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Bacteriophages are viruses that naturally infect and kill bacteria. They are abundant on earth and are continuously evolving. Their general structure consists of genetic material in the form of DNA or RNA and a viral coat protein that encapsulates the genetic material.  They infect bacteria in two ways, through the lytic cycle or the lysogenic cycle. In the first method the bacteriophage multiply within the bacteria ultimately killing it. Some phages follow another method where in, they integrate their genome into the host genome and multiply within the bacteria for a few generations. 
Bacteriophages have various applications and have contributed tremendously to the field of molecular biology and biotechnology.  They have emerged as important delivery vehicles, DNA vaccines, antibodies alternatives, pathogenic bacteria detection, and so on.  Over the years various applications of bacteriophages have evolved. As it is clear that they are viruses with potential applications, our aim was to isolate bacteriophages from environmental samples with the motive to sequence a novel bacteriophage genome. This was achieved by following various methods of collection, processing and analysis.
The samples were collected from three sources of pond (sediment and water), sewage and chicken faeces matter. They were processed using a two stage filtration process. The analysis was performed in two different ways namely culture dependent method and culture independent method. Both revealed different results that are further being processed and ultimately sent for sequencing of the viral genomes.
1. Collection of environmental samples
This experiment used a variety of environmental samples that were taken from different sources. One was a sewage sample that was provided from a sewage processing plant and second was chicken faecal sample provided from a private property in Edinburgh.
The last sample was collected manually from the Blackford pond in Edinburgh. Samples of pond water, pond sediment and sediments from the banks were collected. The various samples were collected in sterile tubes. The pond sediment was collected using a hand-held sediment corer and the sediments from the bank were collected using a trowel. They were stored at 4°C.
2. Processing of environmental samples
The pond samples had to be processed to separate the phage from the environmental debris and contaminants. Sterile phage buffer, prepared by the practical class prep room, was used to re-suspend the environmental samples. Phage buffer consisted of Tris-HCL(3.15g), NaCl(5.84g), MgSO4(1.21g) and made up to 1000ml with distilled water (pH 7.4). The provided chicken faecal sample was also re-suspended to remove debris prior to filtration. Filtration was done in two steps where in the first step uses a Whatmans filter paper and the second step uses a 0.22µm pore size filter. The first stage removes larger solid contaminants and the second filters out the bacteria but allow the bacteriophages to pass through.
Suitable volume of sediment sample from the Reed bed Trowel was added to 40ml sterile sample buffer in a 50ml plastic tube. The sample was mixed thoroughly either by gently vortexing or mixing with a pipette. After re-suspending, the sample was placed in ice. Whatman No. 1 filter paper was folded and placed on a filter funnel and the re-suspended sample was allowed to pass through it. The filtrate was collected in a sterile 50ml tube and was placed in ice. A sterile single use plastic 50ml syringe was fit into single use 0.22µm filter unit and placed on a sterile 50ml tube. The plunger was removed from the syringe and the filtrate was carefully added into the syringe barrel. Then the plunger was placed back in to the syringe and slowly depressed to allow the drop-by-drop flow of filtrate through the pore filter. When all the liquid has passed through the pore filter, the final filtrate is stored in ice.
To concentrate the phage samples, the filtered phage samples are added to a single Centicon Plus-70 Centrifugal Filter Unit. This unit contains a membrane that allows the flow of 100kDa molecular weight particles. This allows larger solutes to pass and retains the phage in the upper portion. The filter units were span in a refrigerated centrifuge at 3,500 *g for 10minutes and reversed and centrifuged at same conditions for 2 minutes. The concentrated phage sample was pipette out into an eppendorf and 10µL of chloroform was added to the final solution and stored in ice.
Some precautions could be taken while processing the samples. The whatman filter can be changed if it is too clogged due to large sediment particles. The syringe should be depressed slowly. The pore filter should not be contaminated and should be held in place while filtering.
3. Phage analysis
Two methods have been used here to analysis the presence of bacteriophages in the collected environmental samples:
Phage titre and specificity analysis: This traditional method will help determine whether the environmental samples have specific phages for different host organisms directly by growing them with the organisms and observing for any lysis or plaque formation on lawns. This is a culture dependent method.
Metagenomic analysis- this is culture independent technique that allows direct separation of phages from samples followed by whole genome amplification of possible viruses. The products of whole genome amplification are then studied through sequencing.
3.1 Culture dependent Phage analysis- Phage titre and Specificity
The phage dependent analysis was done in different stages. First being the amplification of the phage samples in specific organisms in broth followed by filtration and growth of phage lysates on organism lawns to check for plaque formation.
The different host organisms and their growth conditions that have been used are given in the table below and this group worked with B.Fragilis NCTC 9343.
Table 1- Host organisms and their growth requirements
Aerobic/ 37°C/ shaking incubator
Aerobic/ 37°C/ shaking incubator
Aerobic/ 37°C/ shaking incubator
Inoculation of Host cultures with phage lysates
The media used for B.Fragilis was Brain Heart Infusion Agar- Supplements (BHI-S), prepared by the prep room, which had the following composition Brain Heart Infusion Broth (Oxoid)- 37g/L and Supplements- 10ml 5% L-Cysteine (Dissolved in distilled water and autoclaved), 10ml 10% Sodium Bicarbonate (NaHCO3) (Dissolved in distilled water and autoclaved) and 10ml Hemin/ Menadione stock (Hemin stock- 50mg hemin, 1.74g K2HPO4 , 0.4g NaOH made upto 99ml distilled water) (Menadione stock- 5mg/ml in ethanol) (100ml Hemin/ Menadione stock=99ml Hemin + 1ml Menadione)
Three bottles of 5ml of sterile BHI-S broth was taken and 0.2ml of B. fragilis was added into each bottle. Then 0.1ml of phage lysates from sediment 1 (Reed Bed trowel), sediment 2 (trowel 2 bed- filtered by other groups) and sewage (filtered by other groups) was added to one of the media bottles respectively. The bottles were incubated in a static incubator at 37°C to allow anaerobic growth of the organism.
3.1.2 Filtration of amplified phage cultures
The three phage lysates were amplified on B.fragilis NCTC cells in BHI-S media. The samples were poured into a 10ml sterile syringe and filtered through a 0.22µm pore filter. The filtrates were collected into labeled sterile bottles.
3.1.3 Preparation of Lawns of host strains
Host organism lawns were made to detect the presence of phages in the samples by allowing the phage to grow and form plaques. For each of the phage lysate five labeled agar plates were made and labeled 0-5.
Since B. fragilis NCTC 638R (resistance-minus derivative) was the host organism used for the experiment, 15 BHI-S media agar plates were taken (preparation of BHI-S same as mentioned above with 15g/L agar). To form a lawn, top agar (containing 6g/l agar in BHI-S) was taken and 0.1ml of B.fragilis was added to the molten top agar and mixed by shaking the tube. It was then poured onto the base agar plate and immediately swirled to spread the top agar before it solidified.
The top agar used in the experiment was inappropriate and it did not solidify. This could be attributed to wrong agar concentration in the top agar.
3.1.4 Titration of phage samples and phage specificity
Phage samples were serially diluted 10-5 times (dilution factor 105) using phage buffer. Three sets of 5 sterile eppendorf tubes were set up. 0.9ml of sterile phage buffer was added to each eppendorf tube and 0.1ml of phage lysate was added to the first tube. Then 0.1ml sample from the above tube is added to the next tube containing 0.9ml buffer. The process is repeated till the last eppendorf tube had 10ml and all the others have 0.9ml.This is done for all the three samples of phage lysate.
Once the diluted lysates were ready, 5µL from each was spread onto the agar plates where 0 was the undiluted phage sample and 1 was the sample diluted 10times and so on. The plates were supposed to grow in anaerobic conditions at 37°C in a static incubator. But for this experiment since the top agar for the BHI-S plates did not dry, they could not have been plated with the lysate samples.
3.1.5 Analysis of bacteriophage diversity and host range
The agar plates grown with phage lysates were supposed to be examined for plaque formation.
3.1.6 Purification of bacteriophage from plaques
The plaques formed by bacteriophages are known to form a single viral infection just like a bacterium. Therefore, innoculum from one plaque grown on the host media is a suitable technique to purify phage.
The experiment done on the host B.fragilis in BHI-S was unsuccessful due to top agar difficulties. Therefore, there was no plaque formation on these plates. Whereas, other groups that worked with E.coli MG 1655 had found plaques for sewage and chicken faecal phage samples. The experiment was continued with these samples for phage purification.
First stage of purification included the preparation of lawns of E.coli MG 1655 on LB agar plates. Molten top agar was mixed with 0.1ml of E.coli and spread on the base plate and allowed to solidify. To purify the phage plaque, eppendorfs containing 50µl of chloroform and 100µl of phage buffer were set up. A sterile pipette was used to gently stab the plaque to remove it as a plug of agar. Rubber bulb was used to release the agar piece containing the plaque. The tube was vortexed vigorously to re-suspend the phage. This was then span in a microfuge at full speed for 30seconds. This allowed the separation of two phases in the eppendorf. Using a sterile tip, 5µl of sample was pipette out of the top layer and added at three different spots on the LB agar plates with E.coli lawns. The plates were allowed to dry and placed inverted in an incubator. This procedure was done for both the phage samples from sewage and chicken faecal samples from dilution of 104.
3.1.7 Phage DNA preparation
Plaques from the above purified plates were re-suspended in 100µl phage buffer at 4°C and stored overnight. Cultures of E.coli MG 1655 were prepared and grown overnight. The next day, the phage suspensions were added to 500µl of overnight cultures of E.coli and incubated at 37°C for 20minutes. During this time, 100ml of LB broth was prepared and after completion of incubation the above mixture of phage and cells was added to the broth and allowed to incubate at 37°C on the shaker. The cultures were then observed at 30minute intervals for any form of cell lysis. After lysis was observed 500µl of chloroform was added to the culture and incubated for 20minutes in the shaking incubator. The cell debris was then removed by centrifuging the mixture at 75000 rpm at 4°C for 10 minutes. The upper layer was transferred into a fresh tube and stored at 4°C.
After the phage is purified, the phage DNA has to be separated from the contaminating DNA and RNA. RNaseA and DNaseI having final concentration of 1µg/ml were added to the phage lysate and incubated at 37°C for 30 minutes. After contaminating DNA and RNA were degraded, the phage was precipitated using the precipitation solution (20% w/v (200g) PEG 8000, 116.88g NaCl made up to 1000ml with distilled water) and left on ice for one hour. After phage precipitated, it was centrifuged in a refrigerated centrifuge at 10,000 rpm for 4°C for 30 minutes. The supernatant was discarded and the pellet was allowed to air dry. It was then re-suspended in 10ml phage buffer by gentle vortexing and centrifuged at 8000rpm at 4°C for 2 minutes to remove remaining debris. The supernatant was then transferred into a sterile centrifuge tube on ice.
Phage DNA was extracted using Phenol/ Chloroform extraction method. To the supernatant from the above step 10ml of Tris buffered Phenol chloroform (1:1 v/v) was added and vortexed for 1 minute. The layers were separated by centrifuging the tube at 12,000 rpm at 4°C for 5 minutes. The supernatant was carefully transferred into another tube and 10ml of the phenol/ chloroform was added and centrifuged with same conditions. The aqueous layer is transferred to another tube and 10ml of chloroform was added to the sample, vortexed and span again. The upper layer from this was separated into another tube and 10ml isopropanol was added to the mixture and left overnight at 4°C to allow precipitation of DNA.
The next day, DNA was collected by centrifuging the above sample at 12,000rpm at 4°C for 10 minutes. It was washed with 1ml cold 70% ethanol and dried under vacuum using the SpeedVAc for 10 minutes. The purified phage DNA is then suspended into 50-100µL pf TE buffer and stored on ice.
3.2 Metagenomic analysis of the virome from the environmental samples
The other method used to isolate bacteriophages is to directly sequence viral genomes from large volumes of environmental samples after removing contaminating organisms.
Whole genome amplification was performed using the Illustra GenomiPhi V2 DNA Amplification kit manufactured by GE healthcare. This kit uses random primer sequences and bacteriophage Phi 29 DNA polymerase to amplify any DNA present in the sample.
3.2.1 Extraction of phage DNA from phage lysates
For this experiment the initially filtered phage lysates were used. The phage DNA to be amplified had to be separated from the host DNA and RNA. To do this 0.5ml of phage lysate was transferred to sterile eppendorf tubes. The phage lysates used here were sewage, water from pond, chicken faecal, sediment, B.fragilis- sewage sample, B.fragilis sediment 1(reed bed trowel) and B.fragilis sediment 2( trowel 2 edge). Since the phages could not have been grown on B.fragilis, they are directly being checked by whole genome analysis. To the above eppendorf tube, RNaseA and DNaseI were added to a final concentration of 1µg/ml. This was allowed to incubate at 37°C for 30 minutes to allow the complete degradation of host DNA and RNA. Phenol/chloroform extraction method was used to collect the phage DNA. 1ml of Tris buffered Phenol chloroform (1:1 v/v) was added to the above mixture and vortexed for 1 minute. The layers were separated by running them in a microcentrifuge at 16,000 rpm for 2 minutes. The supernatant was carefully transferred into another tube containing 1ml of the phenol/ chloroform for second separation. This was vortexed and span again for 2 minutes at same speed. The upper aqueous layer was separated into another sterile microcentrifuge tube and 1ml of chloroform was added to the sample, vortexed and span again. The upper layer from this was separated into another tube and 1ml isopropanol was added to the mixture and it was made to stand for 5minutes to allow precipitation of DNA. The phage DNA was finally collected by centrifuging this mixture at 16,000 rpm for 5 minutes. The pellet was washed with 100µl cold ethanol and dried under vacuum using SpeedVac for 10 minutes. The DNA was re-suspended in 10µL of TE buffer and stored in ice.
3.2.2 Whole genome amplification reactions
The amplification reaction steps that were illustrated in the Kit protocol were used for the phage lysates. The first step involves the addition of 0.9µL of sterile sample buffer to 0.1µL of phage DNA from different samples, purified in the previous step, in PCR tubes in the PCR cabinet. This phage DNA acts as a template DNA for the amplification reaction. One more tube without any DNA was used as a negative control to check for any contaminants in the procedure. The PCR tubes were then placed in the PCR machine where in they were heated at 95°C for 3 minutes and then cooled at 4°C to allow the phage DNA to denature. While this is cooling, master mixes were prepared in tubes in the PCR cabinet in ice by adding 9µL of reaction buffer with 1µL of enzyme mix. This master mix was added to each of the above phage mixtures and incubated in the PCR machine at 30°C for 1.5hours to allow the amplification of DNA. The final step was the inactivation of the Phi29 DNA polymerase enzyme by heating the samples at 65°C for 10minutes followed by cooling at 4°C. The amplified samples were then stored in the freezer at -20°C for further use.
3.2.3 Analysis of whole genome amplified products
The amplified phage DNA samples were analyzed using agarose gel electrophoresis. A 1% agarose gel was prepared by adding 0.5g agarose in 50ml of 1xTAE buffer and heating the mixture till the agarose dissolved. To visualize the DNA, ethidium bromide dye was used that is an effective stain that intercalates the DNA but is a mutagen and should be handled with care. 3µl ethidium bromide was added to the gel, mixed thoroughly and poured into the gel tray. An appropriate comb was inserted and the gel was allowed to solidify.
Once the gel had solidified, it was placed in the gel tank and 1xTAE buffer was poured to completely cover the gel. Combs were removed and samples mixed with loading dye were loaded into the wells. The first lane had a marker of 1Kb ladder (500µg/ml concentration) followed by the seven amplification samples and the last lane had the negative control. Samples were allowed to run at 70V for 45minutes. The bands were observed under a UV transilluminator via GelDoc system.
RESULT AND DISCUSSION
The environmental samples were successfully collected from their respective sources. The sediment and pond water samples were collected from Blackford pond. The sewage sample was collected from a sewage process plant. The chicken faecal sample was collected from a private property in Edinburgh.
The sediment sample (reed bed trowel) was thoroughly processed using a two stage filtration process by this group. First being a coarse filtration by whatman filter paper and second by 0.22µm pore filter. This method successfully filtered out the debris and cellular contaminants from the samples allowing only phage to be present in the filtrate. The other samples were filtered by the other groups.
Phage analysis was done by two independent methods to allow accurate detection of any phage that may be present in the environmental samples. The first method used was the culture dependent method that observed the growth of phage on host lawns in the form of plaques. Five different cultures were used. The second method was a culture independent method that allows the isolation of any possible phage DNA samples directly from the environmental samples and followed by non- specific amplification and sequencing of phage DNA.
For the first method, phage lysates from sediment 1(reed bed trowel), sediment 2(trowel 2 bed) and sewage samples were successfully amplified on B.fragilis NCTC 9343 cells grown in BHI-S media. After incubation, the media showed considerable cell growth and insignificant cell lysis. However, the phages from these amplified samples were filtered using 0.22µm pore filter and stored in sterile tubes.
The phage lysates were tested on lawns of host organism for specificity and phage titre. Here, the organism used was B.fragilis NCTC 638R (restriction minus derivative). Five agar plates for each lysate were prepared by adding supplements into the base agar and pouring it into the petri dishes. Molten top agar was mixed with organism and poured onto the base agar. The difficulty in this step was that the top agar did not solidify. The plates were left overnight to ensure complete solidification of bacterial lawns. But, even the next day the lawns were not formed. The difficulties during this step could be attributed to the concentration of agar in the top agar preparation. Lower concentration of agar allows slow solidification of agar. Also some human errors could have been involved. The optimum concentration needed for the top agar in 6g agar per l. Therefore, the experiment was discontinued and phage samples were not plated on B.fragilis lawns. Instead, these samples were studied by the other method of whole genome amplification to check if any phages specific to B.fragilis existed.
Among the other organisms, E.coli MG1655 and S.typhimurium SL1344 showed visible plaques formation for varying concentration of samples of sewage and chicken faeces. Hence, the experiment was continued using the plaques formed on E.coli MG 1655 for sewage (dilution 4) and chicken faeces (dilution 4). The plaques from these plates were purified using chloroform and buffer. New plates of E.coli MG 1655 were made and lawns of organism were grown on the plates. The purified phage was poured onto the plates on three different spots on the media. After incubation, isolated plaques were formed on the three spots. Each spot corresponds to one unique bacteriophage specific for E.coli.
Phage from this unique plaque formed by the sewage sample on E.coli was re-suspended into culture containing E.coli in LB broth by this group. Lysis of cells was observed in this sample and also in the other two samples. The results revealed that the lysis was in this order- sewage in E.coli> chicken faeces in E.coli> sewage in S.typhimurium. The samples of phages in E.coli showed significant lysis whereas phages in S.typhimurium did not completely lyse the culture.
The phage DNA from these samples was extracted and precipitated. Further, the DNA present in the samples will be quantitatively tested by gel electrophoresis and nanodrop spectrophotometry to determine the DNA concentration. The samples will later be submitted for sequencing.
The other approach used for analysis of phage in environmental sample was the metagenomic method. In this method, the filtered environmental samples of sewage, pond water, pond sediment and chicken faeces along with sediments that were amplified on B.fragilis were amplified using whole genome amplification kit. The phage DNA from all the eight samples was separated from the contaminating DNA by RNaseA and DNaseI. The phage DNA was extracted using phenol/chloroform solution. The final phage DNA was used as the template for the whole genome amplification reaction. The kit used DNA polymerase from Phi29 bacteriophage. The amplification reaction underwent three stages that had specific conditions maintained for the fixed time period. The stages are denaturation, annealing and elongation. A negative control having no DNA was used to detect any contamination in the method. These amplified samples were stored.
M L1 L2 L3 L4 L5 L6 L7 L8
Fig (1)- Gel image after agarose gel electrophoresis of phage samples; M = Marker (100 bp ladder), L1 = Sewage (unamplified), L2 = Pond Water (unamplified), L3 = Pond Sediment (unamplified), L4 = Chicken Faeces (unamplified), L5 = Sewage (amplified on B. fragilis), L6 = Sediment 1 (amplified on B. fragilis), L7 = Sediment 2 (amplified on B. fragilis), L8 = Negative Control
Some samples were analyzed by running on agarose gel electrophoresis to detect the presence of DNA in the sample. The gel in fig (1) revealed that the samples of sewage and pond water showed detectable DNA in the form of smears on the gel proposing that these samples have significant bactriophages and can be amplified and sent for sequencing. Also the negative control showed no DNA band confirming that the reaction had no external contamination.
In conclusion, both the analysis methods gave independent results which confirm the presence of bacteriophages in the environmental samples collected. The culture dependent method showed presence of E.coli specific phages in sewage samples and chicken faeces samples and S.typhimurium specific phages in sewage sample. This partially corresponds with the whole genome analysis results. The whole genome analysis method reveals presence of phage in sewage and pond water sample. Therefore, all the samples will further be sent for sequencing to determine the sequence of the phages found the environmental samples.
CLARK, JR., MARCH, JB.(2006) Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Trends Biotechnol 24(5):212-218.
HAQ, IU. Et al (2012), Bacteriophages and their implications on future biotechnology: a review, Virology Journal 9:9
STANDARD OPERATING PROCEDURE FOR THE FILTRATION AND CONCENTRATION OF BACTERIOPHAGES FROM THE ENVIRONMENTAL SEDIMENT SAMPLES
TABLE OF CONTENTS
Scope and application
Summary of the procedure
Safety and waste handling
Equipment and supplies
Filtration and concentration procedure
Standard Operating Procedure for the Filtration And Concentration of Bacteriophages from the Environmental Sediment Samples
SCOPE AND APPLICATION
This Standard Operating Procedure describes the filtration and concentration of bacteriophages from the environmental sediment samples. The samples are collected from the Blackford Pond in Edinburgh and filtered using a two stage filtration process. The filtration process included a coarse filter followed by a fine filter. Sample filtrate is concentrated and stored.
SUMMARY OF THE PROCEDURE
The pond samples have to be processed to separate the bacteriophage from the environmental debris and contaminants. Sterile phage buffer is used to re-suspend the environmental samples. Filtration is done in two steps where in the first step uses a Whatmans filter paper and the second step uses a 0.22µm pore size filter. The first stage removes larger solid contaminants and the second filters out the bacteria but allow the bacteriophages to pass through. The filtered phage is then concentrated.
SAFETY AND WASTE HANDLING
Environmental samples are composed of varying contaminants ranging from bacteria to viruses and therefore should be handled with care. It is the responsibility of the user to handle the samples safely. Gloves and lab coats should be worn at all times while handling these samples.
Waste should be disposed as per the rules of the laboratory. The waste should be disposed off in the designated waste bins.
The whatman filter can changed if it is too clogged due to large sediment particles.
The syringe should be depressed slowly. The pore filter should not be contaminated and should be held in place while filtering.
EQUIPMENT AND REAGENTS
Whatmans filter paper and 0.22µm pore size filter
Single Centicon Plus-70 Centrifugal Filter Unit
Phage buffer consisted of Tris-HCLl(3.15g), NaCl(5.84g), MgSO4(1.21g) and made up to 1000ml with distilled water. The pH was adjusted to 7.4.
FILTRATION AND CONCENTRATION PROCEDURE
Suitable volume of sediment sample is added to 40ml sterile sample buffer in a 50ml plastic tube.
The sample is mixed thoroughly either by gently vortexing or mixing with a pipette. After re-suspending, the sample is placed in ice.
Whatman No. 1 filter paper is folded and placed on a filter funnel. The re-suspended sample is allowed to pass through it and the filtrate is collected in a sterile 50ml tube and is placed in ice.
A sterile single use plastic 50ml syringe is fit into single use 0.22µm filter unit and placed on a sterile 50ml tube.
The plunger is removed from the syringe and the filtrate is carefully added into the syringe barrel.
Then the plunger is placed back in to the syringe and slowly depressed to allow the drop-by-drop flow of filtrate through the pore filter. When all the liquid has passed through the pore filter, the final phage filtrate is stored in ice.
The filtered phage sample is added to a single Centicon Plus-70 Centrifugal Filter Unit. This unit contains a membrane that allows the flow of 100kDa molecular weight particles. This allows larger solutes to pass and retains the phage in the upper portion.
The centrifugal filter unit is span in a refrigerated centrifuge at 3,500 x g for 10minutes and reversed and centrifuged at same conditions for 2 minutes.
The concentrated phage sample is pipette out into an eppendorf and 10µL of chloroform was added to the final solution and stored in ice.