الفهرس 
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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.