Coliphages Lytic ; as a potential biological control agent against E . coli ( O 157 : H 7 ) in a mouse model . 1

The aim of this study was to isolate and characterize the phage for use as a potential biological control agent against Escherichia coli O157: H7. A standard strain of E coli O157: H7 (NCTC 12900, China) was used to isolate the lytic phage from bovine faeces and raw wastewater. The spot-assay test was used to check phage activity. Characterization of phage isolates included phage resistance to acidity and alkalinity, electron microscopy and PCR assay for stx1, stx2, and cI genes. Isolate phages were tested where a cocktail was administered to treat mice that was given a single oral dose (10 8 CFU) of Escherichia coli (O157: H7) (NTCC 12900). Three phages (P1, P3, P4) were successfully isolated from the sewage samples as they showed strong lytical activity against E. coli O157: H7 (NCTC 12900). The P1 and P4 phages had icosahedral head and a long, flexible, thin, inelastic tail with the tail fibers while the P3 phage had a less rigid, long and relatively thick tail with the tail fibers. All phages found are resistant to pH 4-9. The PCR results revealed the absence of genomic coding for stx1, stx2, and cI in the isolated phages. All mice treated with the phage gave a negative result for culture of Escherichia coli O157: H7. Introduction Bacteria can be attacked by bacterial viruses (bacteriophages, or simply phages). By nature, they are very specific such that an individual phage may only infect and subsequently kill a specific European Journal of Molecular & Clinical Medicine ISSN 2515-8260 Volume 08, Issue 03, 2021 1307 bacterial strain within a species. This high degree of specificity permits phages to be utilitized against targeted bacteria in a miscellaneous population such as the ruminant gut without disturbing the microbial ecosystem. Bacteriophages are common natural members of microbial ecosystem in the gastrointestinal tract of food animals, including ruminants (Klieve & Bauchop, 1988). Escherichia coli O157:H7 is a causative agent of food-borne disease and this poses a global challenge to public health. Human disease due to E. coli (O157:H7) varies from hemorrhagic colitis to mild watery diarrhea. Cattle are an important domestic animal which are the primary reservoirs for E coli O157:H7 and are the most common sources for foodborne and direct human-animal contact infections (Chapman et al.,2001). The carrier cattle remain healthy, and E. coli serotype O157:H7 is a transient member of the gastrointestinal flora. Cattle are the major carrier of these pathogens (Nastasijevic et al.,2008), and contamination of meat with feces during slaughtering is the principal route for transmission to human beings (Kudva et al.,1999, Chase-Topping et al.,2008). Thus, elimination of E. coli O157:H7 from the bovine gastrointestinal tracts before the slaughtering would be the first barrier required to avoid the enterance of these pathogens into the food chain. Persistence carriage of Escherichia coli (O157:H7) in experimental and natural infection in animals may be continue for days or even months (Cray et al.,1995, Hancock et al.,1997, Besser et al.,1999, Sheng et al., 2004). To reduce the chance of human exposure to E. coli (O157:H7) should be reduce and control the carriage and prevalence of this microorganism in live cattle. There is no animal vaccine or available method to eliminate of Escherichia coli (O157:H7) in living rumenants. A coliophage that lysis the E. coli (O157:H7) is a good method because bacteriophage therapies have been success in animal models against wide range of pathogenic bacteria such as enterotoxigenic and enteropathogenic scherichia. coli (Barrow et al.,1999, Jamalludeen et al.,2009), Staphylococcus aureus (Matsuzaki et al.,2003) and Pseudomonas aeruginosa (Soothill,1992). Therefore, the aim of this study was to apply phage therapy to control and treat the disease caused by E. coli O157:H7 and as a potential biocontrol agent against E. coli O157:H7 in rumenants. This might be reduced or replaced the need for antimicrobials in treatment and prevention of this possible infections. Materials and methods Phage isolation and titration. Standard strain of E coli O157:H7 (NCTC 12900, China) was used to isolate specific lytic phage from bovine feces and raw sewage. Phage was isolated by a standard enrichment procedure (Seeley et al., 2001). Briefly, 15ml of raw sewage or bovine feces were centrifuged at speed 3,500 g, temperature at 10°C and time for 30 minute. Millipore filter (0.45 μm-pore-size) was used to filterate the supernatants, then adding of this filtrate to LB broth (10 ml), and 100 μl contain 10 8 CFU of E. coli (O157:H7) isolate was also added. This mixture was incubated for overnight at 37°C. This mixture was centrifuged at 10,000 g for 10 minutes to remove the bacteria and debris then 0.45 μm-pore-size filter was used to filterate the supernatants. Spot assay was used to test the coliphage activity of the supernatant was tested by put 5μl of coliphage on LB agar inoculated with a lawn of Escherichia coli (O157:H7). After 5 European Journal of Molecular & Clinical Medicine ISSN 2515-8260 Volume 08, Issue 03, 2021 1308 hours at 37°C incubation, the plates were tested for plaques formation. Serial dilution was done to supernatants which give lytic result, then by using a technique of top agar overlay with Escherichia coli (O157:H7) the plaques were isolated and purified (Sambrook and Russel,2001). The coliphage that gave plaques on all E. coli (O157:H7) isolates was selected for further studies. Characterization of isolated phage Resistance of the phage to acidity and alkalinity. lysates of each of phage were subjected to pH values range from (1-11) for a 16 hours period, and then tested for stability. A 100 μl volume of phage lysate (10 9 pfu/mL) was applied to 900 μl of saline set to a certain pH and the mixture was incubated for 16 hours at 37 C (Hazem, 2002). Control tube was made up from 100 μl of phage suspension and 900 μl of normal saline, pH 7.2, was also incubated at 37C for 16 hours. After the end of incubation period, a 100 μl volume of the phage suspension was diluted serially (ten fold), with 100 μl of E. coli (10 9 cfu/ml) and incubated at 37 C for 20 minutes then added to 7% top agar and spread(3 ml) over a plate of LB agar. The titers of the surviving phages were calculted by plaquing ten fold dilutions by the double agar overlay method. Purification of Phage Lysates through Glycerol Gradient. Glycerol gradient protocol was used to yield phage lysate with good purity to be suitable for the subsequent electron microscopy and molecular analysis (Sambrook and Russel,2001). Electron microscopy of coliophages. The morphology of coliphages was examined by transmission electron microscopy. A 10 μl drop of each phage suspension was negatively stained with 2% phosphotungstic acid and added on a copper grid surface then visaulized by transmission electron microscope. Phages have been classified depending on their respective families as set out in the International Committee on Virus Taxonomy guidelines (Walker et al., 2019). Phage DNA Extraction. Phage DNA extraction was achieved by phenol chlorophorm protocol (Sambrook and Russel,2001)). Briefly, after phage had been propagated, bacterial debris was pelleted by centrifugation at 9,000 g, at 4°C for 20 min, , and then treatment of the supernatant with RNase A and DNase I (Thermo Fisher Scientific, USA) to discard any bacterial nucleic acid. The phage was pelleted by centrifugation at 25,000 rpm, at 4°C for 2 hours, and, then, resuspended in SM buffer; sodium dodecyl sulfate and proteinase K were applied to a final concentration of 0.5% and 50μg mL -1 , respectively. After incubation period for 10 min, at 65°C, the protein was removed from the solution. DNA precipitation was performed with sodium acetate and ethanol and then pelleted by a microcentrifuge. After washing with 70% ethanol, the DNA pellet was air dried and resuspended in a Tris-EDTA buffer. Screening for stx1, stx2, and cI genes by PCR. Isolated phages were checked for the presence of stx1 , stx2 and cI genes in isolated phage genome before considered to be used for oral therapy. That was achieved by PCR according to a standard protocol mentioned by (Johansen et al., 2001) using primers designed by Fagan et al. (1999). stx1 and stx2 genes are responsible for shiga like toxin 1 and shiga like toxin 2 respectively while cI gene maintain the phage in lysogenic status. European Journal of Molecular & Clinical Medicine ISSN 2515-8260 Volume 08, Issue 03, 2021 1309 Efficacy of Phage Therapy in Mice. Phage therapy experiments were designed as fulfilled by Tanji et al. (2005) with minor modification. Twelve mice were divided into two groups. Six mice in each group which were given 100 μl of 10 8 CFU Escherichia coli O157:H7 as a single oral dose (NCTC 12900, China) per animal on day 1. Mice in first group (phage group) were given 100 μl of 10 10 PFU/ml from the isolated phages, on days, -1, 0, 1, and 2. Six mice in second group (control group) were not treated with phage. Quantitative analysis of E. coli O157:H7 in faeces. Freshly voided faeces during 2 hrs were weighted and shattered in plain tube after adding of ten fold SM buffer. shattered faeces were centrifuged at 10000 xg for10 min at 4 C and then diluted serially with PBS, then varnished on sorbitol MacConkey agar plate for enumeration of E. coli O157:H7. Fecal samples from all the mice in experiments were tested for presence of E. coli O157:H7 or/and E. coli O157:H7 bacteriophage before the performance of experiment. Results Bacteriophage isolation. Phages were successfully isolated through classical amplification from samples of sewage after several trials (Figure 1). Initially six phages were isolated and of these about four phages were tested. Out of 4 phages only three phages (P1, P3, P4) were selected for further characterization (Phage P2 was not detected after storage). These phages were showing a strong lytic