الفهرس 
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Abstract
Summary
In recent times, there is growing attention to the healthy lifestyle that includes eating fresh vegetables and salads and drinking water periodically. In parallel, many studies have proven a relationship between eating fresh, uncooked foods (vegetables) and the appearance of many poisoning and diseases caused by pathogenic bacteria present in vegetables and water.
In this study, we used some types of vegetables that were brought from different places (markets) in Benha city, Egypt also drinking water samples were collected from the same city to isolate some types of pathogenic bacteria.
The isolated bacteria were plated on the nutrient agar media to study the morphological properties of the bacterial colonies, as well as making a mathematical estimate of the number of bacterial cells per gram of vegetables or per ml of drinking water for each type of isolated bacteria, and then two bacterial isolates were selected for morphological and biochemical identification.
The isolates were plated on a specific media, ”MacConkyˈs agar media”, to study the morphological properties of colonies. Biochemical identification was made using a number of biochemical tests, as well as a microscopic study were done by using gram stain. On the basis morphological, culture, and biochemical characteristics and results obtained by automated identification system VITIEK 2, bacterial isolate no. W102 and no D2007 were identified as Escherichia coli W102 and Aeromonas hydrophila D2007, respectively.
E.coli W102 isolate which isolated from drinking water showed resistance to a large number of antibiotics ,it was resistant to 12 antibiotics out of 14 antibiotics used in this study and showed sensitivity to only two types, Gentamicin and Ampicillin, and bacterial isolate (A.hydrophila D2007) isolated from the dill leaves showed resistance to 8 out of 14 antibiotics and was sensitive to 6 antibiotics which are levofloxacin, Ciprofloxacin, Ofloxacin, Imipenem, Ceftazidime, Gentamicin, and Cefotaxime.
The antibiotics that were used in this study were: Levofloxacin (LEV) 5 µg, Ciprofloxacin (CIP) 5 µg, Cephalexin (CL) 30 µg, Ofloxacin (OFX) 5 µg, Ampicillin–Sulbactam (A/S) 20 µg, Amoxicillin-clavulanic acid (AMC) 30 µg, Imipenem (IPM) 10 µg, Cotrimoxazole (SXT) 25 µg, Gentamicin (GEN) 10 µg, Ampicillin (AMP) 10 µg, Amikacin (AK) 30 µg, Ceftazidime (CAZ) 30 µg, Cefotaxime (CTX) 30 µg and Amoxicillin (AX) 25 µg.
Phages were isolated from sewage in Sendenhur area Qalubia, Egypt and after several stages of purification, three specific phages were isolated and the morphological properties of the plaques of each phage were studied separately. ΦAHP7 phage specific for A.hydrophila D2007 are small 2 mm, round, regular, clear without a center or halo , ΦECP8 phage specific for E.coli W102 bacteria, medium 4 mm, round, regular, clear without a center, and so plaques of ΦECP9 phage specific for E.coli W102 bacteria was large 7 mm circular, regular, clear without a center or halo then the three phages was propagated to reach their concentration to 2.9 x 107for phage ΦAHP7, 2.3 x 106 for phage ΦECP8 and 6.5 x 106 for phage ECP9.
The three phages were defined using the transmission electron microscope, where the ΦAHP7 phage appeared from the family of Myoviridae with a tail and the head dimensions were ( 88.55 ± 0.32 × 114 ± 3 nm), and the tail length (77.25 ± 0.51 nm) and width (26.35 ± 1.35 nm).
Phage ΦECP8 belongs to the long-tail siphoviridae family where the head dimensions were (60.22 ± 1.13nm), the length of the tail (202.25 ± 2.25nm) and its width (23.86 ± 1.14nm). Phage ΦECP9 belongs to the family podoviridae and the head diameters was (66.95 ± 0.45 nm) and its tail length was (16.8 ± 0.2 nm).
The host range of the three phages was studied by using 22 different bacterial isolates. Phage ΦAHP7, infected three Aeromonas hydrophila isolates, Acinetobacter baumanni complex 15 and Klebsiella pneumoniae spp. pneumoniae14 and ΦECP8, and ΦECP9 phages infected multidrug-resistant E.coli isolates and Gram-positive Streptococcus spp. The one step growth curve for the three phages ΦAHP7, ΦECP8 and ΦECP9 showed that the latent periods was 10,20 and 10 minutes, respectively, as well as the average cell burst size of 53.5 ± 0.5, 26.5 ± 0.5 and 67.5 ± 0.5, respectively, and the full cycle of infection took 35,55 and 30 minutes.
The effect of different factors was tested on the three phages, where the phage were exposed to different temperatures (25, 37, 45, 55, 65, and 75 ° C) for different incubation periods (30, 60, and 90 minutes) .ΦAHP7 phage was able to adapted temperatures (25, 37 and 45 ° C), but it lost its activity in temperatures (55, 65 and 75 ° C) and this was the same for all incubation periods. Phages ΦECP8 and ΦECP9 remain active in temperatures (25, 37, 45, 55 ° C) and lost their activity at (65 and 75 ° C) during different incubation periods.
The stability of the three phage was tested under the influence of different pH values (3, 5, 7, 9 and 11) and also different incubation periods were applied (zero time, 30, 60 minutes and overnight ”12 hours”) and the optimum pH value for ΦAHP7 phage was (7) after an hour of incubation. Phages ΦECP8 and ΦECP9 showed suitability for pH 7 and 9 during 60 minutes of incubation.
The three phages were found appropriate for all saline concentrations used (0.1, 0.25, 0.75,1 and 2 Moller).
DNA was not digested with restriction enzymes EcoRI, and HindIII. The inability to digest phages DNA by EcoRI, and BamHI, enzymes could be due to a lack of target sites for the restriction enzymes tested.
The ability of the three phages to inhibit bacterial growth was evaluated by measuring the optical density of a liquid media during the growth of the bacterial host at a temperature of 37 ºC and (MOI = 0.001). When using A.hydrophila D2007 as a host for ΦAHP7 phage , it can to inhibit bacterial host growth to 85.29% after 48 hours of incubation, the ΦECP8 and ΦECP9 phage used E.coli W102 as a bacterial host and were able to inhibit and reduce bacterial growth by 85.88% and 88.23%, respectively, compared to the untreated control. ΦECP8 and ΦECP9 cocktail achieved reduction about 88.25% when compared to the untreated control.
Three separate phages were applied to inhibit the bacterial growth of A.hydrophila D2007 and E.coli W102 bacteria on the surface of tomato and red cabbage that were artificially contaminated, and the results showed a decrease in the number of viable bacterial cells. Treatment of red cabbage with ΦAHP7 reduced the number of viable A.hydrophila D2007 cells by 1.55 log CFU/g after 30 min of incubation and by 1.61 log CFU/g after 24 h of incubation at room temperature. Reductions of viable E.coli W102 counts were 1.68 log CFU/g and 1.76 log CFU/g) after 30 min and 24 h of the incubation period respectively with phage ΦECP8, while reduction was 2.28 log CFU/g after 30 min and 2 log CFU/g after 24 h of incubation period with phage ΦECP9.
Treating the contaminated tomato slices with artificially contaminated with A.hydrophila D2007 by using ΦAHP7 phage at a concentration made reductions by 1.48 log CFU/g and 1.39 log CFU/g after 30 min and 24 h of incubation at room temperature ,respectively. Reductions of viable E.coli W102 counts were 1.38 log CFU/g after 30 min and 1.52 log CFU/g after 24 h of the storage period or incubation period with phage ΦECP8 while, reductions counts were 1.68 log CFU/g after 30 min and 1.53 log CFU/g after 24 h of the storage period or incubation period with phage ΦECP9 (p <0.01).
Also, the three phages were applied separately to inhibit the bacterial growth of the same bacterial isolates in artificially contaminated drinking water. Treatment of the experimentally contaminated drinking tap water with ΦAHP7 reduced the number of viable A.hydrophila D2007 cells by 3.13 log CFU/ml after 30 min of incubation and by 3 log CFU/ml after 24 h of incubation at room temperature. Reductions of viable E.coli W102 counts were 3.08 log CFU/ml and 3 log CFU/ml); after 30 min and 24 h of incubation period respectively with phage ΦECP8, while reductions counts were 3.16 log CFU/ml after 30 min and 3 log CFU/ml after 24 h of the storage period or incubation period with phage ΦECP9(p <0.01).