الفهرس 
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Abstract
Waterborne pathogens can be divided into three main groups:
bacteria (included Listeria monocytogenes, Salmonella spp.), viruses and
parasites. Microbial contamination of different water resources were
happened through discarding treated or untreated sewage in water in
addition to several pollutant sources. The seriousness of the waterborne
pathogens is that they infect both humans and animals and cause serious
diseases that may end in death. When irrigation water is contaminated
with water-borne pathogens, the plants are polluted and become a major
source of foodborne disease. In recent years, due to the increased
presence of bacteria resistant to antibiotics, bacteriophages are used in the
biological control of foods. Phages are high specialization of the host and
are multiply their numbers of self and do not affect the microflora
intestinal.
The aim of this study was to use specific bacteriophages as a safe
alternative to control of some human pathogenic bacteria transmitted
through water polluted with sewage. To achieve this important objective,
the study was carried out as follows:
1. In this study four water samples were collected from different
canals during winter and spring seasons. Al samples were tested
by bacteriological examination. TVBCs were estimated at 22°C,
their counts were 8×104, 4×106, 8.6×108 and 2 ×109 cfu/mL in El-
Esmailia canal, El-Sharkawia canal, Meet Nama canal and El-
Mariotya canal, respectively. It was found that when TVBCs were
estimated at 37°C, their counts were 3×104, 1.3×106, 2×108 and
8.7×108 cfu/mL respectively. The highest number was 2 ×109
cfu/mL at 22°C and 8.7×108 cfu/mL at 37°C in El-Mariotya
canal`s water. While the lowest number was 8×104 cfu/mL at
22°C and 3×104 cfu/mL at 37°C in El-Esmailia canal`s water.
SUMMARY
Yasmer S. Hussein (2018), Ph.D. Fac. Agric., Ain Shams Univ.
147
2. When estimated the MPN of TC found that the highest number
was 1.6×106 cfu/100 mL in El-Mariotya canal`s water and the
lowest number was 9×103 cfu/100 mL in El-Esmailia canal`s
water. FC numbers existed in the four canals` water. The highest
number was 5×105 cfu/100 mL in El-Mariotya canal`s water. The
lowest number was 2.1×103 cfu/100 mL in El-Esmailia canal`s
water. FS numbers existed in the four canals` water. The highest
number was 6×104 cfu/100 mL in El-Mariotya canal`s water. The
lowest number was 2.3×102 cfu/100 mL in El-Esmailia canal`s
water.
3. Listeria bacterial cells were assayed in the irrigation samples and
it was found that the two samples from Meet Nama canal and El-
Mariotya canal were contained Listeria. El-Esmailia canal`s water
and El-Sharkawia canal`s water were free from Listeria. The
irrigation water samples obtained from El-Sharkawia canal, Meet
Nama canal and El-Mariotya canal were contained Salmonella
bacteria while El-Esmailia canal was free from Salmonella
bacteria.
4. The bacterial cultures of L. monocytogenes and S. Typhimurium
were induced by the UV induction method and the results
indicated that, the two bacterial hosts were free from the temperate
phages.
5. Sixteen samples were obtained from different canals and sewage
water from Fac. of Agri., Ain shams Univ., Shoubra El-Kheima
station of sewage water, Passous, El-Gabal El-Asfar waste water
treatment plant, ElKhanka. Phages specific for L. monocytogenes
and S. Typhimurium were detected using spot test method in six
samples in case of L. monocytogenes and six samples in case of S.
Typhimurium. Six virulent phages specific for L. monocytogenes
and six virulent phages specific for S. Typhimurium were isolated
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Yasmer S. Hussein (2018), Ph.D. Fac. Agric., Ain Shams Univ.
148
using single plaque assay. The biologically purified L.
monocytogenes and S. Typhimurium phage lysates were
propagated by the liquid culture method to obtain large amount of
the particles before the ultracentrifugation purification. The
ultracentrifugation was at 30000 rpm for 90 min.
6. Electron microscopy of negatively stained Listeria phages showed
that, the particles have curled non contractile tail with sizes of
269.2 , 260 and 207.7 nm in length and 15.4 ,20 and 7.7 nm in
width and isometric head shape with diameter of about 69.2, 80
and 61.5 nm for ØLG , ØLA and ØLM, respectively, they
belonged to family Siphoviridae. While the others have particles
with contractile tail with sizes of 156 nm, 169.2 and 153.8 nm in
length, 22, 23.1 and 23.1 nm in width and isometric head with
diameter of about 100, elongated head with diameter 84.6 × 92.3
and isometric head 100 nm for ØLN, ØLD and ØLP, respectively,
they belong to family Myoviridae . Electron microscopy of
negatively stained Salmonella phages showed that the particles
have contractile tail with sizes of 145.5, 153.8, 164.3, 157.1, 163.6
and 172.7 nm in length and 27.3, 23.1, 21.4, 21.4, 18.18 and 27.3
nm in width and head with diameter of about 109.1, 92.3×77,
107.1, 92.9, 109.1×100 and 118.2×127.3 nm for ØSM, ØSF, ØSG,
ØSP, ØSA and ØSD, respectively, they belonging to family
Myoviridae.
7. In this investigation, all isolated Listeria phages lost its infectivity
after exposure to 90ºC for 10 min. The isolated Salmonella phages
ØSM, ØSF and ØSG were more stable after thermal treatment at
98ºC for 10 min while phages ØSP, ØSA and ØSD were still had
the ability to lyse Salmonella after thermal treatment at 90ºC for
10 min.
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Yasmer S. Hussein (2018), Ph.D. Fac. Agric., Ain Shams Univ.
149
8. In this study, the results of stability of Listeria and Salmonella
phages at RT and refrigerator, revealed that Listeria and
Salmonella phages were high stable and survived for at least 60
days at RT and refrigerator, when the viral infectivity was assayed
qualitatively weekly by the spot test.
9. Results of this study revealed that, all Listeria and Salmonella
phages didn`t lose its ability to lyse L. monocytogenes and S.
Typhimurium cells at pH ranged from 4.0 to 12.0 .The results
indicated that the Listeria and Salmonella phages are stable in
both the alkaline media and acidic ones.
10. Listeria and Salmonella phages were exposed to UV irradiation at
two different distances far from it. They are at 35 cm from the UV
source for 15, 30, 45, 60, 75 and 90 min, and 30, 60 and 90 min at
53 cm distance from the UV source. Listeria phages didn`t lose
their infectivity after exposure to UV irradiation and Salmonella
phages didn`t lose their infectivity at all.
11. Host specificity of Listeria and Salmonella phages were
determined qualitatively by the spot test against eight bacterial
serotypes and isolates belonging to L. monocytogenes and Listeria
sp. Results indicate that, the isolated that bacteriophages were able
to lyse most of them. This result means that, the isolated Listeria
phages have a narrow host range. While nine bacterial strain,
serotypes and isolates belonging to S. Typhimurium , S. Paratyphi
B, S. Typhi and Salmonella sp. were tested against the infection
with the isolated S. Typhimrium phages. Results indicated that, the
isolated bacteriophages were able to lyse most of them. This result
means that, the isolated Salmonella phages have a restricted host
range.
12. The UV spectra of the purified Listeria and Salmonella phages
were determined by measuring the absorption of UV using
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150
spectrophotometer at wave length from 200 to 320 nm. Data for
Listeria phages showed that, A260/280 ratio was 1.3520, 1.289,
1.347, 1.328, 1.319 and 1.362 for ØLG, ØLN, ØLA, ØLD, ØLM
and ØLP, respectively. Data for Salmonella phages showed that,
A260/280 ratio was 1.3145, 1.3145, 1.2989, 1.3400, 1.3148 and
1.345 for ØSM, ØSF, ØSG, ØSP, ØSA and ØSD, respectively.
The values of A260/280 ratio indicated that the well purification of
virus particles for the phage isolates.
13. Protein quantity of Listeria and Salmonella phages were
determined using Bradford method. The amount of protein in
Listeria phages was 0.190, 0.195, 0.200, 0.230, 0.225 and 0.245
μg/mL for ØLG, ØLN, ØLA, ØLD, ØLM and ØLP, respectively.
The amount of protein in Salmonella phages was 0.212, 0.220,
0.210, 0.232, 0.245 and 0.240 μg/mL for ØSM, ØSF, ØSG, ØSP,
ØSA and ØSD, respectively.
14. Protein properties of the isolated phages were determined using
SDS-PAGE. The results of Listeria phages showed that the 10
total bands contained 5 bands in ØLG with molecular weight ∼87,
70, 45, 37 and 35 kDa. ØLN phage had 5 bands with molecular
weight ∼ 68, 45, 35, 33 and 30 kDa. ØLA phage had 6 bands with
molecular weight ∼ 66, 37, 35, 33, 30 and 28 kDa. ØLD phage
had 6 bands with molecular weight ∼ 45, 37, 35, 33, 30 and 28
kDa. ØLM phage had 5 bands with molecular weight ∼ 66, 45, 37,
33 and 28 kDa. ØLP phage had 6 bands with molecular weight ∼
66, 45, 35, 33, 30 and 28 kDa. The 15 total bands of Salmonella
phages contained 7 bands in ØSM with molecular weight ∼120,
116, 80, 75, 45, 35 and 28 kDa. ØSF phage had 6 bands with
molecular weight ∼75, 45, 37, 35, 28 and 25 kDa. ØSG phage had
7 bands with molecular weight ∼ 76, 45, 37, 35, 30, 28 and 25
kDa. ØSA phage had 6 bands with molecular weight ∼120,100,
76, 45, 35 and 25 kDa. ØSP phage had 8 bands with molecular
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151
weight ∼ 97, 78, 66, 45, 37, 35, 30 and 28 kDa. ØSD phage had 5
bands with molecular weight ∼ 116, 97, 78, 45 and 35 kDa.
15. RAPD-PCR was used to appear the genetic variation between the
six phages of Listeria as Salmonella. A total number of 6 DNA
fragments was amplified. The DNA fragments were ranged from
500 to 2000 bp and distributed among the six Listeria phages as
unique (1), polymorphic (1) and monomorphic (4) fragments,
while six Salmonella phages as unique (1), polymorphic (3) and
monomorphic (2) fragments. For the six phages of Listeria as
Salmonella under investigation, 500, 600, 800, 1000, 1500 and
2000 DNA fragments were flanking via the RAPD-PCR primer
for ØLG, ØLN, ØLA, ØLD, ØLM, ØLP, respectively, ØSM,
ØSF, ØSG, ØSP, ØSA and ØSD, respectively.
16. In this study, numbers of in vitro experiments have been
performed to evaluate the potential of the Listeria and Salmonella
phage cocktails for the reduction the bacterial cell numbers using
different MOI to choose the optimal MOI to use in the biological
control of pathogenic bacteria. The optimal MOI was 10 and 100
which achieved the target about reducing the cells by 1.44 log,
1.60 cfu/mL and 1.75, 2.07 log cfu/mL, respectively after 4 h.
17. Determination the safety of using phages as a food additive was
done by testing the cytotoxicity of phages on liver carcinoma cell
line in vitro. Different concentrations from Listeria and
Salmonella phage cocktails were used. The results indicated that
all phages didn`t show any cytotoxic effect at different
concentrations.
18. In this study, vegetable sample (lettuce) irrigated with polluted
irrigation water and three samples of green salads were obtained
from different local markets (famous and big restaurant, local
small restaurant and street vendor) and analyzed microbiologically
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152
to assay their bacterial load and presence or absence of the
pathogenic bacteria. Results of the microbiological analysis
showed that TVBC in vegetable sample (lettuce) was 2.71×108
cfu/g and total pathogenic bacteria was 2.8×107 cfu/g.
Determination of TVBC in the collecting green salad samples was
2.71×10
11
, 2.58×10
11
and 2.25×10
9
cfu/g, respectively. Determination
of total pathogenic bacteria in the collecting green salad
samples was 2.9×1010 , 2.45×1010 and 1.19×106 cfu/g respectively.
The suggested isolated genera from the green salads were
as following: Escherichia sp., Enterobacter sp., Salmonella sp.,
Listeria sp. and Shigella sp.
19. Microbiological analysis of the three green salads revealed also
that, the green salads contained the phage specific for the main
hosts in this study L. monocytogenes and S. Typhimurium and
isolated three phages.
20. Effects of different concentrations of some food additives to green
salad (salt, lemon, vinegar and spices) were tested on the
infectivity of phages isolated from green salad, Listeria and
Salmonella phages and incubated at room temperature for 6 and
12 h. The results of the spot test indicated that, all phages didn`t
lose their infectivity after treatment by the different food additives.
An applied experiment was designed to use cocktail from Listeria
and Salmonella phages to reduce these microbes’ numbers in green salad.
Green salad was selected for the non-exposure of this type of food to any
thermal treatment chosen because of such foods never exposure to any
thermal treatments. Thus, the microbial load was increased. Cocktail
from phages isolated from green salad, Listeria and Salmonella phages
was used with MOI of 100.
Green salad was processed at the laboratory by selecting vegetables
were fresh, intact, uninjured and healthy appearance (cucumbers,
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Yasmer S. Hussein (2018), Ph.D. Fac. Agric., Ain Shams Univ.
153
tomatoes, onions, green peppers, iceberg lettuce and fresh parsley). They
were sterilized superficially using 70% ethanol and washed several times
using sterile distilled water. The sterilized green salad was divided into
seven groups presents treatments. Three replicates were used for each
treatment.
Inoculation of bacterial culture cocktail 106 cfu/mL was done before
the addition of phages and incubated for 1 h at RT in the third and fourth
treatments.
The addition of phage cocktails 108 pfu/mL was done before the
bacterial inoculation and incubated for 30 min at RT in the sixth and last
treatments. In the sixth and last treatments, Tween 80 (surfactant) was
added to the phage cocktail and used within the steps of salad processing,
to coat plastic bag to put vegetables on it, cutting knife, wooden cutting
board, stainless steel bowls and plastic cases which was to put salad on it
in the final step. The green salad was divided into seven groups and
treated as follows:
1. Green salad was left without any treatment as a control.
2. Green salad was treated by bacterial culture cocktail suspension.
3. Green salad was treated by bacterial culture cocktail followed by
addition of their specific phage cocktail.
4. Green salad was treated by bacterial culture cocktail followed by
addition of their specific phage cocktail, then the food additives
(salt, lemon, vinegar and spices).
5. Green salad was treated by bacterial culture cocktail and their
specific phage cocktail at the same time.
6. Green salad was treated with phage cocktail followed by bacterial
culture cocktail.
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Yasmer S. Hussein (2018), Ph.D. Fac. Agric., Ain Shams Univ.
154
7. Green salad was treated with phage cocktail followed by bacterial
culture cocktail in addition to the food additives (salt, lemon,
vinegar and spices).
Aliquots of the treatments were taken at zero time, 4, 8 and 24 h to
count Listeria and Salmonella numbers on their specific media, also
phage titers were counted.
The optimal treatment was the contamination by bacterial culture
cocktail after the addition of their specific phage cocktails. The numbers
of Listeria were reduced after 4 h from the addition by 1.17 log cfu/g. The
numbers of Salmonella were reduced after 4 h from the addition by 1.6
log cfu/g. Followed by the contamination by bacterial culture cocktail at
the same time with the addition of their specific phage cocktail. Listeria
was reduced after 4 h from the addition by 1.11 log cfu/g. Salmonella was
reduced after 4 h from the addition by 1.5 log cfu/g. Followed by the
contamination by bacterial culture cocktail before the addition of their
specific phage cocktail. Listeria was reduced after 4 h from the addition
by 0.98 log cfu/g. Salmonella was reduced after 4 h from the addition by
1.3 log cfu/g. The food additives were increased the reduction in both
Listeria and Salmonella counts comparing to not addition.
Recommendations:
1. Using bacteriophages to reduce the number of bacteria,
especially since they are harmless and safe.
2. Bacteriophages are added before bacteria during the
manufacturing process.
3. The addition of food additives such as spices increases the rate of
decreasing in the bacterial numbers