الفهرس 
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Abstract
Pathogenic resistance to antibiotics, used for treating diseases, became an
increased phenomenon with increasing pathogens. This study was conducted on
real samples taken from cases admitted to Urology department at El-Mansoura
hospital and did not normally respond to treatments by regular used antibiotics. The
primarily samples investigation concluded (642) urine samples collected from
patients suffered from urinary tract infections (UTI), 458 samples showed
pathogenic bacterial growth on nutrient agar, while 184 samples showed no growth
and considered free from UTI pathogens. The Gram reaction for picked colonies
belonging to these levels revealed that 170 isolated were G-ve, resistant to 2 types of
out of 12 used antibiotics. 19 cases were G+ve, resistant to 3 types. severe infection
with number of pus less than 500 cells per field were represented by 98 cases. The
pathogenic bacteria isolated from these cases were 91 G-ve and 7 were G-ve, resp.
The biochemical characterization of the 485 UTI samples showed 373 cases
were infected with Escherichia coli, 47 cases with Klebsiella pneumonia, 10 cases
with Staphylococcus saprophyticus, 3 cases with Enterobacter faecalis, and 25
cases with other causes. However, E.coli bacteria is one of the most members of the
enteric family that causes about 90% of UTI. Three E.coli strains were selected
from the three infection levels named as: E.coli EMG03 (minor infection level),
E.coli EMG04 (moderate) and E.coli EMG05, which represents the (severe level of
infection). Two reference multi drug resistant strains were used: E.coli EMG01 is
resistant to Ampicillin, Neomycin, and strain E.coli EMG02 is resistant to
Ampicillin and Chloramphenicol.
In this study, the theme oil was used as natural additives to antibiotics. The
minor (GC-Mass) component enclouded the phenolic compounds extracted from
the thyme essential oil; 26 compounds were detected. Thymol, P-Cymene, and the
other Terpinene represented the largest percentage of component by 32.3, 21.8 and
13.9%, resp. While Carvacrol, Cineol and Linalool represented the second
important percentage of component by 5.1, 4.4 and 3.4%, respectively. The other
important component like Camphor, β-Caryophyllene and other Terpen did not
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represent an detectible percentage. While the emulsification process was
synthesized using highly purified thyme crude oil or thymol crystal substance.
The nano-emulsion forms were characterized, the main thyme nanoform was
lie in sized 53,99 nm, the thymol nanoform was lie in 51,09 nm with total zeta
average of 67.64 and 66.05 nm, resp.
The resistance to specific 12 antibiotics was measured using antibiotics
resistance scale according to CLSI,(2017), which assigned three levels of
resistance (resistant, intermediate, and susceptible). The used antibiotics were
Ampicillin and Amoxicillin at a concentration of 10 µg that showed inhibition
zones of 6.41 and 6.73 mm, respectively., and showed 100% resistance.
Cefoxitin at concentration of 30 µg showed inhibition zones of 15.96 mm and
98% resistance. Ten µg concentration of Imipenem showed inhibition zones of
15.92 mm and the strain was 98% resistant. Vancomycin at a concentration of 30
µg, showed average inhibition zones of 11.05 mm and the strain was 95% resistant,
Erythromycin, Gentamycin, Streptomycin, and Neomycin with a concentration of
10 µg showed inhibition zones of 15.03, 13.31 14.64 and 16.81 mm, resp., and
showed 85.4, 86.3, 78.7 mm and 82.6% resistance, resp. the Chloramphenicol of 30
µg revealed inhibition zone of 16.36 mm and the strains were 78.5% resistant.
Ciprofloxacin 5 µg showed inhibition zone of 19.85 mm and strains were 56.5%
resistant. Tetracycline at a concentration of 30 µg showed area of inhibition of
20.54 mm and the strains were 75.9% resistant. Also, the antibiotic susceptibility
tests were conducted against the selected three E.coli strains under study compared
with the reference strains. the three E.coli isolates were resistant to Ampicillin and
amoxicillin as same as reference strains EMG01 and EMG02.
The isolates EMG04 and EMG05 were resistant to Cefoxitin, but EMG03
showed inhibition zone diameter (around 14.23 mm) similar to reference strains.
All isolates as well as reference strains showed similar inhibition zone diameter
(around 15.00 mm) to Imipenem. The isolate EMG05 was resistant to Vancomycin,
but EMG03 and EMG04 was similar to EMG01 and EMG02 in their sensitivity to
Vancomycin as showed inhibition zone ranged between 9.17-10.07 mm. The
isolates EMG05 was resistant to Gentamycin, but EMG03 and EMG04 showed
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average inhibition zone of 12,5 mm similar to those of EMG01 and EMG02. All
isolates as well as reference strains showed similar inhibition zone diameter
(around 14 and 14.7 mm) Erythromycin and Streptomycin, respectively.
On other side, the effect of (1.5, 2 and 2.5%) concentrations of thyme and
thymol either as emulsion or nano-emulsion on the growth of isolated UTI E.coli
were estimated and showed slight inhibition zones around isolated and references
strains. The differences in diameters of inhibition zones between 1.5, 2 and 2.5%
concentrations were insignificant compared with reference strains.
While the increasing on the nano-emulsion of thyme oil concentrations
caused significant increased inhibition zones when used against the three E.coli
isolates, also they were significantly different from those of references strains. It
was noticeable the significant differences among the used concentrations of
nanocrystals against either isolated or reference E.coli strains. Moreover, uses of
2.5% concentrations of nan-form of crystal oil was the most effective among other
concentrations as showed a significant inhibition zone against all isolated and
reference strains.
Besides, the effect of 10 µg of Ampicillin, Amoxicillin, Erythromycin, 30 µg
of Neomycin, Chloramphenicol and 5 µg of Ciprofloxacin were achieved when
mixed with emulsion and nano-emulsion forms of both thyme oil and thymol
crystal at three different concentrations 1.5%, 2.0%, and 2.5% on isolated E.coli
UTI and reference strains. The use of emulsions forms did not show any significant
differences in inhibition zone diameters between isolated and reference strains,
while the use of nano-emulsion forms caused significant differences in inhibition
zone diameters, the nano-emulsion of thyme crystals was the most effective.
For Ampicillin, the inhibition zones diameters were 6.45, 6.32, 6.25 for
EMG03; 6.07, 6.32 and 6.25 for EMG04 and 6.04, 6.25, and 6.15 for EMG05,
resp., compared with 6.27, 6.64 and 6.92 mm for EMG01 and 6.05, 6.17 and 6.25
for EMG02. The effect of nanocrystal form on isolated or reference strains was the
effective, which were 8.35, 11.20 and 16.43 for EMG03; 8.23, 10.88 and 16.25 mm
for EMG04 and 8.11, 10.52 and 16.08, compared with those of reference strains
7.82, 10.64 and 16.70 for EMG01 7.98, 10.72 and 16.54 mm for EMG02, resp.
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The effect of Amoxicillin was 6.22, 6.55 and 7.63 mm for EMG03; 6.19,
6.41 and 7.23 mm for EMG04 and 6.15, 6.24 and 7.05 for EMG05, compared with
those of references strains that recorded 6.89, 6.98 and 7.85 mm for EMG01 and
6.92, 6.95 and 7.91 for EMG02, respectively. The use of thymol nano-emulsion
inhibited the growth of tested bacteria. The inhibition zones were 8.81, 12.82 and
19.87 for EMG03; 8.25, 12.33 and 19.22 for EMG04 and 8.10, 12.13 and 19.14 for
EMG05, compared with reference strain that showed 8.92, 12.67 and 19.90 for
EMG01 and 8.89, 12.72 and 19.76 for EMG02, resp.
For the Erythromycin, the inhibition zones were 15.45, 15.66 and 17.46 for
EMG03; 15.04, 15.32 and 17.18 for EMG04 and 14.79, 15.03 and 16.78 mm for
EMG05, compared with 15.68, 15.75 and17.63 for EMG01 and 15.26, 15.53 and
17.27 mm for EMG02, resp. In case of crystal nano-emulsion were 20.88, 24.87
and 29.86 for EMG03; 20.76, 24.66 and 29.32 for EMG04 and 20.35, 24.57 and
29.13 for EMG05, compared with 20.53 mm, 24.68 and 29.89 mm for EMG01 and
20.19, 24.26 mm and 29.73 mm for EMG02, resp.
Also, the effect of Neomycin showed inhibition zones of 16.93,17.31 and
18.02 for EMG03; 16.65,17.19 and 17.91 for EMG04 and 16.22, 17.98 and 17.86
for EMG05, Resp., compared with 11.83, 11.96 and 12.06 for EMG01 and 16.89,
1723 and 17.93 for EMG02, resp. Although the reference strain EMG01 was
already resistant to Neomycin, it became slightly more sensitive with significant
difference after the addition of emulsions or nano-emulsions, as significant
differences in inhibition zone diameters were found between the rest of tested
E.coli and reference strain EMG02. The inhibition zones diameters recorded for
nano-emulsion of thymol crystals were 22.96, 27.04 and 30.25 for EMG03; 22.72,
26.81 and 29.94 for EMG04 and 22.53, 26.65 and 29.86 for EMG05 compared with
15.09, 17.01 and 19.53 for EMG01 and 22.83, 26.95 and 30.16 for EMG02, resp.
However, the effect of Chloramphenicol showed inhibition zones of 16.58,
17.13 and 17.80 for EMG03; 16.46, 16.98 and 17.72 for EMG04 and 16.01, 16.73
and 17.64 for EMG05, compared with 16.60, 16.71 and 16.44 for EMG01 and
11.65, 11.79 and 11.86 for EMG02, resp.
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Although the reference strain EMG02 was already resistant to
Chloramphenicol, it turned slightly sensitive to used treatments under used various
concentrations, The differences in inhibition zone diameters were significant
between all isolated strains treated with nanocrystal. The inhibition zones were
22.49, 26.78 and 30.02 for EMG03, 22.57, 26.58 and 29.79 for EMG04 and22.35
mm, 26.46 mm, and 29.65 mm for EMG05, compared with 22.31, 26.45 and 29.66
for EMG01 and 14.67 mm, 16.91 mm and 19.35 for EMG02, under used
treatments, resp. Also, the effect of Ciprofloxacin under the nanocrystal treatments,
the inhibition zone diameters were 21.09, 23.14 and 25.62 for EMG03, 21.73,
23.02 and 25.47 for EMG04 and 21.56 mm, 22.86 mm, and 25.35 mm, compared
with 21.24, 23.21 and 25.79 for EMG01 and 20.93 mm, 23.06 mm, and 25.53 mm
for EMG02, resp. Forth more, the increased inhibition zones after adding the nanoforms of oil or crystals are consistent with some evidence that components of
essential oils interact synergistically with antibiotics by interfering with antibiotic
resistance mechanisms.
On other side, the G-ve pathogenic K. pneumonia which isolated from UTI
patient was resistant to Ampicillin and Erythromycin 10 µg, however the addition
of 2.5% concentration of thyme or thymol emulsion significantly affected
differences between all oil forms (emulsion and nano-emulsion) in inhibition zone
diameters. The antibiotics of Neomycin and Chloramphenicol 30 µg showed
similar effects when used with emulsion of thyme oil or thymol crystals.
Also, nano-emulsions forms were similar in their effect, and it was
noticeable the significant of nanoforms.
Also, G+ve S. saprophyticus isolated from UTI patients showed resistance to
Ampicillin (10 µg) and Ciprofloxacin (5 µg). However it became sensitive to these
antibiotics after addition of emulsion or nano-emulsion of thyme oil or thymol
crystals. But the inhibition zones were increased when nano-emulsion forms of
were used, The same effect with the two antibiotics, Neomycin and
Chloramphenicol 30 µg, while the use of Erythromycin with nano-emulsion always
showed significant differences in inhibition zone diameters.
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Moreover, the determination of the inhibition’s zones was dramatically
increased by increasing the natural additives concentrations, and it is normal to
record the highest concentrations levels of minimal inhibitory concentrations and
minimal inhibitory bacterial concentrations (MICs and MBC) which reached to 220
and 440 mg/ml, resp. with bactericidal efficiency (MBC/MIC) by 200% from the
bacteriostatic effects.
It’s clear to noted that, the nano-emulsion forms can sharply decrease the
used amounts of thyme oil or thymol crystal to reach the MIC and MBC by 50%.
The using of 180 to 220 mg/ml of nanoforms sufficient to reach MBCs for the G-ve
bacteria from the G+ve UTI E.coli and K. penumenae genera, while 145 to 180
mg/ml of nanoforms sufficient to reach MBCs for the G+ve UTI S. saprophyticus.
The Transmission electron microscopy was used to study the effect of oil on
the shape and walls of UTI pathogenic E. coli and treated thymol nano-emulsion
2.5%. The images of the cells confirmed the change in the external shape of the E.
coli cells, also showed the presence of damage to the cell walls resulting from the
addition of the natural form of nano thymol, which allows the entry of the antibiotic
into resistant bacteria and enhances its effect when used together as a step to break
the ability of microbes to resist antibiotics.
Conclusion: The study concluded that adding a nanoemulsion of thyme oil
at a concentration of 2.5% to antibiotic treatment for bacteria causing urinary tract
infections increases the effectiveness and effect of antibiotics, strengthens their
action, and reduces bacterial resistance to antibiotics, and thus reduces the doses
used and the duration of treatment and economic cost . .
Recommendations: 1- Using the active compounds (thymol and thyme oil )in
nanometers form at a concentration of 2.5% to support antibiotics.
2- Including thyme flowers an leaves in therapeutic nutrition programs due to its
important components of thymol, cavacrol, phenols, and carbohydrates, and its
ability to resist pathogenic microorganisms.