DETECTION OF THE ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICULES BIOSYNTHESIZED BY STREPTOCOCCUS PYOGENES BACTERIA

This study was aimed to biosynthesized silver nanoparticles by Streptococcus pyogenes bacteria and its antimicrobial activity against (S.aureus ,P. aeruginosa, E.coli, and C. albicans yeast) at different concentrations (20, 40, 60 ,80 and 100) μg/ml by agar well diffusion assay. Fifty sample was collected from Wounds and burns, from Baghdad Teaching City Medicine Laboratories. Samples identified by culture, VITEK 2 Compact system ID-YST kit. The sensitivity of bacterial isolates to antibiotics ware tested and the microbes were more sensitive, resistant and moderate range to antibiotics. Several techniques where used to characterize AgNPs: X-ray Diffraction (XRD), UV–Visible Spectroscopy(UV) and Scanning electron microscope (SEM).The results show that biosynthesized silver nanoparticles are more effective than bacterial supernatant on human pathogenic microbes.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
. It is a killing agent of antibiotic resistant strains, which represent methicillin resistant strains and Vancomycin resistant strains. In addition to Antibacterial properties , Silver Nanoparticles have Antifungal, Anti-inflammatory properties (19) To overcome problem utilizing toxic chemicals and high energy required physical procedures, the nanoparticles have been combined by natural materials which have been used for the union of different metal and oxide nanoparticles. Thus, the biogenic methodology, the use of regular living beings or materials specifically, has offered a dependable, basic, nontoxic and ecoaccommodating technique (7). In addition to different types of plants ,many microorganisms, such as algae, fungi and bacteria have been used in nanoparticles synthesis (1). Biosynthesis of metal nanoparticles can occur either intracellularly or extracellularly (9) (10). In this study, Strept. pyogenes bacteria were selected to the biosynthesis of silver nanoparticles.

MATERIALS AND METHODS Collection of microbial isolates 50 microbial
isolates belong to Strept. pyogenes, E. coli, C. albicans S.aureus and P. aeruginosa were collected from Baghdad teaching city medicine laboratories The isolates were diagnosed by gram stain and biochemical tests .These identified cultures were transferred to nutrient agar slants for preservation and then store in the refrigerator at 4°C (14). Antibiotic susceptibility test: By using disk diffusion assay , by transferred part of colony of microbe grown in BHIB by loop to test tube that contained 5 ml of normal saline and The bacterial suspension was compared to the standard McFarland , then 0.1 ml of the microbe suspension has been spread on the surface of Mueller-Hinton agar, and left to dry. Antibiotic disks were placed on the agar by sterile forceps. After incubation period , The zone of inhibition diameter around the disc is estimated and compared to the CLSI reference table to determine if the organism is susceptible, intermediate or resistant against the antibiotic agents tested (16).

Streptococcus pyogenes
The growing S. pyogenes strains were freshly injected on Muller_Hinton broth and incubated at 37°C for 24hours. The culture was centrifuged at 14,000 rpm for 10 minutes, supernatant added separately for reaction container containing silver nitratea concentration of 10 −3 (1% v/v). The interaction between this supernatant and Ag + ions was carried out in bright status for 24 hours (8).

Characterization of biosynthesis silver nanoparticles
Characterization of Silver nanoparticle were analyzed by (UV-Vis spectrophotometer and X-Ray diffraction) . Color change of the reaction mixtures was checked by estimating UV-visible range of the reaction mixture .SEM used to determination of the morphology and size of nanoparticles and Xray diffractometer by casting (CuKα) over the model to be measured at different angles from 20 ° to 60 ° and measured the results. The Debye-Scherer formula was applied to obtain the size of the synthesized nanoparticles (4). D = K λ / β Cos. D is the particle size; K is a dimensionless shape factor; λ refer to X-ray wavelength; β is the line broadening at half the maximum intensity (FWHM); θ is the Bragg angle (in degrees).

Antimicrobial activity assay
To determine the antimicrobial activity of the biosynthesized AgNPs by using S. pyogenes against other microbial strains that cultured on media and after the plates were permitted to dry, wells ware formed . A 100μL volume of biosynthesis AgNPs was applied in the wells , incubated at 37ºC for 24hrs. the diameter of inhibition zone was estimated around each well , 3 replicate trials were conducted against each microbes (2) . RESULTS AND DISCUSSION Collection of microbial isolates and identification microbial isolates belong to S. pyogenes, isolated from wounds and burns and diagnosed with VITEK ,API strips, gram stain and subjected to biochemical tests for further confirmation (17).

Antibiotic susceptibility test
The results in (Table 1) showed that S. pyogenes bacteria were the most resistant to antibiotics. The number of antagonists was 6 antibiotics of a total of 10 antibiotics, followed by P.aeruginosa, E. coli and S.aureus. The number of antagonists was 4 antibiotics of a total of 10 antagonists, and C. albicans, were resistant to 3 of the 10 antibiotic agents. The results showed that there is a difference in the sensitivity or resistance of microorganisms to antibiotics, due to the common use of some antibiotics for a long time, led to the evolution of strains resistant to them, and the manufacture of new antibiotics working on sensitive sites in bacteria has made the resistance of bacteria have something so that bacteria are sensitive to these antibiotics (5).

Biosynthesis of AgNPs
The results in (Fig.1) shown of the biosynthesis nanoparticles by using supernatant of S. pyogenes with silver nitrate solution on Muller-Hinton broth at a concentration of 1 mM at pH 7 and at room temperature for 24 hrs. The change in the mixture color to brown was refer to a positive synthesis (13).

Characterization of AgNPs
The characterization of nanoparticles show in (Fig.2). By using the UV-Visible spectrophotometer showed the absorption at λmax at 410 nm wavelength, The phenotypic properties of nanoparticles formed by (SEM), Silver nanoparticles appear spherical in shape and Through the X-Ray Diffraction process, the formation of nano-silver at the angle 3θ was shown in degrees (29.39.32.21 and 31.77). By applying Scherrer equation, the calculated nanoparticles were 30.3 nanometers (13).

Figure2. Characterization of silver nanoparticles using A= UV-Visible Spectrophotometer B= SEM image of AgNPs C= X-ray diffraction of biosynthesis nanoparticles Antimicrobial activity of biosynthesis AgNPs
Biosynthensis AgNPs displayed antibacterial properties against bacterial pathogens with close connection of the nanoparticles themselves with the microbial cells. The results showed that the biosynthesized AgNPs by bacteria has a strong inhibitory effect on the growth of pathogenic microbes compared to bacterial supernatant. The supernatant of S. pyogenes showed less effective inhibitory effect on P.aeroginosa bacteria in an inhibition zone reached to 13 mm at concentration of 100% only (Fig. 3A) . The effect of biosynthesized silver nanoparticles was the highest inhibition on the growth of P.aeroginosa with an inhibition zone reached to of 18 mm at concentration of 100%,16mm at concentration 80% ,13mm at concentration 60% (Fig. 3B(. Figure 3.A. The effect of S. pyogenes supernatant on P.aeruginosa, B. The effect of biosynthesized sliver nanoparticles on P.aeruginosa As for S.aureus bacteria where bacterial supernatant showed less inhibitory effect with a 13 mm at 100% conc., and 11mm at 80% conc. (Fig.4A) , but there's no effect for the other conc. The highest inhibition zone recorded to the biosynthesized silver nanoparticles at 26 mm for 100% conc., and 16mm at 80% conc., 14mm at 60% conc., 8mm at 40% conc., and 12mm at 20% conc. (Fig. 4B)   Figure 4. A. The effect of S. pyogenes supernatant on S.aureus , B. The effect of biosynthesized sliver nanoparticles on S.aureus. Followed by E. coli, where bacterial supernatant was less effective inhibition with a 12 mm inhibitory at 100% conc. , and 13mm at 60% conc. (Fig.5A) The highest effect of the biosynthesized silver nanoparticles reached to 21 mm at 100% conc. , 20mm at 60% conc. (  Fig. 5B). Finally, the yeast C. albicans where the effect of bacterial supernatant was less effective inhibition with a 12 mm inhibitory at100% conc. and 12 mm at 80% conc. (Fig.6A), The highest effect of the biosynthesized silver nanoparticles at 100% reached to 23 mm ,13 mm at 60% conc. ,17mm at 60% conc. ,and 14mm at 40% conc. (Fig6B).  The results show that biosynthesis of Silver nanoparticles are more effective than bacterial supernatant. The supernatant of S. pyogenes bacteria have Inhibitory effect on the growth of microbes related to the nature of the substances produced by the bacteria but this effect is weak (3) , But The biosynthesized silver nanoparticles by supernatant of S. pyogenes bacteria have a strong inhibitory effect because silver nanoparticles have a very effective against bacterial and fungal infections. These properties are due to silver nanoparticles having a large surface area of the volume ratio which increases their association with bacterial cell well causing changes in the membrane and thus cell death (18) That can also release silver ions that interfere with thiol groups in biomass enzymes, which inhibit them. Silver ions also inhibit respiratory enzymes and during the inhibition process reactive oxygen species are generated. (ROS) that attack the cell itself and thus die. (11) Silver nanoparticles have the ability to interfere with the sulfur and phosphorus bases of DNA and thus lead to the breakdown of DNA and cell death due to a disturbance in the DNA replication of bacteria and microbes (12). This agreement with that the biosynthesis of Silver nanoparticles by S.pyogenes has great effectiveness against broad spectrum of pathogenic microorganisms (15). The present investigation support the use of biosynthesized AgNPs by S.pyogenes elicited a strong antimicrobial activity. Thus, the biological approach could be an economical alternative to conventional chemical and physical assays of AgNP synthesis and would be suitable for the development of a biological process for commercial large-scale creation. AgNPs have wide application in different fields, such as antibacterial. Therefore, the improvement of their synthesis for nanoparticle production is the main objective of nanotechnology (21).