The eight E. coli isolates confirmed by producing brightpink 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 hosttissue or due to the transfer  method ofhost  leads to loosing or acquiring someproperties  Dean (1990) and Dubreuil etal.

(1991). The morphology of the isolated bacteriashowed short rods, pink coloured, Gram negative bacilli in Gram’s staining. Theeight isolates of E. coli were identified according to biochemicalcharacteristics. In this study  almostall the isolates of E.

coli fermented mannitol, dextrose sucrose,lactose, and maltose with the acid production, while the two E. coli isolatedfrom sewage  did not show maltose fermentation Ali et al. (1998).  There was a little or no difference in thebiochemical characters which stated that such similarity in the isolates mightbe due to presence of some common genetic materials. The results of Catalase, Methylred and indole test of the E.

coli isolates were positive for the allisolates, 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 thethree antibiotics used. One E.coli isolate(E.coli S1) showedmoderate resistance to two antibiotics (streptomycin and ampicillin),and it wassensitive to tetracyclin.

Three isolates (E.coli Af, E.coliW, E.coli A and E.coli U) were sensitive to streptomycin butresistan to ampicillin and tetracyclin.

Three E.coli isolate(E.coliS2, E.coli Ww and E.coli Hw)  showed resistance to the three antibiotics.

 (Onyuka etal., 2011) investigated that E. coli isolates were resistant to tetracycline,ampicillin, cotrimaxazole, chloramphenical and gentamycin. These findings werealmost 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  resistanceof E. coli isolates to ampicillin, tetracycline and gentamicin inMalaysian 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 againsttetracycline, penicillin, erythromycin and chloramphenicol.

They investigatethe resistance of 66-100%  of E.coliisolates against these antibiotics . Tricia et al. (2006) reportswas agree with our results .They investigate the resistance of  of E. coli against ampicillin and hefound that 43% of the isolates were resistant, but they did not ivestigate anyresistance of  the isolates against gentamicin.Daini and Adesemowo (2008) reported 54% and 88%   resistance against gentamicin and tetracyclineof E.

coli strains from Nigeria. A total 30 different water samples were collected and divided in tothree groups accordingto the site from which they originated agreculture drain water, sewage and hospital waste water. 20 samples exhibited presence of coliphage while 10 samples didnot have coliphages according to spot test detection (Armon and Kott ,1993).

The purificationstages resulted in nine different coliphages. It was investigated that the hospital waste water had the highestbacteriophage  levels. The sewage showedlower phage levels. These two areas were suspected to be higher phageconcentration than the the agreculture drain water. This was as expectedbecause this water was the storage site for wastes.  There was not much that could be done withthis water to prevent bacteriophage infestation except ensuring that the wastethat was collected in them did not contain E.

coli. Although bacteria aswell as phages are prevalent in the environment, the most suitable habitat forthe phages is the host bacterium (Primrose, 1990). The spot test  was used to detect phages inthe environmental samples and confirmed with double agar layer (DAL) method, using exponential cultures of E. colias 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 werethose that can infect the E. coli. The formation of plaques on the DALplates was evidence of the presence of active bacteriophages in the samples. Itwas shown that when undiluted bacteriophage samples were used, numerous plaqueswere visible in the plates .

prior to enumeration of plaques dilutions up to  10-10 were necessary. Plaques ofthree size catigories were formed by all these samples after 18 hours ofincubation, and were categorized as small ( 1.5mm), medium (2mm)  and largeplaques ( 3mm) . The plaques formed had turbid zones with clear centers or clearzones with no centers and presence or absence of halo, Since the plaques formedmorphologically different size, shape and other morphological characters. Purificationwas carried out following the isolation of phages from the samples.

DAL wasdone using the samples that have been grouped into three catigories. Theresulting plaques also showed 22 different plaques , and the prevalent plaquesize from each plate was used in this purification step. Each plaque used resultedin mixed plaque sizes and 13different plaque was resulted from the secondpurification step  therefore making itnecessary to repeat purification steps three times. Medium sized plaques werechosen from the mixture as they were more prevalent than the other sizes.

Purification was done to ensure that each bacteriophage sample had one type ofphage in it. After repeating the purification steps three times,nine diffrentsamples with similar sized plaques for the same sample were seen in the DALplates. Purified stocks of bacteriophages werethen enriched with petri dish method . This method was also simple and could be used with ease in anysimply-equipped microbiology laboratory. It also allowed rapid preparation ofpurified stocks of bacteriophages .phages showed variation in host range. Two phageswere able to have abroad host range,four phages failed to infect only one bacterial isolate and three phagesfailed to infect two isolates. The phage  investigates itssutable  host with horizontal gene transfer(HGT) of the host bacteria.

Bacteriophages can forming lysogens in theirhostbacteria by  moving their own genomein their host genomes . Lysogenic conversion, the expression of phage genesfrom the prophage,plays a role in   pathogenesisof several bacterial species (Brüssow et al., 2004; Hyman and Abedon,2008; Fortier and Sekulovic, 2013).The TEM micrographsshowed that there was a single type of phage in each purified sample.  All the bacteriophage samples followed thesame lysis trend and they were all lytic phages,but with diffrent  morphological characters .The nine phagesseemed 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 phagewas 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 inMyoviridae  (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 phagethe burst size  is 420 PFU per cell (Shahrbabaket al.

, 2013). Determinining the host range of the phages fortherapeutic usage was in need of mixing the nine phages to form phage cocktail. That phage cocktailwas capable of causing bacterial killing of eight  E.

coli isolate while the spot test on the four salmonella strains revealed thatproductive infection was only achieved on one bacterial strain of the fourstrains in the collection, however the lysis wasvery weak for the salmonella isolate but the strong lysis of salmonellaisolates may be need to more specific phages for salmonella.The phagecocktail was abetter way for determining the host range because it gave awiderange of infection than using separated phages. The phage cocktail seemed to haveabroad host range. Tt was able to infect two species of bacteria.

( Uchiyamaet al., 2008; Khan and Nilsson, 2015; Yu et al., 2016). The phage cocktailalso was able to infect different strains of the same species E.coli . (Vinodet 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 isdifficult to determine the host range of a specific phage because measuringhost ranges depends on the used technique (Hyman and Abedon, 2010).  The success or failure of  phage adsorption reflrcted on thedetermination of the host range,but the determination of host range for phagetherapy usage is by host cell killing Adams (1959). There are many studiescarried on phages having broad host range or described as polyvalent (Paolozziand 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,butwhen they used two methods depending on the simultaneous multi-host protocol ,they was able to have phages which have abroad host range against severalstrains of E. coli and P. aeruginosa as well as one strain eachof P. putida and P.

syringae. A collection of phages with a hostrange covering bacteria from more than one genus would facilitate to reduce thenumber of necessary phage stocks for phage therapy. There are reports of phagesbeing effective against more than one bacterial species (Hyman and Abedon2010).Present study findings regarding exertion of  influence on the survival of virus by pH ofwater media through affecting the  virusadsorption to other particles. Similarly thefindings of the present study regarding the stability had shown by  the phagecocktail at different pH regimesranging from 4 to 9 . pH finding of the study confirmed Langlet et al.(2007) results which indicated that virus exhibited stability at wide rangeof pH regimes.Temperature is one of the most important environmentalfactor that strongly affects many aspects of the biological systems.

Influenceof temperature upon the biological system is very vivid and it has beenobserved that evolution of phenotypic traits, species distributions, andextinctions in many cases can be traced to changes in temperature regimes (Valeet al.,2008). Present study results are in confirmation with the abovefindings 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 attemperature above 45?C, while on  temperature between 25°C and40°C, theactivity was carried out. , this study showed that at thermophilic temperature45°C,  the phage cocktail developed and performedlysis 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 provedas limiting factor and caused the actual inactivation of the  bacteriophages . Study results regarding theinactivation are in confirmation with those observed by Basdew and Laing(2014) who reported that increase in temperature decreases virus survival andactivity. In the same way, findings by Pope et al. (2004)that indicate an increase in bacteriophage yield till 30°C and39°C corroboratesthe present study results which revealed that 37°C wasideal temperature for bacteriolytic activity of the phage cocktail against E.

coli .The phage cocktail was able to lyse E.coliisolates after incubation in higly saline environment. The cocktail is amixtureof different nine phages belonging to three different families and different characters,SoThe phage cocktail have the ability to adapt with  conditions more hard than individual  phages. Many phages were able to adapt withenvironments with high salinity. Several bacteriophages were isolated frommarine water of different salinities. Wichels et al.

(1998) studied 22phages which they found in water near Helgoland in the North Sea. All of themhad tails and icosahedral heads of 50.2 to 99.

3 nm, and they wereclassified into three different families: 11 phages to Myoviridae, 7 to Siphoviridae,and 4 to Podoviridae. No similarity in DNA structure was shown among phagesbelonging to different families present in this area. There are many conditionswhich may overlap with the phage   andreduce the net concentration of the phage environment  West and Kelly (1962) reported thatmixing of 0.

1ml of 10%NaCl with an equal amount of  the broth culture of the propagating strainof Staphylococcus, followed byimmediate addition of 3 ml. soft agar, quickly diluted the concentration ofNaCl to about 0.8 %. The quantity of NaCl was further decreased onplating the soft agar They had shown that the free phagesdiffer in their NaCl tolerance.Their results disussed the positive results inhigh salt concentrations.    The phage cocktail was able to to lyse bacterial isolates afterexposure to UV rays for different periods of time. Many researches were performedto discuss that UV rays affect the shape and number of paques, but not affectthe survival of phages. Kleczkowski J and Kleczkowski A (1953).

have reported that aculture of irradiated phage produced the same number of plaques ofthe control culture after 24 hour of incubation  , but with  plaques diameter  smaller than  the control. They didnot explain this phenomenon  to be due tophage mutation, because the isolated phages from these small plaques producedplaques of normal size . Luria & Delbriick (1942) reportedthe role U.V in  the inactivation of thephage which made interferance with phage multiplication and also cause harmeffect 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 coulddestroy ability of the interference between the irradiated phage with phagemultiplication , and their capacity of making their host bacteria unable tomultiply also disappeared. The heterologous phages irradiated medium or preparationsif mixed in equal volumes; can slow bacterial multiplication in their liquidcultures. The effect of U.V on the phage inactivation has a common feature  of some polysaccharides or ribonucleaseinhibitors, or due to the interference with phage multiplication in  the host bacteria liquid cultures , whilehaving no effect on the numbers of plaques formed on agar.

This effect on theformer and not the latter, showing an apparent contradiction, has beendiscussed elsewhere (Kleczkowski & Kleczkowski, 1952).  The multiplicity reactivation was observedwith a few coli phages. (Luria &, Dulbecco, 1949)  observed that in the phagepreparations which  irradiated with U.VThenumbers of produced  plaques didnot increase with the increasing concentration at which they were brought intocontact with host bacteria before plating.

 The titre of the phage cocktail afterenrichment steps was 2.2×1011.This titre was high enough for testingits properties, preservation and formulation steps. Titration ofbacteriophages was done by plaque assay. Phageplaques is a traditional method for phagedetection and expressed by  plaque forming unit (pfu). Plaque formation resultedwhen the bacterial species are infected with the sutable phage (Cox2012; Kalni?a et al. 2008).

Traditional plaque assays have been used inthe detection of pathogens, including Staphylococcus aureus (Wallmarket al. 1978),Escherichia coli (Oda et al. 2004), and Campylobacterspecies (Grajewski et al. 1985). The mechanisms ofbacteriophage lysis have been studied for the bacteriophagecontrolof 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 infectioncycle of a lytic bacteriophage (Wang et al., 2000).

The  phage cocktail did lysis from 10-1to 10-10 dilutions ,but in the most high dillutions  10-11 and10-12dilutions the phage cocktail had no lysis activity . It means that at highestdilutions,  coliphages failed to dolysis. Lysis can be produced by phage dilution up to a certain point; as Worley-Morseet al. (2014) reported that bacteriophage concentration is veryimportant for lysis activity. The perservation temperature of bacteriophage is very importantfactor which determines phage activity.

The phage cocktail was stored at threedifferent temperatures 4?C, room temperature and -20?C ,one sample from each temperature was taken and tested for presenceof 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 numberof plaques of the sample preserved at -20?C was higher than the sample preserved at 4?C . Asshown by Ackermann et al,.

(2004), tailed phages were the mostresistant 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 wastewaterphags .This results agree with our results. Bacteriophage samples were preserved in bacterialmedium at 4  ?C and -20  ?C, and in SM buffer. Storage of the samplesat these two temperatures showed that bacteriophages were unstable,but in ourresults the frozen semple showed abetter results than the other samples .

Thiswas 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 fromenvironmental conditions. The royaljelly formula was the best used formula.

itgive more protection to the phage cocktail, however the crude royaljelly wasnot able to lyse bacteria. The royaljelly  gave phages the proper  protection them from protein denaturation. Studies on phage encapsulation have used a variety ofhydrophilic and hydrophobic polymers including agarose  (Bean et al., 2014), alginate ( Tang et al.,2013 and kim et al., 2015), chitosan ( Kaikaboet al.

, 2017 and Ma  etal., 2008), pectin (Dini et al., 2012) , wheyprotein(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 (Markoishviliet al., 2002), polyvinyl pyrrolidone ( Dai et al., 2014), polyethylene oxide/polyvinyl alcohol (Salalha et al.

, 2006), cellulosediacetate (Korehei and Kadla 2014), polymethylmethacrylate (Govenderet al., 2015).Triggers for phage release include polymer solvation, polymer dissolutionand erosion (Korehei and Kadla 2014),  polymer hydrolysis (Katsarava etal.

, 1999), phase inversion induced by temperature (Hathawayet al., 2015), and pH triggered dissolution of polymer and enzyme drivenpolymer degradation (Bean et al., 2014). Developing formulations that incorporate bacteriophagefor therapeutic applications requires an appreciation of the chemical andphysical stresses phage may encounter both during processing as well as duringstorage once formulated. Phage inactivation and long term reduction in phagetitre upon storage is highly undesirableA polymer or lipid may be used to coatan existing structure containing the phage. Murthy and Engelhardt (2009) sprayed phage on dried skimmed milk and thenencapsulated them in a lipid coating. There are many techniques and processesthat may be used for stabilising, immobilising and encapsulating phage.

In thecurrent study the phage cocktail was sprayed on dried skim milk and mixture ofsucrose and corn flour with equal ratio and encapsulated in nun lipid coatingand also give good results. The most common methods are spray-drying, sprayfreeze drying, freeze drying, extrusion dripping methods, emulsion andpolymerisation techniques.  Phages are protein structures and they aretherefore susceptible to factors known to denature proteins; these includeexposure to organic solvents ( Puapermpoonsiri et al.,2009), high temperatures (Briers et al.,2008), pH (Knezevic et al., 2011), ionic strength (Knezevicet al.

, 2011), and interfacial effects. Additionally, mechanicalstresses during formulation or encapsulation including shear stresses duringmixing and agitation and atomisation during spraying (Leunget al., 2016).

One of the most common and successful modes of long-termpreservation of bacteriophage is storage at 4 °C in Trypticase Soy Agarand Brain Heart Infusion broth whereas storage at ? 80 °C requires50% glycerol as a cryoprotectant (Clark 1992) orfreeze drying with excipients (e.g. sucrose or trehalose) as lyophilization andcryoprotectants (Ackermann et al., 2004).Ackermann et al (2004) observed thatphage titres for one of the largest collections of tailed phages typicallytended to drop by 1 log over the course of a year but then remained fairlystable (although there were many individual variations). Clark (Clark1992)  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 andstored for two years at room temperature and at 4 °C as (i) broth lysates;(ii) lysates diluted with 50% glycerol; (iii) saturating filter paper withlysate and then drying; (iv) freeze dried by mixing lysates with an equalvolume of double strength skim milk. Phage titres measured immediatelypost-processing indicated that freeze drying resulted in a significant loss oftitre (between 1 log and 2 log reduction). However, freeze drying didproduce stable phage titres over the course of 2 years when storedrefrigerated. After two years, the titre of phage in the broth lysates werefound to be generally higher than those of glycerol or freeze driedpreparations. Thermally dried preparations generally did not provesatisfactory. Preparations stored at 4 °C showed higher titres than thosekept at room temperature.

this results was agree with our results as ourpreparation stored at 4?C. All titres declined with time regardless of theconditions of preservation.


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