الفهرس 
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Abstract
The appearance of lethal diseases which resulted from many organism not influenced by multiple drugs becomes a great problem and this leads to high morbidity and mortality rate in community. So, the need for discovering of new antimicrobial agent appeared to solve this consequence is required. The use of nanoparticles had attracted big attention as they have special characters such as physical, chemical and optical characters. NPs are particles which have size of (1-100 nm). The most common methods for synthesis are chemical and physical method but they have some draw backs such as toxicity and expensiveness. Biological method is used instead of them as it is environmentally eco friendly, safe, nontoxic and biocompatible.
Biological sources such as bacteria ,fungi and plant are used .In this topic biogenic production of AgNPs by B.subtilis ATCC(6633) and its activity against S.aureus and E.coli had been carried out. .Fifty S.aureus isolates and fourty six E .coli isolates were collected from Al-Qasr Al-Aini at Microbiology laboratories from the period of July 2017 to July 2018 from different sites of infection from male and female patient of varied age and identified by conventional method including, vitek 2 definition , morphological characteristics , biochemical reactions , differential , selective media and molecular identification of mec A gene. Disc diffusion method was the method that used for determination of antibiotics susceptibility test with S.aureus and E.coli.
The antimicrobial susceptibility pattern of S.aureus isolates revealed higher resistance (100%) was observed against amoxicillin/clavulanic acid while 64% sensitive towards trimethoprime-sulfamethoxazole, 24% sensitive to ofloxacin, 22% sensitive to tetracycline 16% sensitive to amikacin. S.aureus isolates showed 100% resistance against cefoxitin antibiotic disk, so this gave evidence that they could be MRSA.
The antimicrobial susceptibility pattern of E.coli revealed that amikacin (54%) sensitive followed by meropenem (52%),tetracyclin (28%), sulfa methoxazole-trimethoprime(19%), streptomycin (13%), amoxacillin –clavulanic acid (4%).All isolated E.coli showed (98%) resistance against Ceftazidime.
MRSA is S.aureus which shows resistance towards all beta-lactam antibiotics. The recognition of mecA gene was tested by fragment amplification of a 293-bp using pair of mec A primer. The chromosomal DNA was made by allowing cultures of bacteria to grow on BHI broth. DNA copying was performed in a PCR thermocycler with the following stages: initial denaturation at 94ºC for 3min, 30s at 94º C, 30s at 60ºC, and 30s at 72ºC. Denaturation, annealing, and extension were performed in 30 cycle followed by an additive cycle of annealing at 60 c for 30s and final extension at 72 ºC for 5 minutes and the result was that among 50 S.aureus isolates that demonstrates resistance to methicillin by the disc diffusion test, only 32 isolates had mecA gene and 18 isolates were S.aureus. The results showed that 64% (32/50) of Staphylococci isolates possessed mecA gene.
The extracellular biosynthesis of AgNPs using supernatants from B.subtilis cultures ATCC (6633) was performed by collecting B. subtilis ATCC (6633) from NODCAR. A loop full was taken from 24 hr incubated cultures was added to 100 ml of Luria Bertani broth in Erlenmeyer flask at 37º C on shaker incubator at 200 rpm for 48 hr. After incubation, supernatant was separated by centrifugation at 5000 rpm for 30 minutes, then 100 ml of cell filtrate in 250 ml Erlenmeyer flask was treated with 1mMAgNO3 in a ratio (1:1) . Incubate the solution on shaker incubator for 72 hr at 200rpm in dark at 40 º C. The flask that contained filtrate of the cell only was used as control (without AgNO3) in the same time with tested flask.
Synthesis of AgNPs was detected after their incubation by the color conversion from pale yellow to dark brown but no color was altered in control flask .The synthesis of AgNPs were confirmed by examination with TEM to determine size and morphology of the formed silver nanoparticle by preparing on copper TEM grids carbon covered. The result showed spherical NPs with size range (20-47nm). Particle size analysis was also carried out by means of laser diffractometry and the results showed that Z-Average (d.nm): was 135.0 nm with 99.2 % of the particles have a hydrodynamic diameter of 188.0 nm (SD= 117.7). The poly dispersity index was 0.246. Zeta potential value was taken to indicate the potential on surface of the silver nanoparticles. The zeta potential analyzer (Malvern zeta seizer 2000, Malvern) was used in measuring surface zeta potential.
The zeta potential value was -17.2mv which indicated good stability due to capping with proteins secreted from bacteria. AgNPs showed good antibacterial, antifungal and antiviral activity.
Agar well diffusion method was done to determine the action of biosynthesized silver nanoparticles against E. coli and MRSAoutside the body using MHA media. Bacterial cell filtrate of B.subtilis was utilized for production of silver nanoparticles outside the cell and acted as negative control.
A single colony of the tested culture was aseptically transferred and grown in MHB and maintained in an incubator at 37º C for 3 hr . The OD was altered to 0.5 McFarland (≈1 × 108 CFU/ml) , the bacterial culture was streaked aseptically on the solid MHA plates. After streaking wells were prepared in which the samples of AgNPs at different concentration were dipped.
Control experiments were prepared using either 50µl of supernatant obtained from B. subtilius culture or 1mM AgNO3 solution in the place of synthesized AgNPs. Finally, the solid MHA plates were placed in incubator at 37º C for 24 hr and the diameters of inhibition zone surrounded the samples were measured in millimeters (mm). The obtained data from the agar well diffusion method showed that the variation in bacteriostatic activity of silver nanoparticles on the target microorganisms was statistically significant and control wells containing the supernatants obtained from the bacterial culture had no inhibition zone.
The MIC of synthesized AgNPs was performed by broth micro dilution method by twofold with application of Mueller Hinton broth following the criteria of CLSI guidelines. For MIC test, broth containing synthesized AgNPs was used at final concentration ranging from 0.1775 to 5.68µg/ml. The MIC value was 1.42µg/ml for MRSA and E.coli and 0.355 µg/ml for standard E. coli ATCC (8739) and standard S.aureus ATCC (6538).
The MBC value was 2.84 µg/ml for E.coli and MRSA and 70 µg/ml for standard E. coli ATCC (8739) and standard S.aureus ATCC (6538). Scanning Electron Microscope is used to detect alteration in shape of bacterial cells with and without treatment with AgNPs.
Results of SEM indicated that control bacterial cells were intact without any noticeable damage, while upon interaction with AgNPs bacterial cells damage was observed as cell lysis. The possible mechanisms that were believed for the antimicrobial activity of Ag NPs was that Positive charged Ag+ ions of AgNPs attracted to the negative charge on surface of cell causing disturbance in its physical and chemical characters of cell membranes which disrupt passing through cell membrane, electron transport and respiration.