Under aseptic conditions, a total of 12 samples, including wastewater and soil (2 samples for each location) were obtained from 6 different locations (Student Village Hostel 1 & 2, Old Jos University Teaching Hospital, JUTH 1 & 2, and Angwa Rukuba 1 & 2), within Jos North Metropolis, Plateau State, Nigeria. Latitude and Longitudes of their various locations were noted as follows: latitude; 9.96565, 9.96571, 9.918695, 9.918323, 9.93922, 9.934003 and longitude; 8.87116, 8.87128, 8.890219, 8.890219, 8.909185, 8.908757 respectively. A 50 ml sterile vials with cover tops were used for this purpose. The containers were immediately disinfected with 70% ethanol at the point of collection, labeled, and kept in a super cool flask for transportation to Microbiology Laboratory Research Unit, Africa Center of Excellence in Phytomedicine Research and Development (ACEPRD) University of Jos, for analysis.
Laboratory Isolation of Environmental Isolates
According to the method of (Ibrahim and Hameed, 2015) modified, a total of 10 ml of each sample (after mixing the wastewater and sand and allowed to decant in a conical flask) was diluted in 90ml of sterile 0.9% NaCl Normal Saline and homogenized. Then, 100 μl of the fourth and fifth diluent of the samples were inoculated on Eosin Methylene Blue Agar (EMB) agar plates for the isolation of enteric bacteria, MacConkey agar plates are used for both lactose and non-lactose fermenters and Sorbitol-MacConkey for E. coli O157:H7, using the spread plate method. All the bacteria plates were incubated at 37˚C for 24 hours.
Total viable count for Environmental Isolates
The total viable count was determined using the spread plate technique on nutrient agar and counting the colonies developed after incubation at 37˚C for 24 hours (Harley and Prescott, 1996).
Identification of Isolates
Gram staining was done by making a thin smear of each isolate on a clean grease-free glass slide, air dried and heat-fixed by passing briefly over flame three times. Each smear was covered with crystal violet stain for 1 minute and rinsed with water, then Lugol’s iodine for a minute too and then washed with water. The dyed smear was then decolorized with acetone for 20 seconds and rinsed with water. Finally, the smear was covered with safranin for 1 minute, washed with water, bloted and air dried. Each slide was then viewed under microscope using oil immersion at X100 objective lens (Forbes et al., 2016).
A straight needle was touched to a colony of a young (18- to 24-hour) culture growing on agar medium. A depth of only 1/3 to ½ inch was stabbed once in the middle of the tube. It was ensured that the needle was kept in the same line it entered as it is removed from the medium. They were incubate at 37°C and examined daily for 7 days. A diffused zone of growth flaring out was observed from the line of inoculation (Forbes et al., 2016). .
After incubating an inoculated blood agar plate, the media around the bacteria growing on it was observed for changes in the opaque, red colour. If the area around the bacteria turns transparent, that strain displays complete haemolysis (Forbes et al., 2016).
A loopful of bacterial culture was inoculated in glucose phosphate peptone water then incubated at 37OC for 24 hours. About eight drops of methyl red were added. A distinct red ring formed at the top of the cryo-vial meant organism was positive.
Voges-Proskauer (VP) test
The isolates were inoculated into 2 ml of sterile glucose phosphate peptone water medium and incubated anaerobically at 37oC for 48 hours. To the culture, a very little amount of creatinin was added and properly mixed. Also added and mixed properly was a 3 ml of the sodium hydroxide (NaOH) reagent. The preparation was left to stand for 1 hr at room temperature as the bottle cap was removed, a positive VP test was a slow development of a pink-red colour.
A loopful of bacterial culture from nutrient slant smeared onto a drop of hydrogen peroxide on a clean grease-free slide. An immediate effervescence of gas bubbles from the culture indicated a positive reaction.
One drop each of normal saline and serum were put on a clean, grease-free slide, and a loopful of a bacterial culture was added and smeared together. The slide was then rocked and observed for agglutination/clumping. Clumping of the culture cells indicates a positive reaction.
A filter paper was soaked with 1% oxidase reagent and a loopful of bacterial culture was then smeared on the treated filter paper and observed for color change. A color change to dark purple or dark blue within 10 to 30 seconds indicated a positive result.
A loopful of bacterial culture was inoculated in freshly peptone water and incubated at 37oc for 24 hours. About 5 drops of kovas reagent were added to the culture and observed for colour formation. Formation of a pink to red ring at the top of the cryo vial indicated a positive result.
Simmons citrate agar was prepared, poured into flavor bottles and then slanted. Each bacterial culture from a nutrient slant was inoculated on the surface of one simmons citrate slant. The slants were then incubated at 37oc for 24 hours. A change of colour from green to Prussian blue on the slant surface and in the medium indicated a positive result.
Christensens’s urea agar was prepared, poured into tubes and slanted. Culture from each nutrient slant were streaked on the slant surface and incubated at 37oc for 24 hours. A colour change of the slant or/ and butt to pink indicates a positive result (Church, 2016; Cheesbrough, 2006).
The medium was allowed to warm to room temperature prior to inoculation. The Purple Broth (with carbohydrate of choice) was inoculated with isolated colonies from an 18-24 hour pure culture of the organism. A control tube of Purple Broth Base was inoculated in parallel with the carbohydrate based media. The inoculated media was incubated aerobically at 37ºC for 3-5 days. Observation was done daily for development of a yellow color in the medium (Forbes et al., 2016).
Triple Sugar Iron (TSI) A Test
The center of a well-isolated colonies obtained from solid culture media was picked with an inoculating needle. With a straight inoculation needle, the top of a well-isolated colony was touched. The TSI was inoculated by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant. The cap on loosely and incubate the tube was left at 35°-37°C in ambient air for 18 to 24 hours. The reaction of medium the medium was examined. (Forbes et al., 2016; Cheesbrough, 2006).
For further identification, the isolates were sent to Westerdijk Fungi Biodiversity Institute (Medical Mycology), Netherlands for Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis.
Preservation of Isolates
The isolates were subcultured on nutrient agar, incubated at 370C for 24 hours. A single colony was inoculated into a sterile nutrient broth, incubated in a shaker incubator (ZHP-100) at 180 rpm for 24 hours at 370C. The isolates were also incubated on a nutrient agar slant at 370C for 24 hours. They were all stored at 40C in a refrigerator.
Antibiotics Susceptibility Profile
The antibiotic susceptibility profile of the test organism and the environmental isolates were determined using the standard Kirby-Bauer disk diffusion method (Bauer, 1966). The antibiotics used has the following disk concentrations: Ceftazidime (10 μg), Cefuroxime (30 μg), Gentamicin (10 μg), Ciprofloxacin (10 μg), Nitrofurantoin (300 μg), Ampicillin (10 μg), Ofloxacin (10 μg), and Augmentin (30 μg). Bacterial culture suspension equivalents of 0.5 tube McFarland turbidity standards were spread on Muller-Hinton agar plates using sterile swabs and incubated aerobically at 37˚C for 24 hours; then, the zone of inhibition diameters around the antibiotic disks was measured. Obtained results were compared with the standard performance chart for antimicrobials disc susceptibility testing provided by CLSI (2012), and the frequencies of sensitivity and resistance were recorded. According to the criteria recommended by (CLSI, 2012), the results were expressed as susceptible or resistant.
(Ezemokwe et al., 2021)
Direct phage isolation from samples
Phage isolation from water/sewage samples
50 ml of water/sewage was centrifuged at 4,200 rpm for 10 min to remove debris and other undissolved particles.The supernatants were divided into portions A and B (saved portion B for enrichment). Portion A was further divided into 2 portions, A1 and A2. To portion A1 0.5 ml chloroform was added to lyse any bacteria sustained. Stored at 4oC for 30 minutes. After 1 hour, it was centrifuged at 21,000g for 10mins and filtered through 0.45 µm filters. The other portion A2 was centrifuged at 21,000g for 10mins and filtered through 0.45 µm filters. Portion B was also centrifuged at 21 000 g for 10mins and filtered with a 0.22 µm filter.
Phage isolation through enrichment
Phage isolation from enrichment of water/sewage samples
The following was added to a conical flask:
- 10 ml of the filtered sample B
- 10 ml double strength LB broth
- 40 µl 1 M CaCl2
- 100 µl of overnight broth culture of bacteria
It was incubated at 37oC up to 48 h with shaking at 50 rpm. After 24 h, 5 ml of the sample (B1) was collected and centrifuged at 4,200 rpm for 10 min. The supernatant was centrifuged at 21,000 g for 10mins and filtered through 0.45 µm filters. After 48 h, the other remaining portion was centrifuged and filtered (Sample B2).
Phage isolation from soils
The following was added to a conical flask:
- 1 g of soil/mud
- 5 ml LB broth
- Mixed continuously by gentle inversion at 20 rpm for 30 min
- Incubated at 4oC for 30 min
For 10 min., the sample was centrifuged at 4,200 rpm. The supernatants were divided into 2 portions, C and D. Portion C was centrifuge at 21,000g for 10mins and filtered through 0.45 µm filters (direct soil phage isolation).
To a flask, the following was added:
- 5 ml double strength nutrient broth supplemented with CaCl2 (see Phage isolation from enrichment of water/sewage samples).
- 5 ml soil/mud centrifuged supernatant D
- 100 ul overnight bacterial culture
It was incubated at 37oC up to 48 h with shaking at 50 rpm. After 24 h, 5 ml of the sample (D1) was collected and centrifuged at 15, 000 x g for 10 min. and was filtered through 0.45 µm filters. After 48 h, the other remaining portion was centrifuged and filtered (Sample D2).
Phage detection from extracted supernatants
Lawns of bacterial isolates were prepared. When the overlays were set, added 10 ul of the filtered samples A1, A2, B1, B2, C, D1 and D2 to a section of the agar plate. They were allowed to dry and incubated at 37oC overnight. The next day, zones of lysis/clearance on the bacterial lawns was observed. Using sterile loop, the lysed zones were scrapped out and transfer to 1 ml SM buffer in Eppendorfs and mixed by inversion. Vortex was avoided and was incubated at 4oC for 30 min. They were centrifuged at 15,000 x g for 10 min and filtered.
1. Filtrates from lysed zones from (B1 and D1 filtrates).
2. Two filtrates were selected to work with while the remaining filtrates were saved as a back-up).
3. One overnight bacterial culture
4. Standardized E. coli O157:H7 (fron NVRI Vom)
4. Warm semi-solid nutrient broth 0.7 % agar
5. SM buffer and nutrient broth
6. Nutrient agar plates
A 10-fold serial dilution of 10-1 to 10-8 of the lysed zones filtrates from two samples of B1 and D1 in cold SM buffer was conducted using 96 wells micro-titer sterile plate. Labelled one nutrient agar plate for each tube marked as 10-1 to 10-8. 100 μl of overnight standardized culture of (E. coli O157:H7 host bacteria) was added to sterile Eppendorf tubes marked 10-1 to 10-8. From the 10-1 serial dilution of the filtrates, 100 μl was removed and add it to the Eppendorf tube containing 100 μl of overnight culture labeled 10-1 and was mixed by pipetting up and down several times. All 200 μl was removed from this tube and added it to 3 ml overlay agar (for 90 mm Petri dishes). Mixed by inversion. The mixture was poured onto the appropriately labeled nutrient agar plate and allowed to set. This was repeated for each serial dilution and incubated aerobically at 37oC overnight.
Day 2, 3, 4
Each plate was visually examined and the one which contains individual distinct plaques were selected. For each distinct plaque morphology present on the plate, an Eppendorf tube with 500 μl of diluent (SM buffer) was prepared. From the selected plate, a pipetteman with pipette tip was selected and gently penetrated the overlay agar in the middle of the plaque. Immediately added the cored plaque to the tube prepared. This was repeated for a total of three times, using the most recently cored plaque in 500 μl of diluent (SM buffer) instead of the enriched phage lysate each time. It was ensured to pick plaques of the same plaque morphology of the parent plaque (i.e. the previous plaque picked).
Where; N = Number of plaques
D = Dilution factor
V = Volume of virus pipetted
Unit = PFU/ml
Where; PFU = Plaque Forming Unit
Phage propagation (Bulking/Multiplication)
The following was added into a sterile conical flask; 2ml E. coli O157:H7 broth, 20ul of the phage lysate, 10ul of nutrient broth and 40ul of 1mM CaCl2. Incubated at 37oC for 24h in a shaker incubator at 180rpm.
(Manohar et al., 2018 modified; Ahmadi et al., 2017; Ateba, and Akindolire, 2019 modified)
Thermal Stability test
A total of 64 nutrient agar base plates were prepared and allowed to stay overnight for sterility test. For the 4 phages isolated, each has 16 plates for the 4 temperature (45, 55, 65, and 75oC). Into sterile test-tubes, 90ul of SM buffer was added. They were heated to temperatures; 45, 55, 65 and 75oC in a water bath and maintained at ± 0.05oC. Water level was maintained 2cm above the treatment with a stirrer. Aliquot of 100ul of the phages for each test-tube of the respective temperature was added to the 900ul and boiled for 60 minutes. A serial dilution to 10-8 was prepared with a sterile micro-titer plate which was added 90ul of SM buffer. For every 15 minutes, 10ul of the sample or treatment phages was pipetted and added into 90ul in micro-titer plate and diluted.
Overlay already seeded with the host bacteria and poured onto nutrient agar plate was spotted with the diluent to 10-8. It was allowed to dry and was incubated by inverting at 37oC for overnight. This procedure was repeated for the individual phages.
pH Stability Test
A total of 24 agar plates were prepared and incubated overnight for sterility test. Nutrient agar over-lay was prepared with 1M NaOH and HCL to yield a pH of 1, 2, 5, 7, 9, 11 using SM buffer and were standardized. 900ul of the individual pH and 100ul of the phages were prepared and incubated at 37oC for 24h. From there, a serial dilution was made (10 folds) i.e. 90ul of SM buffer to 10ul of phage lysate. 10ul of the individual diluents were inoculated into an already solidified overlay agar and incubated at 37oC for 24h.
SM buffer containing NaCl of 0.5%, 5%, 10% and 15% was prepared while using SM buffer that has no salt as a negative control. A total of 20 agar base plates were prepared for the 4 phages each for different percentage. A 10 folds serial dilution was prepared as described above. 90ul SM buffer + NaCl and 10ul of the individual phages were incubated at room temperature for 24h. An overlay seeded with test organism was prepared, allowed to dry. 10ul of the diluents were spotted on them and allowed to dry, incubated by inverting at 37oC for 24h.
Chloroform Stability Test
Eight base plates were prepared. Four base plates for the four phages treated with chloroform and the other four served as a negative control for each of the phages treated without the chloroform. An overlay was prepared, 24h overnight E. coli O157:H7 prepared and 10%v/v of the chloroform using SM buffer prepared. Equal aliquot of 500ul of the lysate and 500ul of the chloroform was mixed in a sterile test-tube and was allowed to stand for 60minutes with slight shaking at intervals. They were centrifuged at 10,000g for 10minutes. A serial dilution to 10-8 as described earlier was carried out. Overlay seeded with the test organism was prepared and allowed to solidify. Individual diluents were spotted (10ul) and allowed to dry under aseptic condition and were incubated by inverting for 24h at 37oC.
Nutrient agar base plates were prepared. Overlay agar seeded with the individual organisms were dispensed against the base agar and allowed to gel. 10ul of the 4 phages were spotted against the overlay. They were allowed to dry, inverted and incubated at 37oC for 24h (Ross et al., 2016)
PRESERVATION/STORAGE OF PHAGES
The phages were stored in 50% glycerol at -80oC.
The mean values of the three replicates were determined by repeating the experiments three times. Populations of the surviving phage obtained in each study, were converted to log10 PFU mL−1. SPSS software version 21 was used to carry out the statistical analysis. To determine the analysis of variance (ANOVA) using Duncan’s multiple range test (JMP v.12 software; SAS Inst., Cary, NC, USA), data were analyzed. P-value less than 0.05 (P < 0.05) were estimated to determine the significant differences.