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Abstract This study focused on the synthesis and characterization of silver nanoparticles (AgNPs) using Artemisia annua extract and evaluating their antibacterial and therapeutic effects on Staphylococcus aureus particularly the Methicillin Resistant Staphylococcus Aureus isolate St.8 in vitro and in vivo .The results can be summarized as follows: A total of 30 clinical samples were collected from 30 patients, including 11 males and 19 females, ranging in age from 21 to 70 years old. The samples consisted of 9 sputum samples, 4 pus wound samples, and 17 blood samples ,which were used for the screening and isolation of S. aureus isolates. The screening of S. aureus isolates involved morphological and biochemical tests to differentiate between Gramnegative bacilli and Gram-positive cocci. Out of the total isolatesm Gram- Negative Bacilli, nineteen isolates number 4, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, and 30 were identified as Gram-negative bacilli, Isolates were characterized as short rods, non-spore forming, and tested negative for the catalase test.Gram-Positive Cocci (Staphylococci),Eleven isolates number 1, 2, 3, 5, 6, 7, 8, 11, 12, 22, and 27 were identified as Gram-positive cocci found in clusters exhibited a positive reaction to the catalase test, indicating a possible affiliation with the Staphylococci family.Among these, nine isolates number 1, 2, 3, 6, 7, 8, 12, 22, and 27 displayed beta hemolysis on blood agar medium,Four isolates number 8, 12, 22, and 27 were confirmed to ferment mannitol and tested positive for the coagulase test. The selected four isolates number 8, 12, 22, and 27 also showed positive results for the DNase test, indicating DNase activity. Assessment of Phytochemical component of Artemisia annua ethanolic extract by GC 1300 mass spectrometer identified major effective compounds of the A. annua ethanolic extract using a GC1300 mass spectrometer revealed nine major compounds, table 6 and figure.(5). These included 4,4-dimethyladamantan-2-ol produced at a retention time (RT) of 27.28 (with an area percentage of 5.13%), β-copaene observed at RT. of 2.68%. Otherwise, nhexadecanoic acid found at RT. of 30.72 (with 12.74%), deoxyartemisinin noticed at RT. of 31.35 (with 3.75%). While 3,4- hexadienal, 2-butyl-2-ethyl-5- methyl was at RT of 32.18 (with 4.88%). Furthermore, 9-octadecenoic acid, methyl ester, (E) determined at R.T. of 32.52 (with 2.31%), oleic acid found at R.T. 33.80 (with 23.28%), octadecanoic acid found at R.T. 34.38 (with 7.30%), and finally 2,3-dihydroxypropyl elaidate recog- nized at R.T. 38.04 (with 1.93%). Silver nanoparticle synthesis and characterization: AgNPs were synthesized using A. annua extract, leading to a change in color to reddish-brown.UV-Vis spectroscopy showed a maximum absorbance at 440 nm, characteristic of Plasmon excitation in silver nanoparticles, Transmission electron microscopy revealed spherical AgNPs with a diameter ranging from 9 to 48 nm, surrounded by a plant extract shell.HR-TEM analysis demonstrated polydisperse nanoparticles forming aggregates, causing disruptions in the cell envelope of isolate no. 8. Scanning electron microscopy confirmed the presence of synthesized nanoparticles, displaying oval and spherical shapes. Elemental analysis using EDX indicated the composition of the AgNPs, with significant peaks for silver, oxygen, carbon, potassium, and calcium. XRD analysis revealed crystalline properties, with peaks corresponding to AgCl and various crystallographic planes of silver. FTIR spectra showed changes in absorption peaks before and after Ag biosorption by the extract.The interaction between functional groups on Artemisia and Ag+ ions during biosorption was evident, involving groups such as amine, C–H, carbonyl, carbonate, and aromatic CH bending. ICP-MS results in the concentration of silver ions in the new green particles decreased as the silver nitrate concentration diminished. Antibacterial Assay: The ethanolic 96% extract of A. annua exhibited a greater antibacterial effect compared to the 70% extract.The new green AgNPs showed higher inhibitory activity against St.8 isolate, with significant effects compared to other treatments. Isolates St.12, St.22, and St.27 exhibited lower antibacterial activity.St.8 isolate was selected for further experiments based on its pronounced response to the new green AgNPs. The minimum inhibition concentration (MIC) values of biogenic AgNPs were lower than those of chemical AgNPs, indicating potent antibacterial activity.St.8, identified as MRSA, exhibited resistance to certain antibiotics but showed sensitivity to others. Biogenic AgNPs demonstrated superior antibacterial activity compared to AgNO3, particularly against MRSA. Molecular identification using MALDI-TOF MS and 16S rRNA gene sequencing confirmed St.8 (RM-Ph8) as Staphylococcus aureus with Accession number OQ421819 , the phylogenetic tree showed high genetic relationship 98.12% with the reference strains of Staphylococcus aureus. In Vivo assessment of Antibacterial Activity of A. annua extract and green nanoparticles , The experimental protocol was ethically approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, University of Sadat City with an approval number (VUSC-012-1-22). Hematological Effects : In MRSA-infected rats, treatment with A. annua and A. annua AgNPs led to the normalization of hematological parameters such as hemoglobin concentration, red blood cell count, hematocrit percentage, and lymphocyte count. Total leukocyte count, neutrophils, and platelet count decreased with the supplementation of A. annua and A. annua AgNPs. 14.Oxidant/Antioxidant Biomarkers:MRSA infection increased malondialdehyde (MDA) levels, indicating oxidative stress, which was reduced by A. annua and A. annua AgNPs. Catalase (CAT) and superoxide dismutase (SOD) activity decreased with infection but increased with treatment. Histological Assessment : Lung tissues of MRSA-infected rats showed severe pathological changes, including interstitial tissue thickening, inflammatory cell infiltration, and necrotic areas. Treatment with A. annua and A. annua AgNPs improved lung histology, indicating a recovery process. Immunostaining Analysis:Immunostaining for TNF-α and iNOS in lung tissues showed strong expression in the MRSA-infected group, which decreased with A. annua and A. annua AgNPs treatment. Liver and Kidney Function Biomarkers MRSA infection led to increased levels of ALT, AST, urea, and CRP in the liver, which were normalized by A. Annua and biogenic AgNPs.Liver histology revealed vacuolar degeneration, necrosis, and inflammation in infected rats, with improvements seen with treatment. The immunohistochemical findings of TNF-α and iNOS in liver tissue revealed distinct patterns among different treated groups. group A, group B, and group C exhibited very weak to no immune-reactive cells for both TNF-α and iNOS. In contrast, group D showed strong expression of both markers. Notably, Groups E and F displayed a moderate to weak positive immune reaction for TNF-α and iNOS in a few cells. |