The eight E. coli isolates confirmed by producing bright
pink colonies on Mackonkey agar and black centered colonies on the EMB agar.
The differences in the morphological characters of the colonies in  the isolates may be due to the choice of host
tissue or due to the transfer  method of
host  leads to loosing or acquiring some
properties  Dean (1990) and Dubreuil et
al. (1991). The morphology of the isolated bacteria
showed short rods, pink coloured, Gram negative bacilli in Gram’s staining. The
eight isolates of E. coli were identified according to biochemical
characteristics. In this study  almost
all the isolates of E. coli fermented mannitol, dextrose sucrose,
lactose, and maltose with the acid production, while the two E. coli isolated
from sewage  did not show maltose fermentation
 Ali et al. (1998).  There was a little or no difference in the
biochemical characters which stated that such similarity in the isolates might
be due to presence of some common genetic materials. The results of Catalase, Methyl
red and indole test of the E. coli isolates were positive for the all
isolates, while Voges-Prosequare test was negative which are in agreement with  Buxton and Fraser (1977) and Honda et al.
(1982).

 

The eight E.coli isolates showed different responces to the
three antibiotics used. One E.coli isolate(E.coli S1) showed
moderate resistance to two antibiotics (streptomycin and ampicillin),and it was
sensitive to tetracyclin. Three isolates (E.coli Af, E.coli
W, E.coli A and E.coli U) were sensitive to streptomycin but
resistan to ampicillin and tetracyclin. Three 
E.coli isolate(E.coli
S2, E.coli Ww and E.coli Hw)  showed resistance to the three antibiotics.  (Onyuka et
al., 2011) investigated that E. coli isolates were resistant to tetracycline,
ampicillin, cotrimaxazole, chloramphenical and gentamycin. These findings were
almost similar to our results for the first two cases. (Nthenge et al.,
2008) reported that Escherichia coli found resistant to ampicillin,
nalidixic acid and Kanamycin which was almost similar to our results in  the case of ampicillin . (Apun et al.,
2008) reported 11-95% range  resistance
of E. coli isolates to ampicillin, tetracycline and gentamicin in
Malaysian broiler chicken . Rahman et al., (2008) reported37-87.5%
 resistant to chloramphenicol,
ampicillin, ciprofloxacin, tetracycline and streptomycin of E. coli  isolates 
in layer poultry and broiler in Bangladesh. (Islam et al. 2008)
reported the multy drug resistance of E.coli from poultry in Bangladesh against
tetracycline, penicillin, erythromycin and chloramphenicol. They investigate
the resistance of 66-100%  of E.coli
isolates against these antibiotics . Tricia et al. (2006) reports
was agree with our results .They investigate the resistance of  of E. coli against ampicillin and he
found that 43% of the isolates were resistant, but they did not ivestigate any
resistance of  the isolates against gentamicin.
Daini and Adesemowo (2008) reported 54% and 88%   resistance against gentamicin and tetracycline
of E. coli strains from Nigeria.

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A total 30 different water samples were collected and divided in to
three groups according
to the site from which they originated agreculture drain water, sewage and hospital waste water. 20 samples exhibited presence of coliphage while 10 samples did
not have coliphages according to spot test detection (Armon and Kott ,1993)
. The purification
stages resulted in nine different coliphages. It was investigated that the hospital waste water had the highest
bacteriophage  levels. The sewage showed
lower phage levels. These two areas were suspected to be higher phage
concentration than the the agreculture drain water. This was as expected
because this water was the storage site for wastes.  There was not much that could be done with
this water to prevent bacteriophage infestation except ensuring that the waste
that was collected in them did not contain E. coli. Although bacteria as
well as phages are prevalent in the environment, the most suitable habitat for
the phages is the host bacterium (Primrose, 1990). The spot test  was used to detect phages in
the environmental samples and confirmed with 
double agar layer (DAL) method, using exponential cultures of E. coli
as the host bacterium. This method is widely used in bacteriophage isolation (Lu et al.,
2003). Bacteriophages are prevalent in the environment (Wommack et al.,
1992), therefore the only bacteriophages of interest for this study were
those that can infect the E. coli. The formation of plaques on the DAL
plates was evidence of the presence of active bacteriophages in the samples. It
was shown that when undiluted bacteriophage samples were used, numerous plaques
were visible in the plates . prior to enumeration of plaques dilutions up to  10-10 were necessary. Plaques of
three size catigories were formed by all these samples after 18 hours of
incubation, and were categorized as small (

1.5mm), medium (2mm)  and large
plaques (

3mm) . The plaques formed had turbid zones with clear centers or clear
zones with no centers and presence or absence of halo, Since the plaques formed
morphologically different size, shape and other morphological characters. Purification
was carried out following the isolation of phages from the samples. DAL was
done using the samples that have been grouped into three catigories. The
resulting plaques also showed 22 different plaques , and the prevalent plaque
size from each plate was used in this purification step. Each plaque used resulted
in mixed plaque sizes and 13different plaque was resulted from the second
purification step  therefore making it
necessary to repeat purification steps three times. Medium sized plaques were
chosen from the mixture as they were more prevalent than the other sizes.
Purification was done to ensure that each bacteriophage sample had one type of
phage in it. After repeating the purification steps three times,nine diffrent
samples with similar sized plaques for the same sample were seen in the DAL
plates. Purified stocks of bacteriophages were
then enriched with petri dish method . This 
method was also simple and could be used with ease in any
simply-equipped microbiology laboratory. It also allowed rapid preparation of
purified stocks of bacteriophages .phages showed variation in host range. Two phages
were able to have abroad host range
,four phages failed to infect only one bacterial isolate and three phages
failed to infect two isolates. The phage  investigates its
sutable  host with horizontal gene transfer
(HGT) of the host bacteria. Bacteriophages can forming lysogens in their
hostbacteria by  moving their own genome
in their host genomes . Lysogenic conversion, the expression of phage genes
from the prophage,plays a role in   pathogenesis
of several bacterial species (Brüssow et al., 2004; Hyman and Abedon,
2008; Fortier and Sekulovic, 2013).

The TEM micrographs
showed that there was a single type of phage in each purified sample.  All the bacteriophage samples followed the
same lysis trend and they were all lytic phages,but with diffrent  morphological characters .The nine phages
seemed to belong to three different phages. Four phages (Ec.ph1, Ec.ph3, Ec.ph6 and Ec.ph9) were among the family Myoviridae, three phages(Ec.ph1, Ec.ph5 and Ec.ph8)   belonged to Siphoviridae and two phages
(Ec.ph4 and Ec.ph7) were among the family Podoviridae   Ackermann (2009)

 

The one-step growth curve of Ec.ph4 phage
was performed to calculate burst size of the phage. The latent period was only 10
, which considered shorter than the latent periods of most phages of Myoviridae
(21–120 min) . The short latent period help the phage for fast replication. The
 burst size of our phage was  (90 and 200) PFU, which ranging from  50to100 PFU/cell for most phages in
Myoviridae  (Chang et al.,
2005; Raya et al., 2006; Bao et al., 2011; Park et al.,
2012). A few Myoviridae phages have very large burst sizes.For PhaxI phage
the burst size  is 420 PFU per cell (Shahrbabak
et al., 2013).

Determinining the host range of the phages for
therapeutic usage was in need of mixing the nine phages to form phage cocktail. That phage cocktail
was capable of causing bacterial killing of eight  E.coli 
isolate while the spot test on the four salmonella strains revealed that
productive infection was only achieved on one bacterial strain of the four
strains in the collection, however the lysis was
very weak for the salmonella isolate but the strong lysis of salmonella
isolates may be need to more specific phages for salmonella.The phage
cocktail was abetter way for determining the host range because it gave awide
range of infection than using separated phages. The phage cocktail seemed to have
abroad host range. Tt was able to infect two species of bacteria. ( Uchiyama
et al., 2008; Khan and Nilsson, 2015; Yu et al., 2016). The phage cocktail
also was able to infect different strains of the same species E.coli . (Vinod
et al., 2006; Gupta and Prasad, 2010; Anand et al., 2015; Xu et al., 2016).
The phage cocktail was able to infect eight E.coli isolates  and one salmonella isolate. It is
difficult to determine the host range of a specific phage because measuring
host ranges depends on the used technique (Hyman and Abedon, 2010).  The success or failure of  phage adsorption reflrcted on the
determination of the host range,but the determination of host range for phage
therapy usage is by host cell killing Adams (1959). There are many studies
carried on phages having broad host range or described as polyvalent (Paolozzi
and Ghelardini, 2006) made studies on  Mu phage which was  able to infect species of E.coli, Enterobacter,
Shigella sonnei, Citrobacter freundii, and Erwinia.  Yu et al,. (2016) failed to achive broad host range for their phages,but
when they used two methods depending on the simultaneous multi-host protocol ,
they was able to have phages which have abroad host range against several
strains of E. coli and P. aeruginosa as well as one strain each
of P. putida and P. syringae. A collection of phages with a host
range covering bacteria from more than one genus would facilitate to reduce the
number of necessary phage stocks for phage therapy. There are reports of phages
being effective against more than one bacterial species (Hyman and Abedon
2010).

Present study findings regarding exertion of  influence on the survival of virus by pH of
water media through affecting the  virus
adsorption to other particles. Similarly the
findings of the present study regarding the stability had shown by  the phagecocktail at different pH regimes
ranging from 4 to 9 . pH finding of the study confirmed Langlet et al.
(2007) results which indicated that virus exhibited stability at wide range
of pH regimes.

Temperature is one of the most important environmental
factor that strongly affects many aspects of the biological systems. Influence
of temperature upon the biological system is very vivid and it has been
observed that evolution of phenotypic traits, species distributions, and
extinctions in many cases can be traced to changes in temperature regimes (Vale
et al.,2008). Present study results are in confirmation with the above
findings as during the experiment it was observed that yield of  phage cocktail was highly temperature dependent.The phage cocktail was unable to develop and perform lysis on E.coli at
temperature above 45?C, while on  temperature between 25°C and
40°C, the
activity was carried out. , this study showed that at thermophilic temperature
45°C,  the phage cocktail developed and performed
lysis on his host bacteria and support the results of Pollard and Woodyatt
(1964), who reported that bacteriophage developed at 41.2°C.
While, temperature regimes from 50°C,
to 70°C proved
as limiting factor and caused the actual inactivation of the  bacteriophages . Study results regarding the
inactivation are in confirmation with those observed by Basdew and Laing
(2014) who reported that increase in temperature decreases virus survival and
activity. In the same way, findings by Pope et al. (2004)
that indicate an increase in bacteriophage yield till 30°C and
39°C corroborates
the present study results which revealed that 37°C was
ideal temperature for bacteriolytic activity of 
the phage cocktail against E. coli .

The phage cocktail was able to lyse E.coli
isolates after incubation in higly saline environment. The cocktail is amixture
of different nine phages belonging to three different families and different characters,So
The phage cocktail have the ability to adapt with  conditions more hard than individual  phages. Many phages were able to adapt with
environments with high salinity. Several bacteriophages were isolated from
marine water of different salinities. Wichels et al. (1998) studied 22
phages which they found in water near Helgoland in the North Sea. All of them
had tails and icosahedral heads of 50.2 to 99.3 nm, and they were
classified into three different families: 11 phages to Myoviridae, 7 to Siphoviridae,
and 4 to Podoviridae. No similarity in DNA structure was shown among phages
belonging to different families present in this area. There are many conditions
which may overlap with the phage   and
reduce the net concentration of the phage environment  West and Kelly (1962) reported that
mixing of 0.1ml of 10%NaCl with an equal amount of  the broth culture of the propagating strain
of Staphylococcus, followed by
immediate addition of 3 ml. soft agar, quickly diluted the concentration of
NaCl to about 0.8 %. The quantity of NaCl was further decreased on
plating the soft agar They had shown that the free phages
differ in their NaCl tolerance.Their results disussed the positive results in
high salt concentrations.

 

 

 

 

The phage cocktail was able to to lyse bacterial isolates after
exposure to UV rays for different periods of time. Many researches were performed
to discuss that UV rays affect the shape and number of paques, but not affect
the survival of phages. Kleczkowski J and Kleczkowski A (1953). have reported that aculture of irradiated phage produced the same number of plaques of
the control culture after 24 hour of incubation  , but with  plaques diameter  smaller 
than  the control. They did
not explain this phenomenon  to be due to
phage mutation, because the isolated phages from these small plaques produced
plaques of normal size . Luria & Delbriick (1942) reported
the role U.V in  the inactivation of the
phage which made interferance with phage multiplication and also cause harm
effect of the bacterial multiplicity, while  the other inactivated phage did not act so.
This results was not agree with our results.The excess of U.V irradiation could
destroy ability of the interference between the irradiated phage with phage
multiplication , and their capacity of making their host bacteria unable to
multiply also disappeared. The heterologous phages irradiated medium or preparations
if mixed in equal volumes; can slow bacterial multiplication in their liquid
cultures. The effect of U.V on the phage inactivation has a common feature  of some polysaccharides or ribonuclease
inhibitors, or due to the interference with phage multiplication in  the host bacteria liquid cultures , while
having no effect on the numbers of plaques formed on agar. This effect on the
former and not the latter, showing an apparent contradiction, has been
discussed elsewhere (Kleczkowski & Kleczkowski, 1952).  The multiplicity reactivation was observed
with a few coli phages. (Luria &, Dulbecco, 1949)  observed that in the phage
preparations which  irradiated with U.VThe
numbers of produced  plaques did
not increase with the increasing concentration at which they were brought into
contact with host bacteria before plating.

 

The titre of the phage cocktail after
enrichment steps was 2.2×1011.This titre was high enough for testing
its properties, preservation and formulation steps. Titration of
bacteriophages was done by plaque assay. Phage
plaques is a traditional method for phage
detection and expressed by  plaque forming unit (pfu). Plaque formation resulted
when the bacterial species are infected with the sutable phage (Cox
2012; Kalni?a et al. 2008). Traditional plaque assays have been used in
the detection of pathogens, including Staphylococcus aureus (Wallmark
et al. 1978),Escherichia coli (Oda et al. 2004), and Campylobacter
species (Grajewski et al. 1985). The mechanisms of
bacteriophage lysis have been studied 
for the bacteriophage

control
of bacterial virulence in animals and human (Young et al., 2000).
It has been found that lysis of the bacterial host is the final event in the infection
cycle of a lytic bacteriophage (Wang et al., 2000). The  phage cocktail did lysis from 10-1
to 10-10 dilutions ,but in the most high dillutions  10-11 and10-12
dilutions the phage cocktail had no lysis activity . It means that at highest
dilutions,  coliphages failed to do
lysis. Lysis can be produced by phage dilution up to a certain point; as Worley-Morse
et al. (2014) reported that bacteriophage concentration is very
important for lysis activity.

 

The perservation temperature of bacteriophage is very important
factor which determines phage activity.The phage cocktail was stored at three
different temperatures 4?C, room temperature and -20?C ,one sample from each temperature was taken and tested for presence
of phages. The droup of the number of plaqes of the first sample (stored at 4?C)
was faster than the other two samples . At the end of the experiment the number
of plaques of the sample preserved at -20?C was higher than the sample preserved at 4?C . As
shown by Ackermann et al,. (2004), tailed phages were the most
resistant to storage and showed the longest survivability.This results  agrees with our result. Olson et al. (2004)
recommend 4°C as the optimum temperature for short phage storage in wastewater
phags .This results agree with our results. Bacteriophage samples were preserved in bacterial
medium at 4  ?C and -20  ?C, and in SM buffer. Storage of the samples
at these two temperatures showed that bacteriophages were unstable,but in our
results the frozen semple showed abetter results than the other samples . This
was also observed by Rossi (1994).

The three formulas were performed and tested over about seven weeks.
The results were expected because the capsulation material protect phages from
environmental conditions. The royaljelly formula was the best used formula.it
give more protection to the phage cocktail, however the crude royaljelly was
not able to lyse bacteria. The royaljelly  gave phages the proper  protection them from protein denaturation. Studies on phage encapsulation have used a variety of
hydrophilic and hydrophobic polymers including agarose  (Bean et al., 2014), alginate ( Tang et al.,
2013 and kim et al., 2015), chitosan ( Kaikabo
et al., 2017 and Ma  et
al., 2008), pectin (Dini et al., 2012) , whey
protein(Samtlebe et al., 2016 and Tang et al.,
2015) , gelled milk protein (Samtlebe et al.,
2016), hyaluronic acid methacrylate (Bean et al., 2014), hydroxypropyl methyl cellulose (HPMC) (Alfadhel et al., 2011), poly (N-isopropylacrylamide) (Hathaway et al., 2015), Poly(dl-lactide:glycolide)
( Puapermpoonsiri et al., 2009), polyesteramide (Markoishvili
et al., 2002), polyvinyl pyrrolidone ( Dai et al., 2014), polyethylene oxide/polyvinyl alcohol (Salalha et al., 2006), cellulose
diacetate (Korehei and Kadla 2014), polymethyl
methacrylate (Govender
et al., 2015).
Triggers for phage release include polymer solvation, polymer dissolution
and erosion (Korehei and Kadla 2014),  polymer hydrolysis (Katsarava et
al., 1999), phase inversion induced by temperature (Hathaway
et al., 2015), and pH triggered dissolution of polymer and enzyme driven
polymer degradation (Bean et al., 2014). Developing formulations that incorporate bacteriophage
for therapeutic applications requires an appreciation of the chemical and
physical stresses phage may encounter both during processing as well as during
storage once formulated. Phage inactivation and long term reduction in phage
titre upon storage is highly undesirableA polymer or lipid may be used to coat
an existing structure containing the phage. Murthy and Engelhardt (2009) sprayed phage on dried skimmed milk and then
encapsulated them in a lipid coating. There are many techniques and processes
that may be used for stabilising, immobilising and encapsulating phage. In the
current study the phage cocktail was sprayed on dried skim milk and mixture of
sucrose and corn flour with equal ratio and encapsulated in nun lipid coating
and also give good results. The most common methods are spray-drying, spray
freeze drying, freeze drying, extrusion dripping methods, emulsion and
polymerisation techniques.  Phages are protein structures and they are
therefore susceptible to factors known to denature proteins; these include
exposure to organic solvents ( Puapermpoonsiri et al.,
2009), high temperatures (Briers et al.,
2008), pH (Knezevic et al., 2011), ionic strength (Knezevic
et al., 2011), and interfacial effects. Additionally, mechanical
stresses during formulation or encapsulation including shear stresses during
mixing and agitation and atomisation during spraying (Leung
et al., 2016). One of the most common and successful modes of long-term
preservation of bacteriophage is storage at 4 °C in Trypticase Soy Agar
and Brain Heart Infusion broth whereas storage at ? 80 °C requires
50% glycerol as a cryoprotectant (Clark 1992) or
freeze drying with excipients (e.g. sucrose or trehalose) as lyophilization and
cryoprotectants (Ackermann et al., 2004).
Ackermann et al (2004) observed that
phage titres for one of the largest collections of tailed phages typically
tended to drop by 1 log over the course of a year but then remained fairly
stable (although there were many individual variations). Clark (Clark
1992)  working for the American Type Culture Collection (ATCC)
evaluated storage stability of a wide variety of bacteriophages (16 in total)
for long term preservation and distribution. Phage specimens were treated and
stored for two years at room temperature and at 4 °C as (i) broth lysates;
(ii) lysates diluted with 50% glycerol; (iii) saturating filter paper with
lysate and then drying; (iv) freeze dried by mixing lysates with an equal
volume of double strength skim milk. Phage titres measured immediately
post-processing indicated that freeze drying resulted in a significant loss of
titre (between 1 log and 2 log reduction). However, freeze drying did
produce stable phage titres over the course of 2 years when stored
refrigerated. After two years, the titre of phage in the broth lysates were
found to be generally higher than those of glycerol or freeze dried
preparations. Thermally dried preparations generally did not prove
satisfactory. Preparations stored at 4 °C showed higher titres than those
kept at room temperature.this results was agree with our results as our
preparation stored at 4?C. All titres declined with time regardless of the
conditions of preservation.

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