AEDNet Interim Report

Project Name Anthrax Environmental Decontamination Network (AEDNet)

Report compiled by Dr. Fatih BUYUKWith contributions from Dr. Dogan AKCA

Reporting periodInterim report between 3rd of November and 3rd of December, 2015

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 stil 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 maximises 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 results

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AEDNet Interim Report

AEDNet contains several work packages, tasks and deliverables for four years. It has 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 characterisation 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 there own contaminated sites.1.1.2. Characterisation: It is essential that any bacteriophages which will be employed as an environmental decontaminant are fully characterised 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 recognised different binding sites on the surface of B.anthracis. Thus training will be provided in how to identify bacteriophages which recognised 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

Purification of Bacillus anthracis specific bacteriophages from soil samples of Georgian territories

1. SamplingThree sites at which Bacillus anthracis contaminated animals had been either buried or autopsied were sampled. Samples were taken randomly from each focus without using any systematic method. Samples were harvested from the top 5-10 cm of

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topsoil and with a wight of 15-20 g in duplicate; one copy for spore and the other for specific bacteriophage isolation of B. anthracis. Samples were then transferred to clean 300 ml plastic pots and returned to the laboratory for processing (Table 1 and Figure 1).Table 1. Characteristics of sampling areas.

Focus information Focus 1 Focus 2 Focus 3

Location Close to X Village In X Village Next to the Baku-Ceyhan-Tbilisi Pipeline - X Region

HistorySeveral dead animals, mostly cattle, were burned and buried in it

Contaminated with infected animal carcass - one fatal human case

Contaminated with infected animal carcass

Focus characteristics

A hole surrounded by concrete blocks-about 3mX3m area

A garden in front of the barn

Periphery of a concrete block next to real contaminated restricted area

Coordinates X X X

Altitude 463 468 390

Contamination time 2004 2004 2004

Soil samples used for phage purification

# 1# 2 # 5 # 9

# 12

Figure 1. Representative photos from soil sampling areas.

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2. Cultivation, Propagation and CharacterisationFirstly, 50 g soil was dissolved in 450 ml of sterile distilled water in a 1l cell culture flask. The mixture was shaked well and waited overnight at room temperature for the soil will have settled down to the bottom of the flask.The next day, the supernatant was filtered through ordinary filter paper to remove all debris. Roughly 150-200 ml filtrate was harvested from each samples. Totally 45 ml of each sample filtrate was add onto 5 ml of 10XBrain-Heart Infusion (BHI) broth. 500 ul of fresh host (and mixed host) B. anthracis vaccine strain culture that was prepared as below (Table 2) was added on this mixture and incubated at 37 ◦C overnight.Bacterial host culture preparation:

Three vaccine strain of B. anthracis were used as host. These are: B. antharcis STI: A Georgian/Russian live anthrax spore vaccine, B. anthracis 55: An equivalent type of vaccine is used in Russia and Eastern

Block countries, B. anthracis 34F2: Sterne strain, has naturally lost its pXO2 plasmid, and

consequently its ability to produce a capsule, B. anthracis I17(Ikhtiman): I-17 is pXO2 negative and were received by the G.

Eliava IBMV collection in 1967. The host bacterial growth from slants were washed with 4,5 ml of BHI broth and vortexed kindly. The mixture of three bacterial culture was adjusted by using 1 ml of each bacterial growth in a tube and mixed well. 0,5 ml of this mixture used for each individual phage suspension.

Table 2. The alignment of sample and host combinations.Phage number Sample number Prefered host combination

1 1 Ba 34F2 + Ba 55 + Ba STI

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2 1 Ba 553 1 Ba STI4 2 Ba 34F2 + Ba 55 + Ba STI5 2 Ba 556 2 Ba STI7 5 Ba 34F2 + Ba 55 + Ba STI8 5 Ba 559 5 Ba STI

10 9 Ba 34F2 + Ba 55 + Ba STI11 9 Ba 5512 9 Ba STI13 12 Ba 34F2 + Ba 55 + Ba STI14 12 Ba 5515 12 Ba STI

The following day, falcon tubes containing 50,5 ml mixture were centrifugated at 4000 rpm for 35 minutes at +4 ◦C. The supernatant was filtered through 0.20 um syringe filter and about 10-15 ml presumptive phage solution was obtained. The phage supernatant was stored at +4 ◦C for future tests.Duble-layer agar method (composed of BHI broth and 0.5 g agar bacteriological) was used for plating of phage suspensions on three different host bacteria (Ba 55, Ba STI, Ba 34F2). Fresh bacterial slants were washed 4,5ml BHI broth and vortexed briefly. 100 ul of these bacterial suspensions were mixed about 3 ml soft agar with temperature of 40-45 ◦C. All suspensions were poured on the solid BHI agar plates and wait 5-10 minutes for dry. 10 ul from each phage suspension was dropped on a unique BHI agar plates that were seeded with bacteria and permitted to dry for 10-15 minutes. Plates containing host strain and phage were incubated at 37 ◦C for overnight.Plates were evaluated and photographed (Figure 2). Sample #8 on Ba 55 host and sample #15 on Ba STI were chosen representing potentially different phage pattern (Table 3).

Figure 2. First phage results on certain bacterial host.

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During the propagation step of these phages (#8 and #15) duble-layer agar method was used for plating. Five hundred ul of these phage stock was mixed with 4,5ml broth and vortexed well. A hundred ul certain bacterial strain (Ba 55 for #8 and Ba STI for #15) was added onto 1 ml of ten times diluted phage. About 3ml soft agar at 40 ◦C was added onto bacteria-phage mixture and was plated on BHI solid agar plates. Plates were incubated at 37 ◦C overnight.Table 3. Host-phage interaction results.

Phage number

Sample number

(#)Host characteristic

Host bacteria using for preying of phages

Ba 55 Ba STI Ba 34F2 Ba I17

1 1 Ba 34F2 + Ba 55 + Ba STI - - - -2 1 Ba 55 - - - -3 1 Ba STI - - - -4 2 Ba 34F2 + Ba 55 + Ba STI - - - -5 2 Ba 55 - - - -6 2 Ba STI - - - -7 5 Ba 34F2 + Ba 55 + Ba STI + + + +8 5 Ba 55 + + + +9 5 Ba STI + + + +

10 9 Ba 34F2 + Ba 55 + Ba STI + + + -11 9 Ba 55 - - + +12 9 Ba STI - + + -13 12 Ba 34F2 + Ba 55 + Ba STI - - + -14 12 Ba 55 + + + +15 12 Ba STI + + + +

12 Ba I17 +

The following day, plates were evaluated and decided which phage plaque is representing potentially different pattern (Figure 3).

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Figure 3. Phage plaque on host bacterial culture.

Two different plaques were picked up from sample #15 and one plaque from sample #8 and suspended in 1ml broth. Phage plaques were vortexed kindly and was mixed with 100 ul related host and of 3ml soft agar and incubated at 37 ◦C overnight.

The next day, plates were evaluated; the phages lysed corresponding bacteria. For future purification from each phage plates, soft agar layer was skimmed off in a 50 ml Falcon tube and was suspended 1ml broth, vortexed briefly and centrifugated at 4000 rpm for 30 min. The supernatant was filtered 0.20 ul filter and phage filtrate was stored at +4 ◦C for future purification.

Figure 4. Host-selected phage interaction on solid media.

The next day, three filtrated phage suspensions were diluted serially for plating on solid media for subsequent purification (to obtain individual phage lines) (Figure 5).

Ten-fold sub-dilutions (10-1 to 10-8) of purified phages with a final volume of 4,5 ml were prepared

Phage sub-dilutions of 10-6 and 10-8 were plated. 1 ml of each dilution was transferred in a tube and mixed 100 ul their host bacterial culture. 3 ml soft agar was added on tube and the mixture was poured on solid agar plates. The plates were incubated at 37 ◦C overnight.

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Figure 5. Phage plaque morphology on solid agar.

The last step, two droplets of chloroform was added onto the tubes containing phage picked up the day before and vortexed briefly (chloroform is expected to clear host bacterial residues. 500 ul phage suspension (carefully harvested without taking chloroform) was subdiluted and plated with bacterial cultur and soft agar.

After last purification and sufficient propagation of 5 different phages, ten-fold sub-dilutions were prepared and plated with soft agar to calculate the pure phage titer as pfu/ml.

Table 4. Summary of 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 2x109

Ba I17 #12 small Focus 3 12 B. anthracis I17 5x109

Ba I17 #12 big Focus 3 12 B. anthracis I17 8x109

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

Ba STI #15 big Focus 3 12 B. anthracis STI 4x109

As a summary, 5 phages with a different pattern (host, size, etc.) specific to B. anthracis were obtained after cultivation of soil samples from two contaminated areas (Facus 2 and 3) of Georgian territories. Phages were stored +4 ◦C for future use.

The training was informative and powerful to detecting anthrax specific bacteriophages from contaminated areas such as soil. We are able to improve our skill on phage isolation and characterization which was great opportunity for Turkish researchers.

Researchers from Kafkas University:Fatih BUYUKDogan AKCA

Coordinator name of G. Eliava Instituteof Bacteriophages, Microbiology and Virology:

Mzia KUTATELADZE

Trainers from the Eliava Institute:Lika LESHKASHELIDarejan BOLKVADZE

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