الفهرس 
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Abstract
Nanobacteria act as a potential nucleus for aggregating
different minerals in physiological conditions which
subsequently causes widespread pathological calcifications
(as kidney stone formation, arterial heart disease,
Alzheimer’s disease, polycystic kidney disease, gall stones
and gallbladder inflammation, prostatitis and cancer.
This study aimed to inhibit nanobacterial growth by
combination treatment of antibiotics and gamma irradiated
medicinal plants.
In this study 54 kidney stones were collected from male
and female patients suffering from urolithiasis. The
chemical analysis of the studied stones indicated that there
were five types of stones(COM & CaP (44.44%), followed
by CaP& CaOX (24.07%), while MgAmP, AmU& P and
COD & CaP were (22.22%), (5.56%) and (3.71%),
respectively.
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Growth of nanobacteria showed that 22 samples
(40.74%) were negative for nanobacterial infection. While
32 samples (59.26%) were positive for nanobacterial
infection. During growth it is observed a white growth of
dense particles touching near the bottom of the vessel after
1 weak. After two weeks, they had become bigger and
formed visible growth aggregates, then after one month,
many were in clumps and attached to the bottom.
The present study included collection of fifty four kidney
stones from 54 males and females patients, among these
collected stones, 7 (21.88 %) were from females and 25
(78.12 %) were from males, with variable ages between 30 -
60 years.
Additionally, microscopic examination showed Gram
negative cocci, coccobacilli and bacilli surrounded by
amorphous shells. Also, it was noticed that NB isolates were
negative for urease activity. SEM analysis of nanobacteria
revealed that its size ranged from 375 nm to 600 nm and
showed clusters of coccoid and bacilli form.
Besides that, TEM micrographs noticed nanobacteria
with spherical coccoid shape, thick cell walled structures and
reproducing by binary fission.
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Detection of nanobacterial protein exhibited two major
bands distinctive for NB with MW of 71.66 and 65.31 kDa.
Also, FTIR spectrum of NB observed abnormal peak
between 1000 and 1200 cm-1 which belonged to phosphate
absorption and the band at 3272 cm–1 was due to the presence
of O-H group and absorption at 1636 cm-1 was due to
vibration of C=O.
EDX analysis of nanobacterial pellet showed featured
absorption peaks related to nanobacterial shield as C, 22.41-
33.27%; K, 15.25-6.25%; N, 10.57–13.47% and Ca, 3.39–
2.44%. While, Mg content was 2.22–0.95%. Finally, the Na
and P contents were 1.11–0.86% and 1.05–0.78%,
respectively.
The 32 nanobacterial isolates were subjected to test their
ability to produce the biofilm quantitatively by ELISA
method and it was found that all thirty two isolates (59.26%)
were strong biofilm producers where their optical density’s
ranged from 0.272 to 1.372.
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The most potent biofilm producer no.21 was identified by
PCR technique using genomic DNA as template the result
revealed that DNA band of about 1400 base pair in length.
All the strongest nanobacterial biofilm producers were
subjected to antibiotic susceptibility using 6 different types
of antibiotics. It was found that Streptomycin, Trimethoprim
/ Sulfamethoxazole and Doxycycline have antinanobacterial activity against all isolates but penicillin G.,
Ciprofloxacin and Cefepime haven’t any effects on the
nanobacterial growth.
The phyto-preparations of medicinal plants have
gained specific interest as alternative products that could
resolve problems concerning to the appearance of resistant
strains to classical antibiotics.
Medicinal plants such as khella, halfa bar, thyme, neem,
fennel, parsley, dill and thistle have been used in traditional
Arabic medicine for treatment of kidney stones.
The tested samples were exposed to increasing doses of
gamma radiation from 1 to 10 kGy. Three plants (thistle,
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parsley and dill) were free from microbial contamination,
while, thyme, neem and fennel were free from only fungal
contamination.
Screening of the effect of medicinal plant extracts and
essential oils on growth of the strongest nanobacterial
biofilm producers obviously cleared that oils (fennel, thyme
and sage), aqueous extract of (irradiated and non-irradiated
khella, fennel and non-irradiated parsley) and EEs
(irradiated and non-irradiated khella, halfa bar and thyme)
have anti nanobacterial activity on growth and biofilm
formation ability.
Determination of minimum inhibitory concentration
(MIC) of the most effective antimicrobials on growth of the
strongest biofilm producer isolates showed that MICs of the
most effective tested antibiotics ranged from 3.9 to 15.6 µg/
ml. Several studies proved that sulfamethoxazoletrimethoprim (SXT) antibiotic cause nephrotoxicity and a
deterioration of kidney function in patients.
So, it is excluded and streptomycin and doxycycline
were choiced and checked out for their anti-biofilm
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formation ability towards the strongest biofilm producer
isolates and the results showed that DO was the most agent
which had the ability to reduce the biofilm production and
change power of adherence of nanobacteria from strong to
week.
The results showed that aqueous extract of irradiated
khella at 6.0 kGy and fennel at 7.0 kGy and non-irradiated
parsley had the lowest MIC values ranged from 3.9 to 15.6
µg/ml. On the other hand, MICs of EEs of all medicinal
plants ranged from 3.9 to 15.6 µg/ml and there was no any
noticeable change in MICs after irradiation.
The results disclosed that aqueous extract of irradiated
khella appeared to be the most effective antinanobacterial
agent and was chosen for further studies.
Also, the anti nanobacterial activity of six essential oils
were preliminary evaluated, the results indicated that out of
them 3 essential oils had antinanobacterial activity. MIC of
fennel and thyme oils ranged from 3.9 to 15.6 µg / ml.
whereas; MIC of sage was 3.9 to 31.2 µg / ml.
As a result, a combination treatment was applied using
doxycycline and aqueous extract of irradiated khella. The
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results indicated that combination treatment inhibited
nanobacterial growth and affected significantly the biofilm
production ability. Also, combination treatment caused
disappearance of the specific protein bands of nanobacteria.
Release of cell constituents in control and treated
cultures was assayed indicating increase in ODs reading of
the cells subjected to combination treatment.
For quantitation of total protein content, the results
revealed that the strength of the nanobacterial cell
membranes changed after incubation with doxycycline
and aqueous extract of irradiated khella. Also, there was a
large change in ultrastructure of cells with complete
dissolution of the cell wall with treatments as shown by
TEM.
The in vivo study, included estimation of kidney
functions as well as, histopathological changes in kidney
tissues were studied. Our data showed that raise of urea,
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creatinine concentration after NB inoculation and decrease
in albumin concentration.
The results also showed that NB group caused a
significant decrease in GSH and a significant increase in
MDA and NO levels.
The biochemical results confirmed the histological
examination as we observed large area of interstitial
hemorrhage and calcification in kidney tissues of treated
animal groups which could be due to damage induced by
these bacteria. The combination treatment of NB injected
rats with DO and aqueous extract of irradiated khella
ameliorated the renal function tests and histological
alternations.