. AgNP-F included phosphorus 2.8%, potassium 15.2%, and AgNP-S had calcium 7.5%, pottassium 15.5% elements. In contrast, AgNP-W contained all the elements including the elements that differed in AgNP-F and AgNP-S. Interestingly, the silver element in AgNP-F had the highest content of 82% compared to AgNP-W and AgNP-S which had a silver content of 79% and 77% respectively. Another observation on EDX analysis revealed that AgNP-W, AgNP-F, AgNP-S did not show the presence of nitrogen peak, this indicates that trace ions from AgNO3 are absent in the samples. The size of biosynthesized AgNP-W, AgNP-F and AgNP-S was determined with Atomic Force Microscopy (AFM). Figure 8 show that the size of nanoparticles differed, for instance, AgNP-W had a size of 70 nm, AgNP-F with size 33 nm and AgNP-S with size 131 nm. Figure 8 (A-C, E'G and I-K) represents the two dimensional images of AgNP-W, AgNP-F and AgNP-S. Figure 8 (D, H and L) shows the three dimensional image of AgNP-W, AgNP-F and AgNP-S respectively. The different composition of plant organs, such as stem, flower and whole plant could be the reason for the observed variability in, color change, UV'Vis absorption, EDX, FTIR. In addition, the results of AFM data and XRD show that the synthesis of AgNP can be manipulated with different plant organs.
Figure 5XRD spectra of (a) AgNP-W, (b) AgNP-F, (c) AgNP-S. Peaks are appeared at 111, 200, 220, 311 and 222.
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Figure 6PCCS analysis: particle number distribution of synthesized AgNP-W (A), AgNP-F (B) and AgNP-S (C).
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Table 2 Elemental composition of the synthesized silver nanoparticles by Carduus cripus.Full size table
Figure 7EDX spectra for (A) AgNP-F, (B) AgNP-S and (C) AgNP-W along with SEM image area (inset).
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Figure 8Atomic force microscopy images (2D and 3D) of silver nanoparticles on siliconized cover slide; AgNP-W (A'D), AgNP-F (E'H) and AgNP-S (I'L).
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Antibacterial activity
The antibacterial activity of silver nanoparticles was studied against pathogenic bacterial strains of gram-negative E.coli and gram-positive M.luteus using the well diffusion method (Fig. 9). Standard antibiotics such as Penicillin G and Chloramphenicol, plant extracts, AgNO3 and distilled water were chosen as the control group. The results of the antibacterial activity showed that all synthesized silver nanoparticles had efficient antibacterial activity against both gram-negative E.coli and gram-positive M.luteus bacterial strains. The inhibition zone of AgNP-F, AgNP-W and AgNP-S against E.coli and M.luteus were 6.5'±'0.3, 6'±'0.2, 5.5'±'0.2 and 7.5'±'0.3, 7'±'0.2, 7.7'±'0.4 mm respectively. The plant extract and AgNO3 did not reveal any antibacterial activity against both E.coli and M.luteus, which can be interpreted that AgNP-W, AgNP-F, and AgNP-S are solely responsible for the antibacterial activity. The mode of action of AgNPs against bacteria is not completely understood yet. However, several hypotheses are explaining the antibacterial activity of silver nanoparticle: (1) generation of reactive oxygen species; (2) release of Ag'+'ions from AgNPs denaturize proteins by bonding with sulfhydryl groups; (3) attachment of AgNPs on bacteria and subsequent damage to bacteria4,11,24. The multiple published reports on the antibacterial activity of silver nanoparticles against gram-negative and gram-positive bacteria showed that silver nanoparticles had a slight antibacterial activity on gram-positive bacteria6,22,31,36. Interestingly, AgNP synthesized by Carduus crispus exhibited effective inhibition on both gram-positive and gram-negative bacteria which can be interpreted that the antibacterial activity of silver nanoparticles (AgNP-W, AgNP-F and AgNP-S) is not affected by the difference in the bacterial wall.
Figure 9Petri dishes showing the zone of inhibition of synthesized AgNP-W on (A) M. luteus and (B) E. coli, and AgNP-F on (C) M. luteus and (D) E. coli, AgNP-S on (E) M. luteus and (F) E. coli (AgNP: silver nanoparticle, AgNO3: silver nitrate, DW: distilled water, PE: plant extract).
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In-vitro cytotoxicity assay
Cytotoxicity is considered as an important indicator for cell viability, therefore in this study we employed crystal violet assay to investigate the effect of different concentration of AgNP-W, AgNP-F and AgNP-S on the adherent human hepatoma cell line HepG2 (Fig. 10). The liver is an important organ with detoxifying effect, additionally, it is considered as an accumulation site for AgNPs51. In this study, the untreated HepG2 cell lines revealed significant adherence to the well plate. On the other hand, the treated cells with nanoparticles exhibited small decrease in cell viability after 24 h incubation at 3 to 17 µg/ml. The cell viability of these treated groups with AgNP-W, AgNP-F and AgNP-S were 87.93'±'4.87%, 92.24'±'1.21% and 86.20'±'2.43% at 17 µg/ml. The toxicity of AgNPs to bacteria and human cells is widely known, however, the result of our study suggests that AgNPs synthesized by medicinal plant Carduus crispus with concentration of 3 to 17 µg/ml have low toxicity on HepG2 cell line (Fig. 10A,B). In addition, biosynthesized silver nanoparticles possessed efficient antibacterial activity against Gram-negative and Gram-positive bacteria (Fig. 9). The antibacterial activity of the synthesized AgNPs and their low toxicity to human cells may enable further application in biomedical field. The low toxicity of biosynthesized AgNPs to adherent human cells are similar to other published reports52.
Figure 10A microscopic pictures of HepG2 cells treated with AgNPs for 24 h in cell culture: control (A), AgNP-W (B), AgNP-F (C) and AgNP-S (D). After 24 h, the cell toxicity effect was examined with Crystal Violet (E).
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In Vitro Antimicrobial Activity of Green Synthesized Silver ...
Silver nanoparticles (AgNPs) used in this study were synthesized using pu-erh tea leaves extract with particle size of 4.06 nm. The antibacterial activity of green synthesized AgNPs against a diverse range of Gram-negative foodborne pathogens was determined using disk diffusion method, resazurin microtitre-plate assay (minimum inhibitory concentration, MIC), and minimum bactericidal concentration test (MBC). The MIC and MBC of AgNPs against Escherichia coli , Klebsiella pneumoniae , Salmonella Typhimurium, and Salmonella Enteritidis were 7.8, 3.9, 3.9, 3.9 and 7.8, 3.9, 7.8, 3.9 μg/mL, respectively. Time-kill curves were used to evaluate the concentration between MIC and bactericidal activity of AgNPs at concentrations ranging from 0×MIC to 8×MIC. The killing activity of AgNPs was fast acting against all the Gram-negative bacteria tested; the reduction in the number of CFU mL -1 was >3 Log 10 units (99.9%) in 1'2 h. This study indicates that AgNPs exhibit a strong antimicrobial activity and thus might be developed as a new type of antimicrobial agents for the treatment of bacterial infection including multidrug resistant bacterial infection.
Introduction
Recently, nanotechnology has emerged as a dynamically developing area of scientific interest in the world. Nanoparticles are defined as a nanoscale particle of size ranging from 1 to 100 nm. Among the metallic nanoparticles, silver nanoparticles (AgNPs) have gained increasingly attention due to its unique physical, biological and chemical properties. AgNPs are well-known to exhibit a strong antimicrobial activity against various microorganisms such as bacteria, viruses, and fungi due to its smaller in size and large surface area (Franci et al., ). AgNPs are also widely used as anti-fungal (Medda et al., ), anti-inflammatory (Hebeish et al., ), and anti-viral properties (Bekele et al., ).
Green synthesis of AgNPs employing either biological microorganisms or plant extracts has emerged as a simple and alternative to chemical synthesis. Green synthesis method provides advancements over chemical methods as it is environmental friendly and cost effective. Plant extracts-mediated synthesis of AgNPs can be advantageous compared with other biological processes as it does not require the process of maintaining the cell cultures and aseptic environments (Loo et al., ). Several studies on the green synthesis of AgNPs using plant extracts have been reported (Medda et al., ; Ahmed et al., ; Dhand et al., ; Selvam et al., ).
Foodborne illnesses have emerged as a major public health concern around the world. WHO () reported that there is about 30% of the population in industrialized countries affected by foodborne diseases every year. The consumption of foods contaminated with foodborne pathogens such as bacteria, fungi, viruses, and toxins are often recognized as the main source of foodborne illness in humans. Food especially minimal-processed food can be contaminated during pre-harvesting, post-harvesting, processing, transport, handling, or preparation. The most common foodborne pathogens found in food are Salmonella spp. (Lee et al., ; D'Ostuni et al., ), Listeria spp. (Ferreira et al., ; Välimaa et al., ), Escherichia coli O157 (Heiman et al., ), Campylobacter spp. (Kaakoush et al., ), and Clostridia spp. (Chukwu et al., ).
The presence of multidrug resistance pathogens have increased the number of infectious disease and became the main cause of death in the world (WHO, ; Tanwar et al., ). Widely misuse and abuse of antibiotics are the leading cause of antibiotic resistance in the bacteria (O'Bryan et al., ). Multidrug resistant bacteria infection may lead to several impacts including increase of mortality and morbidity rates, prolong of hospitalization period, and economic loss (Patel et al., ). Woh et al. () detected multi-drug resistant non-typhoidal Salmonella among migrant food handlers, which may cause cross-contamination to the food products. Thus, the development of a new and natural antimicrobial agent is needed as there is a growing concern in multidrug resistant foodborne pathogens.
The aim of this study is to determine the antibacterial activity of green synthesized AgNPs against a diverse range of Gram-negative foodborne pathogens by using disk diffusion method, resazurin microtitre-plate assay minimum inhibitory concentration (MIC), minimum bactericidal concentration test (MBC), and time-kill curve assay.
Materials and Methods
Preparation of Silver Nanoparticles
The synthesis of AgNPs using pu-erh tea leaves extracts was done using the method as described previously (Loo et al., ). Ten gram of tea leaves was weighed in a beaker. The tea leaves was added with 100 mL of distilled water and maintained at 60°C for 10 min. After 10 min, the tea extract was filtered using 0.45 μm Millipore membrane filter and followed by 0.2 μm Millipore membrane filter. For synthesis of AgNPs, 12 mL of tea extracts was added into 100 mL of AgNO3 (1 mM) in Erlenmeyer flask at room temperature. Color changes of the solution were observed. The synthesized AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM).
Bacteria Strains Preparation
Escherichia coli ATCC (E. coli), Klebsiella pneumoniae ATCC (K. pneumoniae), Salmonella Typhimurium ATCC (S. Typhimurium), and Salmonella Enteritidis ATCC (S. Enteritidis) were obtained from the American Type Culture Collection (Rockville, MD, United States). All the bacteria strains were cultured in Mueller Hinton broth (MHB) (Merck, Germany) at 37°C for 24 h with 200 rpm agitation.
Preparation of Resazurin Solution
The resazurin solution was prepared at 0.02% (wt/vol) according to Khalifa et al. (). A 0.002 g of resazurin salt powder was dissolved in 10 mL of distilled water and vortexed. The mixture was filtered by Millipore membrane filter (0.2 μm). The resazurin solution can be kept at 4°C for 2 weeks.
In Vitro Susceptibility Test
Disk Diffusion Method
The antibacterial activity of AgNPs against the selected Gram-negative foodborne pathogens was carried out using Kirby'Bauer Disk Diffusion Susceptibility Test method (Bauer et al., ). The bacteria strains were spread on the Mueller-Hinton agar (MHA) (Merck, Germany) using sterile cotton swab. Sterile blank antimicrobial susceptibility disk was used in the test. The disks were loaded with 10 μL of tea leaves extracts, silver nitrate solution (1 mM), and solution containing tea leaves mediated synthesized AgNPs separately. The disks were then placed on the agar plate and incubated at 37°C for 24 h. The zone of inhibition was observed after 24 h of incubation.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Evaluation
The MIC and MBC of green synthesized AgNPs were done using the method described in the guideline of CLSI (). The MIC test was performed in 96-well round bottom microtiter plate using standard broth microdilution methods while the MBC test was performed on the MHA plates. The bacterial inoculums were adjusted to the concentration of 106 CFU/mL. For the MIC test, 100 μL of the synthesized AgNPs stock solution (500 μg/mL) was added and diluted twofold with the bacterial inoculums in 100 μL of MHB started from column 12 to column 3. Column 12 of the microtiter plate contained the highest concentration of AgNPs, while column 3 contained the lowest concentration. Column 1 served as negative control (only medium) and the column 2 served as positive control (medium and bacterial inoculums). Each well of the microtiter plate was added with 30 μL of the resazurin solution and incubated at 37°C for 24 h. Any color changes were observed. Blue/purple color indicated no bacterial growth while pink/colorless indicated bacterial growth. The MIC value was taken at the lowest concentration of antibacterial agents that inhibits the growth of bacteria (color remained in blue).
The MBC was defined as the lowest concentration of the antibacterial agents that completely kill the bacteria. MBC test was performed by plating the suspension from each well of microtiter plates into MHA plate. The plates were incubated at 37°C for 24 h. The lowest concentration with no visible growths on the MHA plate was taken as MBC value.
Time-Kill Curve
Time-kill assay was done in MHB medium as described by Zainin et al. () and Lau et al. (). The bacterial inoculums were adjusted to 106 CFU/mL. The AgNPs solution was diluted with MHB media containing bacterial inoculums to obtain the final concentration of 0× MIC, 0.5× MIC, 1× MIC, 2× MIC, 4× MIC, and 8× MIC for each type of bacteria in the total final volume of 1 mL. The cultures were then incubated at 37°C with 150 rpm agitation. The cultures (100 μL) were spread on MHA plates at time 0, 0.25, 0.5, 1, 2, and 4 h. The experiment was carried out in triplicate. The number of colonies on the MHA plates was quantified in CFU/mL after incubation at 37°C for 24 h. For statistical analysis, SPSS (v.19) statistical package was used to determine the significant (P < 0.05) difference among the tested foodborne pathogens.
Results and Discussion
This study was aimed to determine the antibacterial effect of green synthesized AgNPs. The green synthesized AgNPs used in this study were characterized by UV-vis spectroscopy, XRD, FTIR spectroscopy, and TEM. The XRD patterns for synthesized AgNPs showed that five main characteristic diffraction peaks for Ag were observed at 2θ = 38.4, 44.5, 64.8, 77.7, and 81.7, which correspond to the (111), (200), (220), (311), and (222) crystallographic planes of face-centered cubic (fcc) Ag crystals. The UV-vis absorption spectrum of the synthesized AgNPs showed a broad peak at 436 nm which is a characteristic band for Ag. Three infrared bands were observed at 3,271, 1,637, and 386 cm-1 in FTIR measurement. The band at 3,271 and 1,637 cm-1 indicated that the presence of protein as capping agent for AgNPs which increases the stability of the nanoparticles, while the broad peak at 386 cm-1 corresponded to the Ag metal. TEM image revealed that the AgNPs is spherical with the particle size of 4.06 nm (Loo et al., ).
The antibacterial activity of AgNPs was determined against four species of Gram-negative foodborne pathogens: E. coli ATCC , K. pneumoniae ATCC , S. Typhimurium ATCC , and S. Enteritidis ATCC . The results for disk diffusion test, MIC and MBC of the AgNPs are summarized in Table 1. For the disk diffusion test, the presence of clear zone around the AgNPs disk suggesting that the AgNPs possessed antibacterial activity which is able to inhibit the growth of the Gram-negative foodborne pathogens. As previous study by Guzman et al. (), reported that AgNPs employed antibacterial activity on Gram-negative bacteria. The visible clear zone produced by AgNPs against four different species of Gram-negative bacteria is showed in Figure 1.
TABLE 1
TABLE 1. The diameter of zone inhibition (mm), MIC value (μg/mL), and MBC value (μg/mL).
FIGURE 1
FIGURE 1. Visible clear zone produced by tea leaves extract mediated AgNP against four species of foodborne pathogens: (A) E. coli ATCC , (B) K. pneumoniae ATCC , (C) S. Typhimurium ATCC , and (D) S. Enteritidis ATCC .
Disk diffusion test was described as the preliminary study in screening the antibacterial activity of an antimicrobial agent; therefore, a further evaluation in determining the antibacterial activity of AgNPs using MIC value was needed (Burt, ). MIC was defined as the lowest concentration of the antibacterial agent to inhibit the growth of bacteria by serial dilution. As showed in Table 1, the MIC values of AgNPs against the foodborne pathogens were ranged from 3.9 to 7.8 μg/mL. K. pneumonia, S. Typhimurium and S. Enteritidis showed the MIC value of 3.9 μg/mL while E. coli showed the MIC value of 7.8 μg/mL. MBC is the lowest concentration of antibacterial agent to kill the bacteria (showed no growth on the agar plate). In the study, MBC for K. pneumoniae and S. Enteritidis were 3.9 μg/mL while S. Typhimurium and E. coli showed the MBC value of 7.8 μg/mL. The MIC and MBC value of E. coli showed that E. coli was less susceptible to AgNPs. This may due to the positive charges of AgNPs trapped and blocked by lipopolysaccharide, thus make E. coli less susceptible to AgNPs (Lara et al., b).
Resazurin dye was used in the study as an indicator in the determination of cell growth, especially in cytotoxicity assays (McNicholl et al., ). Oxidoreductases within viable cells reduced the resazurin salt to resorufin and changed the color from blue non-fluorescent to pink and fluorescent. According to McNicholl et al. (), resazurin dye has been applied for decades to check for the bacterial and yeast contamination in milk.
The time kill activity of four foodborne pathogens is shown in Figure 2. The bactericidal activity of AgNPs is effective against the selected Gram-negative pathogens; the reduction in the number of CFU/mL was '3 Log units (99%). The bactericidal endpoint of AgNPs for E. coli was reached after 2 h of incubation at 4× MIC (31.2 μg/mL) and 8× MIC (62.4 μg/mL); while for K. pneumoniae, the bacteria was killed after 2 h of incubation at 2× MIC (7.8 μg/mL), 4× MIC (15.6 μg/mL), and 8× MIC (31.2 μg/mL). S. Typhimurium was killed after 1 h of incubation at 4× MIC (15.6 μg/mL) and 8× MIC (31.2 μg/mL). The bactericidal endpoint of AgNP for S. Enteritidis was reached after 2 h of incubation at 2× MIC (7.8 μg/mL) and 4× MIC (15.6 μg/mL); however, the end point reached faster after 1 h of incubation at 8× MIC (31.2 μg/mL). No significant differences (P > 0.05) were found among the tested Gram-negative foodborne pathogens. This indicates that AgNPs are broad spectrum antimicrobial agents which exert the same effect to all Gram-negative bacteria strains.
FIGURE 2
FIGURE 2. Time-kill plots of AgNPs against (A) E. coli ATCC , (B) K. pneumoniae ATCC , (C) S. Typhimurium ATCC , and (D) S. Enteritidis ATCC at different concentration and time-length.
Silver nanoparticles are well-known as the most universal antimicrobial substances due to their strong biocidal effect against microorganisms, which has been used for over the past decades to prevent and treat various diseases (Oei et al., ). AgNPs are also widely used as anti-fungal (Kim et al., ), anti-inflammatory (Nadworny et al., ), and anti-viral properties (Lara et al., a). Recently, non-hazardous AgNPs can easily be synthesized using a cost-effective method and tested as a new type of antimicrobial agents.
In this study, the application of AgNPs as an antimicrobial agent was tested against selected Gram-negative bacteria on agar plate and liquid medium. The results showed that the tested bacteria could completely inhibit by AgNPs. The inhibition of bacteria growth was reported affected by the concentration of AgNPs and bacteria used in the experiments (Sondi and Salopek-Sondi, ). The green synthesized AgNPs in this study are able to inhibit the high concentration of bacteria (approximately 106 CFU/mL). This indicated that AgNPs showed an excellent antimicrobial effect as the high CFU concentration of bacteria used in this study are rarely appeared in real-life systems.
The antibacterial activity of AgNPs has been reported by many researchers. However, the MIC values from the previous studies showed the range through a large extent of variation. Therefore, the comparison of the results is difficult as there is no standard method for determination of antibacterial activity of AgNPs and different methods have been applied by the researchers (Zarei et al., ). In this study, AgNPs exhibit a good antibacterial activity against the tested foodborne pathogens. Based on the results, the tested bacteria were able to kill in a shorter time at low concentration of AgNPs. This may due to the cell wall structure of Gram-negative bacteria. The characteristic cell wall structure of Gram-negative bacteria is different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains known as the peptidoglycan layer.
The rapid reproduction time of bacteria is one of the main causes of bacteria's infectivity (Lara et al., b). However, the reproduction time of the bacteria could be an ideal way to prevent the viable infection as AgNPs were effective in inhibiting and killing the bacteria in a dose and time dependent manner as shown in the time-kill assays. Zhang et al. () reported that the smaller size of AgNPs could cause more toxicity to the bacteria and show better bactericidal effect compared to the larger particles as they have larger surface area. Previous study by Agnihotri et al. () found that the antibacterial efficacy was increased for the AgNPs with less than 10 nm size. They also concluded that AgNPs with the size of 5 nm have the fastest antibacterial activity compared to others size of AgNPs.
Silver nanoparticles have emerged as antimicrobial agents against multidrug resistant bacteria due to their high surface-area-to-volume ration and unique chemical and physical properties. AgNPs have particle size ranging from 1 to 100 nm. The surface area-to-volume ratio of AgNPs increases as the particles size decreases. Morones et al. () reported that AgNPs with the size of 10-100 nm showed strong antimicrobial effect against both Gram-positive and negative bacteria. The small particle size enables AgNPs to adhere to the cell wall and penetrate into the bacteria cell easily, which in turn improves their antimicrobial activity against bacteria. The antimicrobial effects of AgNPs against multidrug resistant bacteria have been studied by many researchers and it was proved that AgNPs are effective against multidrug resistant bacteria such as multidrug resistant E. coli (Paredes et al., ; Kar et al., ), multidrug resistant strain of Pseudomonas aeruginosa (Durairaj et al., ), methicillin-resistant Staphylococcus aureus (MRSA) (Paredes et al., ; Yuan et al., ), and extended-spectrum β-lactam (ESBL) producing bacteria (Doudi et al., ; Subashini et al., ).
On the other hands, AgNPs are advantageous compared to conventional chemical antimicrobial agents as the major problem caused by the conventional chemical antimicrobial agents is multidrug resistance. The effectiveness of the chemical antimicrobial agents depends on the specific binding of the microorganisms with the surface and metabolites of the antimicrobial agents. However, the chemical antimicrobial agents are limited to use especially in medical field as various microorganisms have developed multiple resistance traits over a period of generations. Thus, the development of AgNPs could be an alternative way to overcome the multidrug resistance microorganisms as bacteria are less likely to develop resistance to metal nanoparticles compared to the conventional antibiotics.
The exact mechanisms of AgNPs against bacteria still remain unknown. However, there are some researchers proposed that the action of AgNPs on bacteria may due to its ability to penetrate into the cell (Sondi and Salopek-Sondi, ), the formation of free radicals (Danilczuk et al., ; Kim et al., ), the inactivation of proteins in the cell by silver ions (Rai et al., ) and the production of reactive oxygen species (ROS) (Dakal et al., ). Besides that, there are also some factors in affecting the bactericidal mechanisms of AgNPs such as the concentration of AgNPs and bacteria class (Kim et al., ; Zhang et al., ), shape (Pal et al., ; Meire et al., ), size (Martinez-Castanon et al., ), and the combination of various antibiotics (Fayaz et al., ; Singh et al., ).
Conclusion
Silver nanoparticles showed significant antibacterial activity against the selected Gram-negative foodborne pathogens. Thus, AgNPs might be a good alternative to develop as antibacterial agent against the multidrug-resistant strains of bacteria. The applications of AgNPs may lead to valuable findings in various fields such as medical devices and antimicrobial systems.
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Author Contributions
YL, BC, YR, and RS developed the study design. YL and CK carried out the confirmation for the selected foodborne pathogens. MN provided culture media and technical advice in the study. YL interpreted the data, drafted the manuscript, and revised the manuscript. YR, M-A-RN-K, CK, BC, and RS checked on the manuscript. All authors read and approved the final version of the manuscript.
Funding
This research was funded by a Research University Grant Scheme Initiative Six (RUGS 6) of Universiti Putra Malaysia (GP-IPS ) and Fundamental Research Grant Scheme (FRGS) of Ministry of Higher Education (MOHE), Malaysia (02-01-14-FR) and, in part, by the Kakenhi Grant-in-Aid for Scientific Research (KAKENHI ), Japan Society for the Promotion of Sciences and grant-in-aid of Ministry of Health, Labor and Welfare, Japan.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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