Antimicrobial activity of silver nanoparticles synthesized using honey and gamma radiation against silver-resistant bacteria from wounds and burns

Silver nanoparticles (AgNPs) are promising antimicrobial agents for treatment of wounds and burns. We synthesized AgNPs using honey at different pH values or with different gamma irradiation doses. The resulting nanoparticles were characterized by UV–vis spectroscopy, TEM, DLS and FTIR. Their antimicrobial activity, against standard bacterial strains and silver-resistant clinical isolates from infected wounds and burns, was evaluated in vitro through determination of their minimum inhibitory concentration (MIC). AgNPs prepared using 30 g of honey exposed to 5 kGy gamma radiation had the best physical characters regarding stability and uniformity of particle size and shape. They recorded the lowest MIC values against both the standard and silver-resistant isolates. In conclusion, honey and gamma radiation can be used in synthesis of highly stable pure AgNPs, without affecting the physico-chemical and antimicrobial activity of honey. This offered an advantage in terms of inhibition of silver-resistant bacteria isolates.

its synthesis in the nanoparticle form. Silver nanoparticles (AgNPs) have greater efficiency regarding their microbial inhibitory activity in comparison with other silver compounds [6,7]. The synthesis of Ag in its nanostructure was done by using several physico-chemical protocols, such as metal ablation [8], micro emulsion [9], chemical reduction [10] and electrochemical method [11], which involve the use of hazardous materials that adversely affect human health and environment. Other safer techniques were proposed such as using natural polymers [12], microorganisms [13] and plant extracts [14] in production of AgNPs.
Honey has also been used in the green synthesis of AgNPs. It contains ingredients such as glucose and fructose that allowed its use as both capping and reducing agent in the synthesis process. In addition, it has extraordinary healing properties that are attributed mainly to its acidic pH and the presence of hydrogen peroxide. However, the reduction of silver ions into AgNPs, using honey, occurred only at alkaline pH [15] where these valuable properties of honey were lost.
Gamma radiation has also been used in the synthesis of metallic NPs; such as silver, nickel and copper [16][17][18]. It has several important advantages, including: (i) controlled reduction of metal ions without using excess reducing agent or producing any undesired oxidation products from the reductant, (ii) reducing agent is produced uniformly in medium, (iii) provides metal NPs in fully reduced, pure and highly stable form, (iv) no interfering impurities like metal oxides, (v) synthesis can be carried-out at ambient conditions [16,19].
In this study AgNPs were synthesized using honey, as capping and reducing agent, in combination with different doses of gamma radiation. The antimicrobial activity, of the resulting nanoparticles, was evaluated against standard bacterial strains as well as against different silver-resistant clinical bacterial isolates obtained from wounds and burns.

Microorganisms
Standard bacterial strains, namely Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, were obtained from VACSERA, Cairo, Egypt. Also, 150 clinical bacterial isolates were obtained from different wound sepsis and burns. They were collected from the Central Laboratories, EL-Demerdash Hospital, Ain-Shams University, Cairo, Egypt. These bacterial isolates were identified as S. aureus (n = 38), E. coli (n = 22), Klebsiella pneumoniae (n = 32), P. aeruginosa (n = 21), Acinetobacter spp. (n = 14), Enterobacter spp. (n = 8) and Proteus spp. (n = 15). A total of 19 bacterial isolates were silver-resistant with a silver nitrate minimum inhibitory concentration (MIC) ⩾512 µg ml −1 .  [15], 20 g of honey was dissolved in 80 ml of deionized water. Then 15 ml of this honey solution was added to 20 ml of AgNO 3 solution (1 mM) and stirred well for 1 min. To initiate the reduction of Ag ions, the pH of the mixture was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 using NaOH. The same procedure was repeated using different honey weights (10 g, 30 g, 40 g and 50 g). Changing the color of the system from transparent to yellow indicated the formation of AgNPs. The synthesis was done using the same above procedure; however, the reduction was initiated by exposure to different gamma radiation doses (1, 5, 10, 15, 20, 25 and 30 kGy). The same above procedure was repeated using different weights of honey (10 g, 30 g, 40 g and 50 g). The original pH of honey was kept throughout the preparation at 3.9. After irradiation, the formation of AgNPs was indicated by changing the solution color from transparent to yellow.

Characterization of AgNPs
The physical properties of the synthesized AgNPs were characterized using: 2.4.1. Ultraviolet-visible (UV-vis) spectroscopy. The optical properties of the AgNPs were determined by measuring the absorbance in the range from 190 to 600 nm at a resolution of 1 nm using UV-vis spectrophotometer (JASCO-Japan, model V-560). The honey solution was used as a blank.

Transmission electron microscope (TEM).
The produced AgNPs' structure was analyzed using JEOL 1010 transmission electron microscope, at accelerating voltage 80 kV and 120 kV. The TEM samples were prepared by drop-casting the reaction mixtures on carbon-coated grids and allowed to dry at room temperature, prior to measurement.

2.4.3.
Dynamic light scattering (DLS). The particle size, the size distribution and the peak intensity of the AgNPs was determined by DLS using Zetasizer nano series Nano ZS, Malvern Instrument, UK. The DLS measurements were carried-out for size ranges from 0.1 to 100 nm.

Fourier transform infrared (FTIR).
For the identification of the possible biomolecules responsible for the capping and stabilization of the nanoparticles in the honey solution; measurements were carried-out using FTIR (FTIR spectrum one, Perkin Elmer, Germany). The spectra were scanned in the 400-4000 cm −1 range at a resolution of 4 cm −1 .

Determination of the antimicrobial activity of AgNPs
The antimicrobial activity of the different AgNPs preparations, that were confirmed to have pure, fully reduced and uniform properties, was tested against the standard bacterial strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923) and against the nineteen 19 clinical silver-resistant bacterial isolates. AgNPs solutions were prepared to give a concentration range of 0.84-108 µg ml −1 . Their MIC was determined using broth macro-dilution method [20]. The MIC was recorded as the lowest concentration yielding no visible growth.

Synthesis of AgNPs
3.1.1. Using honey at different pH. It was found that the synthesis of AgNPs was achieved only at alkaline pH = 9, 9.5 and 10. However, no AgNPs were produced at lower pH values. The increase in the used honey concentration allowed the production of AgNPs at the pH = 9 and 9.5. Thus, the color change, and hence the production of AgNPs, was observed with the following combinations of honey/pH values: 20 g/pH 10, 30 g/pH 9.5, 30 g/pH 10, 40 g/pH 9, 40 g/pH 9.5 and 40 g/ pH 10 (figures 1(a)-(c)). While no color change was observed when using either 10 g or 50 g of honey at any tested pH.

Using honey combined with different doses of gamma
radiation. Exposure to different gamma radiation doses stimulated the reduction of Ag + ions to AgNPs. The color change, and hence AgNPs production, was observed when 20 g of honey combined with 25 kGy and when weights of 30 g and 40 g were combined with gamma radiation doses of 5, 10, 15, 20 and 25 kGy (figures 1(d) and (e)). However, no AgNPs were produced using either 10 g or 50 g of honey at any radiation dose tested.

Characterization of AgNPs
3.2.1. Ultraviolet-visible spectroscopy studies. The result of the UV-vis spectroscopic analysis revealed that the AgNPs prepared using 20 g, 30 g and 40 g of honey at pH = 10 produced absorption spectra with sharp peaks (surface plasmon resonance, SPR) at 400 and 460 nm; 415, 435, 455 nm; and 400, 415, 435, 450 nm, respectively. While in case of AgNPs, prepared using gamma radiation, the strong sharp peaks SPR appeared only with the following combination of honey/ gamma radiation; 30 g/5 kGy, 40 g/20 kGy and 40 g/25 kGy at 415 nm, 415 nm, and 400 and 420 nm, respectively. The peak  intensity of the UV-vis spectra was higher in case of the preparations using gamma radiation compared to that prepared at different pH. Other NPs formulae showed broad peaks indicating the formation of low-yield AgNPs with different particle sizes (table 1). The AgNPs prepared using 30 g of honey irradiated with gamma radiation dose of 5kGy produced a very sharp peak with the highest intensity (9.999) indicating the highest yield of AgNPs with a highly uniform particle size (figure 2).

3.2.2.
Transmission electron microscope studies. The transmission electron microscope (TEM) analysis revealed that the AgNPs prepared with 30 g of honey irradiated with 5 kGy gamma radiation yielded the highest well-dispersed spherical NPs over the honey matrix. The TEM image of its honey matrix clearly showed the absence of agglomeration with roughly-spherically shaped NPs and smooth edges (figure 3(a)) compared with that prepared using the same honey weight 30 g/pH 10 ( figure 3(b)).

Dynamic light scattering studies.
The results of dynamic light scattering (DLS) analysis revealed that AgNPs prepared with 30 g of honey irradiated at gamma radiation dose of 5 kGy had the best properties regarding their particle size range and predominant particle size (table 2). They had a particle size range from 2.69-10.10 nm with most predominant particle size of 4.187 nm and good size distribution intensity ( figure 4(a)). However, AgNPs prepared using the same honey weight at pH = 10 had a particle size range of 7.5-21 nm with most predominant particle size of 11.7 nm ( figure 4(b)).

Determination of the antimicrobial activity of AgNPs
The MIC of different AgNPs preparations tested against the standard bacteria and the different silver-resistant bacterial isolates (n = 19) indicated that the AgNPs synthesized using 30 g of honey irradiated with gamma radiation at 5   kGy had the best activity against all tested organisms. The MIC ranged from 1.6875 to 3.375 µg ml −1 and 1.6875 to 6.25 µg ml −1 against the standard bacteria and tested clinical isolates, respectively (table 3). It was also revealed that the synthesized AgNPs were more active against the tested Gramnegative bacteria than Gram-positive ones.

Discussion
The synthesis of AgNPs was successful only at alkaline pH. This was in accordance with that reported by Haiza et al [15] where NaOH solution was used as a pH regulator. The addition of NaOH increased the pH of the solution. At the same time, it allowed the production of more gluconic acid from glucose resulting in a rapid reduction of Ag ions and formation of a large number of small NPs. This accounted for the decrease in the size of produced AgNPs with NaOH addition and pH increase. The use of gamma radiation allowed successful production of AgNPs, in presence of honey and maintained its acidic pH. AgNPs were produced at gamma radiation doses ranging from 5 to 25 kGy, depending on the used honey weight. This was in accordance with the study of Rao et al [16] where AgNPs were successfully produced using gamma radiation in a range 1-24 kGy along with gum acacia. Also, our results indicated that the use of higher honey weight led to smaller size of AgNPs. According to Spinks and Woods [22], the exposure of aqueous solutions to γ-radiolysis allowed the production of the solvated electrons and H· atoms, which act as strong reducing agents. They effectively can reduce Ag + ions, which is the main process in the formation of NPs. Therefore, gamma radiation's use allowed the maintenance of the acidic honey pH, which is highly advantageous for the healing and antimicrobial activities of honey.
The physical characterization of AgNPs proved the superiority of AgNPs prepared using 30 g of honey at 5 kGy radiation dose. In the UV-vis spectrophotometric analysis, the colloidal AgNPs exhibit absorption peak in the wavelength from 400-450 nm range depending on the complex di electric constant of the metal, the cluster size and the environment [16]. Also, the spectroscopical range depends strongly on the size, shape and functionalization of the metallic NPs [6].The AgNPs synthesized using 30 g honey irradiated with gamma rays at 5 kGy exhibited a sharp peak at 415 nm only with the highest intensity. This indicated the formation of a uniformly sized AgNPs in spherical or nearly spherical shape with highest yield, compared to other tested preparations.
The superiority of the physical characters of the AgNPs prepared using honey at 5 kGy gamma irradiation dose was confirmed using the TEM and DLS. TEM confirmed the absence of agglomeration of AgNPs in this preparation. Also, DLS results revealed the homogeneity and small particle size of the produced nanoparticles. Sizes and shapes of NPs are influenced by number of factors including pH, precursor concentration, reductant concentration and time of incubation, temperature as well as method of preparation [23]. The superiority of this AgNPs preparation indicated that the use of gamma radiation allowed the production of AgNPs with super ior physical properties compared to those produced using the same amount of honey at elevated pH.
The results of FTIR indicated the importance of honey ingredients in stabilizing the produced nanoparticles where different types of honey biomolecules were responsible for the stabilization and capping of the AgNPs within the matrix. The stabilization of 30 g/5 kGy occurred through both the free amine groups and the -COO − (carboxylate ion) groups of amino acids residues with free carboxylate groups in the protein. On the other hand, other AgNPs preparations were stabilized only through the-COO − (carboxylate ion) groups of amino acids residues with free carboxylate groups in the protein. The binding of honey proteins to AgNPs using both the free amine groups and carboxylate ion of the amino acid residue was previously reported [15,24,25].
Also, the AgNPs synthesized using 30 g of honey irradiated at 5 kGy gamma irradiation dose recorded the highest antimicrobial activity against both the standard and the silverresistant clinical bacterial strains obtained from wounds and burns. This can be accounted for in terms of the superior physical properties of this preparation where the effect of AgNPs is highly influenced by their size, shape and concentration [7]. The smaller particle size the larger surface area to volume  ratio and the higher the antimicrobial activity [26]. Its superiority can also be attributed to the presence of honey at acidic pH. Where, it is well-known that the antimicrobial activity of honey depends mainly on its acidic pH [27] and the use of gamma radiation doses in a range from 5 to 15 kGy keeps the physical and chemical properties of honey unchanged, especially its low pH [28]. Therefore, the use of gamma radiation is highly advantageous in allowing the production of AgNPs with the smallest particle size (4.187 nm) and maintaining the antimicrobial activity of the honey. In accordance with previous studies, the synthesized AgNPs were more active on Gram negative bacteria than Gram positive ones [29,30]. This difference in activity may be attributed to the difference in the cell structure between the Gram positive and negative bacteria, where the thicker cell wall of Gram positive allow less silver entry to the cytoplasmic membrane than the Gram negative [31].

Conclusion
In conclusion, the green synthesis of AgNPs using honey and gamma radiation allowed the production of nanoparticles with a uniform small particle size in a highly-stable state without using harsh conditions. This method kept the physico-chemical and antimicrobial properties of honey unchanged. These AgNPs were highly active against the different silver-resistant bacterial isolates obtained from wounds sepsis and burns. Therefore, they could be used as an effective treatment in resistant wound sepsis and burn infections.