Characterization and Utilization of Podoviridae Phages for Controlling Pathogenic <em>Escherichia coli</em> Serovar O168 from Ducklings in Egypt

Special Article - Escherichia coli

J Bacteriol Mycol. 2018; 5(6): 1079.

Characterization and Utilization of Podoviridae Phages for Controlling Pathogenic Escherichia coli Serovar O168 from Ducklings in Egypt krusei

El-Daly RA1,3, Merwad AMA²*, Barakat AB¹, Hassan SE³ and Askora A4

¹Department of Microbiology, Faculty of Science, Ain Shams University, Cairo 11566, Egypt

²Department of Zoonoses, Faculty of Veterinary Medicine, Zagazig University, 44519, Zagazig, Egypt

³Animal Health Research Institute, Zagazig, Sharkia, Egypt

4Department of Microbiology and Botany, Faculty of Science, Zagazig University, 44519 Zagazig, Egypt

*Corresponding author: Abdallah MA Merwad, Elzerah Square, Zagazig, Faculty of Veterinary Medicine, Zagazig University, Egypt

Received: August 13, 2018; Accepted: September 12, 2018; Published: September 19, 2018


This study was carried out to isolate and characterize lytic bacteriophages against the multidrug resistant and pathogenic Escherichia coli serotype O168 isolated from ducklings in Egypt; and also to study efficacy of single phage and cocktail phages on In vitro inactivation of E. coli O168. One hundred and fifteen samples from ducklings, including caecal contents, skin, minced breast, gizzard, swabs from mouth, cloacae and inner surface of liver, were collected, preenriched in peptone water and streaked MacConkey agar, and Eosin-Methylene Blue agar. The presumptive isolates of E. coli were subjected for biochemical and serological identifications. Antimicrobial susceptibility testing was performed by the disk diffusion method. Phages against E. coli serotype O168 were isolated from sewage samples using plaque assay test. An evaluation of phages efficacy was achieved separately and in cocktail to control the most multidrug resistant serotype E. coli O168. Three different single plaques with different plaque morphologies and diameters designated as ECa1, ECb1, and ECc1 were picked and chosen for further purification, amplification and characterization. The recovered phages were belonged to the family Podoviridae. The use of cocktail phages (ECa1/ECb1/ECc1) was significantly effective (reductions of 7.4 log CFU/ml 12hrs post treatment) than the use single phage suspensions. This study confirmed the higher efficacy of phage cocktails in controlling the infection of ducklings with multidrug resistant E. coli O168. These phages will reduce mortality in ducklings, and also protect human health from adverse side effects of antibiotic residues.

Keywords: Escherichia coli Serovar O168; Podoviridae Phages; Multidrug Resistance; Ducklings; One-Step Growth Curve; Phage Cocktails


Duck meat is the most popular type of food in Egypt and various parts of the world. Ducks are excellent sources of animalderived high quality proteins that contain essential and nonessential amino acids. Pathogenic Escherichia coli infecting poultry is the causative agent of colibacillosis, one of the most important causes of economic losses in industry of poultry worldwide. In Egypt, five serotypes of E. coli (O86, O127, O114, O26 and O78) were identified from gizzard, heart, spleen and muscles of ducks [1]. Some bacterial diseases such as salmonellosis, colibacillosis, and pasteurelliosis infect a variety of organ systems with involving the alimentary tract. Escherichia coli is a food borne pathogen, and has a public health concern [2]. Recently, there has been attention that some multidrug resistant E. coli have represented a worldwide multiple food borne disease disorders related to the contaminated food consumption [3]. Also, the misuse of antimicrobial agents in poultry production for growth promotion and treatment purposes increases the major interest for the multidrug resistance that are frequently seen among serovars of E. coli and Salmonella [4]. Antimicrobial-resistant food borne pathogens is recognized as an essential public health in the developing countries and this resistance reduces the therapeutic options for treatment of human salmonellosis [5]. These aspects lead to a dangerous threat to public health and the searches for alternative strategies are crucial to overcome the spread of resistant E. coli and their evolution becomes an absolute necessity. Many studies have suggested that bacteriophages are considered as potential therapeutic agents for the biocontrol of multi- drug resistant bacteria in poultry [6,7]. Bacteriophages are viruses, which attack only bacterial cells and assume a dynamic part in the biology of indigenous habitats, affecting the dynamic of prokaryotic population [8]. The advantages of bacteriophages include the interaction ability with its target host cells, its lytic ability and its capability to multiply throughout the process of infection. Therefore, this study was aimed to isolate and characterize different bacteriophages against the multidrug resistant strains of pathogenic E. coli serovar O168 isolated from ducklings in Egypt as well as to evaluate the efficacy of phages separately and in cocktail on the In vitro inactivation for the growth of E. coli O168.

Materials and Methods

Ethical statement

The study was approved by Institutional Animal Care and Use Committee, Zagazig University (ZU-IACUC).

Collection and preparation of samples

One hundred and fifteen samples from ducklings with an age range (1-14 days) were collected from three commercial duckling farms and from Animal Health Research Institute (AHRI), Sharkia, Egypt. The samples included gizzard, liver and spleen and swabs from mouth and cloacae. All samples were collected from euthanized ducklings. Samples were directly transported to the Laboratory under aseptic conditions and kept in an insulated box with ice packs.

Isolation of Escherichia coli, Salmonella species and Pasteurella species

Isolation of E. coli from swab and tissue samples of ducklings was carried out according to the microbiological method listed in Bacteriological Analytical Manual with minor modifications [9]. The swabs from mouth and cloacae were pre-enriched in buffered peptone water; while 25g of each liver, spleen and gizzard was added to 225ml of buffered peptone water, then homogenized using a stomacher at 230rpm for 5min. The pre-enriched swabs and tissue mixtures were incubated at 37°C for 18hrs. One loopful of enriched culture was subjected to streaking on MacConkey agar plates, then followed by an incubation at 37°C for 18-24 hrs. Afterwards, reinoculation of lactose fermenting colonies was performed on the Eosin Methylene blue (EMB) agar plates, then plates were incubated at 37°C for 18-24 hrs. The presumptive E. coli isolates appearing as metallic green colonies on EMB agar plates were subjected for biochemical identification as previously described [10]. Serotyping of E. coli isolates was done using commercial antisera kits ((Difco, Detroit, MI, USA) at the Serology Unit, Animal Health Research Institute, Dokki, Giza, Egypt. Stock cultures of the isolates were stored in 50% glycerol at -80°C.

The isolation of Salmonella species from swab and tissue samples of ducklings was carried out according to the ISO-6579 method [11]. Briefly, 0.1ml of pre-enriched cultures was added to 10ml of Rappaport-Vassiliadis soy peptone (RVS, OXoid CM0669) broth then incubated at 41.5°C for 18-24 hrs. A loopul from enriched broth was streaked on the surface of Xylose Lysine Desoxycholate (XLD, Oxoid, CM0469) agar plate followed by incubation at 37°C for 24hrs. The isolates of salmonellae were biochemically identified. Serotyping of Salmonella isolates was carried out according to [12]. The preenriched swabs were streaked on 5% sheep blood agar plates for isolation of Pasteurella spp., and then incubated at 37°C for 24hrs. The identification of Pasteurella species was done as previously described [13].

Antimicrobial susceptibility testing

A total of 22 bacterial isolates including Escherichia coli serotype O168 (n=6), Salmonella Typhimurium (n=13) and Pasteurella anatipestifer (n=3) were tested for antimicrobial susceptibility against 10 antibiotics by the disk diffusion method [14]. The used antibiotics and their concentrations were including: Amoxicillin-clavulanic acid (30μg), gentamycin (10μg), chloramphenicol (30μg), doxycycline (30μg), imipenem (30μg), streptomycin (30μg), rifampin (10μg), ciprofloxacin (30μg), ampicillin (30μg) and sulfadimethoxineormetoprim (2μg. The test was performed by applying a bacterial inoculum of approximately 2×108 CFU/ml to the surface of Mueller- Hinton agar plate. The disks of antibiotics were distributed on the surface of inoculated agar plate, and incubated at 37°C for 18-24 hrs. The inhibition growth zone around each antibiotic disk is measured to the approximate millimeter. The diameters zone of each antibiotic was explained according to criteria documented by the European Committee on Antimicrobial Susceptibility Testing [15].

Isolation of lytic phages against Escherichia coli serotype O168

Duckling’s droppings from the same retail stores mentioned above and sewage water were collected from several stations in Zagazig, and 10th of Ramadan, then homogenized in 100ml of Luria-Bertani (LB) broth supplemented with 10ml mol/L CaCl2. Those homogenates were subjected to inoculation with overnight cultures (100ml) of 9 E. coli isolates as host cells, and then followed by incubation at 37°C for 24h. The inoculated samples (5ml) were harvested and centrifuged at 12,000x g for 5min. The supernatant was collected and filtrated through 0.45mm pore size filter (Millipore, France) to generate phage lysate. For screening of the existence of lytic phages, the lysate (10ml) was spotted onto the lawn of bacteria prepared from 9 bacterial hosts after overnight culture of on the double layer LB agar plates (Oxoid). These plates were dried at room temperature for 10 minutes, and then were exposed to overnight incubation at 37°C. Lysate showing clear lytic zones at the application point on the double layer LB plates were serially diluted in SM buffer (0.05mol/L Tris- HCl buffer, pH 7.5, containing 0.1mol/L NaCl, 0.008mol/L MgSO4, and 0.01% gelatin). The dilutions (100ml) were subjected to incubation with 100ml of corresponding host(s) at 37°C for 20min, mixed in 4ml molten agar, plated on LB and incubated at 37°C for 24hrs. The existence of plaques was checked in the plates. For phage isolation, we picked up the one clear and the most separated plaque that appeared on the double layer agar plates, then followed by serial dilution in SM buffer and incubated with bacterial host culture and plated on the double layer LB as previously stated. For the isolation of every phage, this procedure was repeated at least 3 times at least to prove the phage purity. The storage of final lysates was performed in SM buffer at 4°C until use.

Electron microscopy

A drop of three purified bacteriophage particles of a highly concentrated suspension (109PFU/ml) was spotted on the carboncoated copper grid, left to adsorb for 2min, and then followed by negative staining with 2% (w/v) uranyl acetate. Phage morphology was observed with electron microscope (Hitachi, H600A) at University of Mansoura, Egypt.

Phage adsorption

Exponential host bacterial cultures of E. coli isolate serotype (O168) were adjusted to a 0.8 O.D. at 600nm (corresponding to a cell density of 109CFU/ml). Phage suspensions (10μl) were added to 10ml of E. coli isolate to obtain a multiplicity of infection (MOI) of 0.001 and then incubated at 25°C [16]. Aliquots of mixture were gathered after zero, 5, 10, 15, 20, 25, 30, 40, 50, 60 and 70 minutes of incubation and chloroform (1% as a final volume) was added. The mixture was centrifuged at 12.000g for 5 minutes and supernatants were immediately filtered by using 0.2 (Millipore, France). Dilution and titration of unadsorbed phages were performed. Incubation of plates was carried out at 37°C followed by examination of plaques after 4-8 hrs. The adsorption was expressed as the percentage decrease of phage titer in the supernatant when compared to the zero time. The phage suspensions without any host cells were utilized as nonadsorption standard for calculations [16]. Three independent assays were performed.

Determination of Phage host range and analysis for Efficiency of Plating (EOP)

After phage isolation, the bacterial strains used in the present study were listed in (Table 3). The stocks of phages were prepared and the host range of isolated phages was detected by the spot testing as previously explained [17]. Each isolated phage was tested against 39 strains of pathogenic bacteria to determine the host range of each phage [18]. Briefly, phage stocks (10ml) were spotted onto each bacterial lawn, then it dried in a clean bench for 10 min and exposed to incubation at 37°C for 24hrs. The lytic zones of phages being visible at the application point were labeled as positive for lytic activities. Two categories of pathogenic bacteria were differentiated according to the clarity of spot including: Clear lysis zone (+), no lysis zone (-). Using the double-layer agar method, the EOP was determined for bacteria with positive spot tests (occurrence of a clear lysis zone) [17]. For each phage, three independent experiments were performed. The EOP was calculated by the following equation: Average PFU on target bacteria/average PFU on host bacteria with the standard deviation for the three measurements as previously explained [19].

Effect of temperature and pH on phages stability

For thermal stability test, the suspensions of phages were added to LB broth at different temperatures of 28, 37, 45 and 65°C, then exposed to incubation at the respective temperatures for one hour. For pH stability test, the suspension of phage was inoculated into the LB liquid medium with a pH range of 3-11 and the phage titer was determined after incubation at 37°C for one hour.

One single-step growth experiments

Adjustment of exponential host bacterial cultures of E. coli strain was done to a 0.8 O.D. at 600nm (corresponding to a cell density of 109 CFU/ml). The phage suspensions (10μl) were mixed with 10ml of the bacterial culture to have a MOI of 0.001 and followed by incubation at 28°C [20]. The mixture was centrifuged at 12.000g for 5 minutes, and then pellet was re-suspended in LB (10ml) at 28°C and then followed by perfect dilution and titration. During incubation at 37°C for 30-minutes, samples were removed intervals up to 5hrs and the phage titers were determined by the double-layer plaque assay. The first set of samples was subjected to dilution before titration. The second set of samples was treated with chloroform (1% V/v) for the release of intracellular phages to determine the eclipse period. Incubation of plates was performed at 37°C and examination of plates was done for presence of plaques after 4-8 hrs [20]. Three independent assays were done.

Challenge of E. coli O168 with three phages separately and in phage cocktail

The phages ECa1, ECb1 and ECc1 were separately tested then phage cocktails were tested (two or three phages mixed together at the same concentration). The two phage cocktails were: ECa1/ECb1, ECa1/ECc1, ECb1/ECc1 and ECa1/ ECb1/ECc1 phages. E. coli O168 was used as host at a MOI of 100 (based in preceding studies using different MOI: 1, 10, 100 and 1000, data not shown). To reach a MOI of 100, 2.5μl of E. coli serotype O168 culture (≈108 and 20μl of phage suspension (109 were added to LB medium (30ml) and then followed by incubation at 37°C without agitation. Two control samples were included in each assay: The bacterial control (BC) and the phage control (PC). The inoculation of BC was not done with the phages and the inoculation of PC was not performed with the phages but without the bacteria. One milliliter of test samples and BC and PC was withdrawn after 0, 2, 4, 6, 8, 10, 12 and 24 hrs of incubation. For all assays, determination of phage titer was carried out in duplicate using the double agar layer method after incubation at 37°C for 4-8 hrs. The reductions in bacterial counts were calculated by the differences in viable counts between the control and phage-treated samples.


Bacterial isolation and identification

In this study, the overall presumptive isolates of E. coli, Salmonella spp. and Pasteurella spp. were detected in 140 of 150 (93.3%) samples of ducklings by plating onto selective media. The identification of all isolates showed that 101 isolates were confirmed as E. coli and 29 isolates were Salmonella spp. and 10 strains were identified into Pasteurella spp. (Table 1). The O-serogrouping of 27 E. coli strains showed that 15 out of 27 (55.6%) strains were O-serogroup untypeable (OUT); while the remaining12 out of 27 (44.4%) were typeable into 9 different groups; O1, O55, O78, O111, O114, O125, O127, O168, and O169 (Table 3). The serotyping of Salmonella isolates was ranging between serotype S. Typhimurium, S. Enteriditis. Also, Pasteurella anatipestifer was identified from ducklings.