Anthrax Environmental Decontamination Network · Web viewadditional phages isolation and characterization from burial sites in Georgia and Turkey sharing of best practices and to link

AEDNet Interim Report

Project Name Anthrax Environmental Decontamination Network (AEDNet)

Report compiled by Mustafa Reha COSKUNWith contributions from Dr. Fatih BUYUKReporting period 15th of January to 12th of February, 2017

Home InstituteKafkas University, Faculty of Veterinary Medicine, Department of Microbiology, 36100, Kars, TURKEY

Host InstituteG.Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, GEORGIA

Section one: SummaryAnthrax, caused by the spore forming bacterium Bacillus anthracis, is a disease of animals which can infect humans either directly through contact with infected animals or indirectly as a consequence of bioterrorism. Contamination of large areas due to the ability of the spores to survive in soil for many years is the main abutment of anthrax outbreak. Due to the lack of effective preventative and control programmes anthrax is still prevalent in countries such as Eastern Europe and Central Asia and to have a major impact on human and animal health. Anthrax Environmental Decontamination Network (AEDNet) is the project of to link related anthrax study group and projects together in a manner which maximizes the current investment and will deliver an environmentally friendly decontamination system.AEDNet aims to the followings,• reducing of spore numbers with ecologically friendly agents-bacteriophages• additional phages isolation and characterization from burial sites in Georgia and

Turkey• sharing of best practices and to link researchers in neighboring countries facing

similar problems• maximize the current investment• deliver an environmentally friendly decontamination system• provide training opportunities for researcher• increase individual mobility and the career prospects of each person involved• facilitating transfer of knowledge between research groups and to maximize the

dissemination and impact of the resultsAEDNet contains several work packages, tasks and deliverables for four years. It has

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also plenty of secondments to undertake of workload during this four years period. One of them is as follows:

Task 1.1 : The isolation and characterization of B.anthracis specific bacteriophages

1.1.1. Isolation: A range of approaches have been employed by members of the network to isolate B.anthracis specific bacteriophages from B.anthracis spore contaminated animal burial sites in Georgia (EIG) and Turkey (KAU). As part of this effort we will compare and contrast the effectiveness of these methods and will provide training to groups seeking to isolate bacteriophages from their own contaminated sites.1.1.2. Characterization: It is essential that any bacteriophages which will be employed as an environmental decontaminant are fully characterized with regards its physical, genetic and biological properties. The group at Eliava are the world experts in this area and will provide training in how to undertake these studies.1.1.3. Phage cocktail: The ability of bacteria to develop resistance to a particular bacteriophage means that any future decontamination approach is likely to be based on a mixture of bacteriophages which recognized different binding sites on the surface of B.anthracis. Thus training will be provided in how to identify bacteriophages which recognized different receptors (EIG, KAU and CU).1.1.4. Binding avidity: We will also provide training in the use of laboratory methods developed in Cardiff to increase the binding specificity of individual phages.Section two: Activities and Progress

Identification and Measurement of Some Biological Values of Anthrax Phages

1. Phage concentration and titer determination for future analysisDuring the last secondment (between 3rd of November and 3rd of December, 2015) we have been isolated and purified 5 different phages from topsoil samples of Georgian territories (Table 1). Ba STI #15 small is noticed to have the same morphology with Ba STI #15 big phage and this these were assumed as a unique phage for future characterization. In this manner, 4 field phages were included in this study and the

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order of these is: Ba 55 #8, Ba I17 #12 small, Ba I17 #12 big and Ba STI #15 big. These phages titers were propagated with soft agar method and determined final titers as 2X1010, 3X1010, 2X1010 and 1X1010, respectively. To preparation of phage samples for SDS-PAGE analysis, the 10-5 and 10-6 phage dilutions plates like a spider web image were collected from soft agar with drigalski and added the same volume broth and centrifuged at 4000 rpm for 35 min. After that, supernatants were filtered through 0,22 um filter and the filtrates were used for SDS-PAGE.Table 1. Summary of the isolated phages from soil samples.

Phage code Location Soil sample number Host Phage titer

(pfu/ml)Ba 55 #8 Focus 2 5 B. anthracis 55 2x1010

Ba I17 #12 small Focus 3 12 B. anthracis I17 3x1010

Ba I17 #12 big Focus 3 12 B. anthracis I17 2x1010

Ba STI #15 small Focus 3 12 B. anthracis STI 2x109

Ba STI #15 big Focus 3 12 B. anthracis STI 1x1010

2. SDS-PAGE analysis of the purified phagesThe phage proteins were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) on 12% acrylamide. Due to the solidification error of SDS-PAGE gel this method was not successfully performed.

3. Restriction fragment analysis of phage genomic DNAs3.1. Genomic DNA extraction of phagesPhages were propagated and their genomic DNAs were extracted by phenol-choroform-isoamyl alcohol method. In brief, 5 ul DNase and 5 ul RNase were added on 100 ul phage suspension and the mixture was incubated at room temperature for 30 min. 5 ul proteinase K (50mg/ml) was added and tubes were incubated at heat-block at 56 ºC for 15 min. After heat blocking, 15 ul lysis buffer (100 ul 10% SDS, 100 ul 0,5M EDTA, 800 ul ddH2O) was added and incubated for 10 min. 500 ul phenol was added and the tubes were kindyl shaked by hand for 10 min. Tubes were centrifugated at 10000 g for 10 min and about 500 ul of top-DNA-phase was harvested with pipette tip. 500 ul phenol-chloroform-isoamyl alcohol (25:24:1) was added and tubes were kindyl shaked by hand for 10 min and then were centrifugated at 10000 g for 10 min. The toplayer suspension was harvested and mixed with 500 ul

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chloroform-isoamyl alcohol (24:1) and tubes were kindyl shaked by hand for 10 min and then were centrifugated at 10000 g for 10 min for future purification. After centrifugation, top DNA layer was harvested into a microcentrifuge tube with a volume of 500 ul and 300 ul isopropanol and 10 ul 5M NaCl2 was added and tubes were shaked kindy to check visible DNA. Tube were centrifugated at 10000 g for 10 min to get DNA pellet and this step was repeted with adding 1 ml 70% ethanol on DNAs. The purified DNAs were final suspended with 100 ul TE buffer and kept for future use.

DNA concentrations were determined with agarose gel before the restriction analysis. For this, DNAs were run on 1.5% agarose gel and finally it was detected that phage Ba 55 #8 and Ba I17 #12 small had good amount of DNA but others had low quantities (Figure 1).

Figure 1. Gel electrophoresis images of extracted phage DNAs.

3.2. Restriction fragment analysisPurified phage DNAs were separately digested with EcoRI and EcoRV restriction endonucleases and subsequently subjected to electrophoretic analyses. A reaction mixture was preparet as shown in Table 2. Tubes were incubated at 37 ºC for 2 hours. After incubation, the restricted products were analysed on 1.5% agarose gel.

Table 2. A reaction mixture of restriction analysis of phages.Phage code DNA amount

(ul)Enzyme Enz. amount

(ul)Buffer Buff. amount

(ul)H2O (ul)

Ba 55 #8 2 EcoRI 1 EcoRI 2 15

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bufferBa I17 #12 small

2 EcoRI 1 EcoRI buffer

2 15

Ba I17 #12 big6 EcoRI 1 EcoRI

buffer2 11

Ba STI #15 small

6 EcoRI 1 EcoRI buffer

2 11

Ba STI #15 big6 EcoRI 1 EcoRI

buffer2 11

Ba 55 #82 EcoRV 1 EcoRV

buffer2 15

Ba I17 #12 small

2 EcoRV 1 EcoRV buffer

2 15

Ba I17 #12 big6 EcoRV 1 EcoRV

buffer2 11

Ba STI #15 small

6 EcoRV 1 EcoRV buffer

2 11

Ba STI #15 big6 EcoRV 1 EcoRV

buffer2 11

The gel image was not satisfying for make a profile analysis of restricted DNA (Figure 2) and it was thought that the reason of this was poor qualty or low concentration of DNAs.

Figure 2. Gel electrophoresis image of restricted DNAs of phages.

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4. Measurement of some biological activities of phages4.1. Measurement adsorption rateAdsorption is the first step of viral infection. It is also important to select phage with a best adsorption rate to future use. For learn adsorption rate the following goods were prepared;

host bacteria (Ba STI) with a titer as 108 cfu/ml and testing phage (Ba STI #15 small) with a titer as 107 pfu/ml (reaction tube)

test control with a titer as 107 pfu/ml (control tube) 9.9 ml cold broth (at +4◦C) (cold shock to stop adsorption) soft agar, agar and broth water bath (37◦C) filter (0,22 µm)

Two ml phage and host bacteria with given concentrations was mixed and put in a water bath at 37◦C for the following time-point sampling. 100 ul of sample was taken at 5, 10, 15, 20, 25 and 30th minutes of incubation. Sample was transferred in a reaction tube including 9.9 ml cold broth and was kindly vortexed and filtered (cold-shock). 100 ul of filtrate was added to 900 µl broth (was diluted ten times). 100 µl diluted phage, 100 ul bacteria and 3ml soft agar were kindly mixed and poured on solid media to count phage plaques. For test control, phage-bacteria from control tube that was synchronously incubated with test tube at water-bath and at the end of the last time point (30 min) 100 µl was added on 9,9 ml broth without any cold-shock step and seeded on solid agar. Plates were incubated at 37 and the results were read after 24 hours. To get confidential results adsorption test was repeated three times.

Table 3. The plaque number of time point sampling and adsorption rate of phage.Time point Number of phage plaque Rate of non-adsorbed phage

(%)Adsorption rate (%)

5 min 52 5,1 ≈%9510 min 23 2,3 ≈%9815 min 9 0,8 ≈%9920 min 10 0,8 ≈%9925 min 3 0,2 ≈%10030 min 0 - -

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The adsorption rate was calculated by considering the plaque number of control tube as 1016 (Table 3). The plaque number of each time point was divided to the number of control tube plaque and multiplied a hundred times. Thus, non-absorbed phage rate was detected. The absorbed phage rate (so the absorption rate) was obtained by subtracting the non-adsorbed phage rate from a hundred.

During the second and third experiments, the incubation time in water bath were shortened and different time points were selected as 2, 4, 6, 8, 10, 13 and 15 min due to the short time requirement of phage to sufficient adsorption. As the result of three runs we chose a more constant adsorption rate (maximum adsorption rate in minimum time) of the phage Ba STI #15 small as 98% after the 6 minute of incubation.

4.2. One step growth experimentA lytic phages in a host bacteria can multiply with different phases and the shape of the curve originated from the multiplication would look step-wise and is called “one-step phage growth curve” (Figure 1). The initial period (eclipse) is the time period taken for the appearance of first intracellular phages and phages can not be detected during this period as the phages are being uncoated and phage DNA is being injected for replication. The phage growth curve starts with a latent or eclipse period. The next phase is called as maturation or release stage when new phage particles are assembled and released.Synthetic period is the time during intracellular particles are being produced. As in the eclipse period, there are no phage particles released during the synthetic period.The first two periods are combined in the third period known as latent period. The latent period is a time period prior to the release of infection particles or appearance of extra cellular phages.Rise period is a period lysis occurs and extracellular phages appear and they increase in number of concentration of bacteriophages rises.

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Figure 1. The one-step phage growth curve of a general lytic bacteriophage.

A one-step growth experiment was conducted to determine the latent time period and burst size of the phage Ba STI #15 small. In brief, reaction tube like adsorption experiment (108 cfu/ml bacteria+ 107 pfu/ml phage) was incubated at water-bath at 37 ºC for 6 minutes. Then, 100 µl reaction tube content was transferred into 9,9 ml broth (stock dilution tube) and thus it was subdiluted 1 to 50 (dilution 1, rate is 5.000 time). 100 µl stock dilution tube content was transferred into 9,9 ml broth (dilution 2, rate is 500.000 time). Two different time point with dilution sets were performed for calculation of burst size. The first, 100 µl sample was taken from first dilution tube at 15th to 60th min for every 5 minutes intervals and the second from dilution tube at 20th

to 120th minute for every 10 minutes intervals. Samples were plated with soft agar method with the given amount of host bacteria and soft broth.

Table 4. Phage plaque amount of different dilution tubes.Time point of dilution 1

Number of phage plaque

Time point of dilution 2 Number of phage plaque

15 min. 161 20 min. 020 min. 131 30 min. 225 min. 108 40 min. 130 min. 91 50 min. 235 min. 117 60 min. 140 min. 132 70 min. 145 min. 133 80 min. 5

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50 min. 124 90 min. 3655 min. 145 100 min. 21

60 min. 97 110 min. 36120 min. 20

The fixed values of three time points which were close to each other were chosen to burst size calculation. For this purpose, the plaque number of 40, 45 and 50 min were considered for dilution tube 1 and of 90, 100, 110 and 120 min were for dilution tube 2 (Table 4).

Dilution1=132+133+1243 =129,9

Dilution2=36+21+36+204 =28,2

A formula displayed as below was used by considering the dilution 1 and 2 data for burst size calculation.

Burst ¿Dilution2×100

Dilution1−( non−adsorbed phage5 )=28,2×100

130−(335 )= 23

The burst size (viral yield) was determined from the ratio of the mean yield of phage used for bacterial infection to the mean of phage particles liberated after infection. Thus, the burst size of the phage Ba STI #15 small was calculated as 23 pfu per infected bacteria.

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Figure 2. The one-step phage growth curve of phage Ba STI #15 small.

The separate time calculation of eclipse, synthetic and rise period is not performed in this experiment. But, the latent period (represents the time after penetration through release of mature phages) of phage Ba STI #15 small was calculated as 80 minutes (Figure 2). The stationer phase at the end of the rise period is not constant, thus it looks need to repeat one-step growth experiment much more.

5.Phage MorphologyIt would be good to put here the details of imaging method and electron microscope photos of phages.

The training was informative and powerful to characterizing anthrax specific bacteriophages isolated from contaminated areas of Georgian territories. We are able to improve our skill on phage isolation and characterization which was great opportunity for Turkish researchers.

Researchers from Kafkas University:Mustafa Reha COSKUNFatih BUYUK

Coordinator name of G. Eliava Institute

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of Bacteriophages, Microbiology and Virology:Mzia KUTATELADZE

Trainers from the Eliava Institute:Lika LESHKASHELIDarejan BOLKVADZE

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Anthrax Environmental Decontamination Network · Web viewadditional phages isolation and characterization from burial sites in Georgia and Turkey sharing of best practices and to link