The antimicrobial activity of silver nanoparticles in vitro

Silver nanoparticles possess a high potential as an antimicrobial substance against a wide spectrum of bacteria, including antibiotic-resistant strains. Antimicrobial properties of silver nanoparticles with 30 nm in diameter synthesized according to the original protocol have been determined in this study. In in vitro study using the serial dilutions method in the solid medium the minimal inhibition concentration (MIC) of silver nanoparticles against such test-strains as Staphylococcus aureus MRSA ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 2592, Shigella sonnei, Salmonella typhimurium 144 was equal to 33.46 μg/ml. MIC against B. subtilis ATCC 6633 was 133.8 μg/ml. The antimicrobial activity of silver nanoparticles has been studied on clinical isolates with multiple drug resistance isolated from wounds, urine, endocervical and faucial scrapings in surgical patients with Klebsiella ozaenae 4348, Citrobacter freundii 4369, Escherichia coli 4358, Enterobacter aerogenes 2476, Proteus mirabilis 4363, Staphylococcus aureus 4312 and Pseudomonas aeruginosa 283. The total inhibition of the microorganisms growth under the action of both doses of silver nanoparticles studied – 10 μg and 20 μg has been observed.

The search for effective antimicrobial substances is an important task of pharmacology nowadays. The appearance of antibiotic-resistant bacterial strains requires the use of antimicrobial agents with principally new properties compared to traditional drugs, which are able to overcome more successfully the resistance of the causative agents of certain diseases. Metal nanoparticles, and especially silver nanoparticles are in the focus of attention of researchers. It is known that silver nanoparticles are characterized by a pronounced antimicrobial activity. Based on them medicines in the form of creams [6,9], gels [14], as well as such medical products as catheters [20] and dressings [8,11] have been developed and introduced into practice. At the Pharmacology Department of O.O.Bohomolets National Medical University the research of pharmacological and toxicological properties of different metal nanoparticles (copper, iron, silver) is carried out. In terms of continuing to study antimicrobial properties of nanosilver an experimental substance of silver nanoparticles (AgNP) with the size of 30 nm has been studied.

Materials and Methods
Silver nanoparticles were obtained by means of the chemical reduction method in the aqueous medium according to the original protocol developed in F.D. Ovcharenko Institute of Biocolloidal Chemistry of NAS of Ukraine. They were characterized by size using dynamic light scattering (Zetasizer-3, "Malvern Instruments Ltd", Great Britain) and shape using transmission electron microscopy (JEM-1230, "JEOL", Japan).
The evaluation of the antimicrobial activity of silver nanoparticles was carried out in in vitro studies using two methods. The inoculation doses of test-strains were 10 3 , 10 4 and 10 5 CFU/cm 3 . The antimicrobial effectiveness of AgNP was studied in the final concentrations of 133.8 μg/ml, 100.38 μg/ml, 66.9μg/ml, and 33.46 μg/ml in the nutrient medium (Mueler-Hinton agar). A sterile water dispersion of AgNP was introduced into a sterile Mueler-Hinton agar cooled to 50°C, then mixed and poured on Petri dishes. Cultivation of microorganisms was carried out in thermostat at the temperature of 37°С for 24 h.
2. The second study was performed on a solid nutrient medium using the method of dosed droplets. Microorganisms involved in the study were multiple drug resistant clinical isolates from wounds, urine, endocervical and faucial scrapings in surgical patients with Klebsiella ozaenae 4348, Citrobacter freundii 4369, Escherichia coli 4358, Enterobacter aerogenes 2476, Proteus mirabilis 4363, Staphylococcus aureus 4312, Pseudomonas aeruginosa 283. Microorganisms were inoculated on Mueler-Hinton agar in the concentration of 10 5 and 10 7 CFU/cm 3 to form a bacterial lawn. Suspensions of microorganisms were prepared using 0.9% saline solution. After 30 min of drying of Petri dishes with inoculated bacterial cultures droplets of aqueous dispersions containing nanoparticles with the concentration of 800 μg/ml by metal were applied onto the agar surface. The droplets were 12.5 and 25 μl in volume and contained 10 and 20 μg of silver nanoparticles, respectively. Bacterial cultures were then cultivated in thermostat for 24 h at the temperature of 37°С. The calculation of results was carried out by measuring the diameter of growth inhibition zones. After measurements of growth inhibition zones Petri dishes were stored for 15 days at the room temperature for detecting any secondary growth.

Results and Discussion
Previous studies proved safety of the AgNP studied, namely genotoxicity, mutagenic action, effect on probiotic bacteria of the gastrointestinal tract [1].
Transmission electron microscopy confirmed that AgNP have a spherical shape (Fig. 1). Results of dynamic light scattering measurements are shown in Fig. 2.
According to the graph it is seen that the size distribution of nanoparticles is monomodal, it means that the colloidal solution has no other fractions. The ZAve parameter shows the average size of nanoparticles. Its average value was 31.8 nm and the absolute error was 0.8 nm.
In Tab. 1 the results of in vitro study of the antimicrobial activity of silver nanoparticles against test-strains of microorganisms are given. All strains of microorganisms studied were susceptible to silver nanoparticles. The results obtained indicate a pronounced antimicrobial activity against Staphylococcus aureus MRSA ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Esche richia coli ATCC 2592, Shigella sonnei, Salmonella ty phimurium 144 in the concentration of 33.46 μg/ml. Complete growth inhibition of Bacillus subtilis ATCC 6633 was observed at a higher concentration of AgNP -133.8 μg/ml. This result is important since it is known that B. subtilis is a component of the human normal microflora [13]. It is known that bacteria from Bacillus genus may develop resistance to silver nitrate and can be used in the process of biological synthesis of silver nanoparticles [10]. The mechanism of appearance of Bacillus sp. resistance is uncertain and may be associated with the presence of nitrate reductase enzyme in the bacteria [16].
A pronounced antibacterial activity of AgNPs was also revealed against antibiotic-resistant clinical isolates,  The ZAve parameter shows the diameter of nanoparticles , "+/-" -is the absolute error. and inhibition of bacterial growth was observed in all cases. The results obtained showed that clinical isolates such as Pseudomonas aeruginosa 283 and Klebsiella ozaenae 4348 which were resistant to the majority of antibiotics, appeared to be susceptible to silver nanoparticles. Diameters of growth inhibition zones for P. aeru ginosa were the largest among all strains tested. Both gram-negative bacteria from Enterobacteriaceae family (K. ozaenae, E. aerogenes, C. Freundii, E.coli, P. mi rabilis) and gram-positive coccus of S. aureus appeared to be susceptible to silver nanoparticles (Tab. 2). Effective concentrations of silver nanoparticles varied from Table 1 The antimicrobial activity of 30 nm silver nanoparticles against test-strains of microorganisms Notes: "Ø" -complete inhibition of growth; "++++" -intensive growth; "+++" -weak growth inhibition; "+" -only the growth of single colonies was observed. Table 2 The antimicrobial activity of silver nanoparticles (30 nm) against antibiotic-resistant clinical isolates 0.03 μg/mm 2 to 0.13 μg/mm 2 calculated with reference to the surface area of the nutrient medium (for detailed information see Tab. 3). There was no secondary growth observed in all growth inhibition zones in 15 days of observation.
The mechanism of the antibacterial action of silver nanoparticles is insufficiently studied. The opinion that effect of silver nanoparticles is associated with generation of reactive oxygen species inside the cell is widespread [7,12,18,21]. According to the data [11,17] a possible mechanism of action of silver nanoparticles includes the complex of the following factors: • Silver nanoparticles are adsorbed on the surface of the membrane of microorganisms. • Nanoparticles destroy molecules of lypopolisaccharide and form "sites" of high permeability in the membrane. Silver nanoparticles penetrate inside the cell releasing the Ag + -ions, which cause the following effects: -silver ions interact with cytochromes and block the respiratory chain; -silver can also interact with DNA inhibiting its replication. It has been reported that antimicrobial properties of AgNP depend on size and geometry of particles. According to Kahru A., Dubourguier H.-C. [15] the inhi-biting action of nanoparticles against nitrifying bacteria was more pronounced if the size of nanoparticles was less than 5 nm. Pal S. et al. [19] have found that there is dependence between the effect of silver nanoparticles and their geometrical parameters. Thus, AgNP with a triangular shape revealed higher activity than spherical shaped nanoparticles. Researchers explain this regularity by high density of silver atoms in triangular nanoparticles, which along with a high specific surface area provide more active interaction with bacterial cells.