Category Archives: Uncategorized

2016 Secondment and Technical Reports

a-tskhvediani-secondment-reports-uhoh-2016

williams-secondment-report-eig-2016

zakalashvili-secondment-report-kas-nov-2016

abazashvili-secondment-report-kas-jun-0216

abazashvili-secondment-report-nov-2016

askilashvili_-secondment-report-cardiff-2016

bolkvadze_-secpndment-report-cardiff-2016

brown-secondment-report-eig-2016

l-leshkasheli-secondment-report-to-uhoh-2016

martashvili-secondment-report-kas-nov-2016

martashvili-secondment-report-kas-jun-2016

rhiannon-probert-secondment-report-eig-2016

Technical reports

d4-17-month-30-internal-technical-report

d4-21-month-36-internal-technical-report-completed

milestone-7-progress-report-2016

modebadze-secondment-report-kas-jun-2016

m8-report-on-the-efficacy-of-field-trials-progress-report

m9-report-on-the-genetic-diversity-of-banthacis-isolates-in-the-region-progress-report

2015 Secondment Updates

Summary and technical reports of 2015 secondments

Technical Report Mariam Zakalashvili form NCDC to KAU 2015

NCDC ro KAU 03-07-2015-report-of-research-activity-nino-modebadze

Bolotin and Koshheliev from NSC IECVM to UHOH 2015

Beyer from UHOH to NCDC Sept 2015

Emanuele Campese to NCDC 2015

Baillie from CU to NCDC 03-07-2015

Coskun from KAU to NCDC 2015 (1)

Technical Report of trip in Cardiff-Adam Kotorashvili DEcember 14-27[1]

Technical Report of trip in Cardiff-Adam Kotorashvili

Report of Research Activity – Lia Askilashvili

Report of Research Activity – Darejan Bolkvadze

Technical ReportE.Khmaladze from NCDC to WiHiE 2015

03-07-2015-report-of-research-activity-from-eig-to-izspb-natia-skhirtladze-2

Technical Report T. Imnadze to NCDC from WiHie 2015

Technical Report K. Sidamonidze from NCDC to WiHiE 2015

Secondment report Beyer

Secondment reporrt Rohmer from UHOH to EIG 2015

Report AEDNet Project- Emanuele Campese

Report Buyuk and Akca KAU to EIG 2015

Technical Report of trip in Cardiff-Adam Kotorashvili-1

Anthrax decontamination Ukrainian

 

Abstracts and Posters

Recent Conference abstracts and Posters

Investigation on Bacillus anthracis circulating in Bangladesh through the analysis of soil samples in areas at risk and imported animal feed –  Anthrax in Bangladesh 

Anthrax Environmental Decontamination Network Abstract Abstract Anthrax ADEnet-4 and Poster Poster_Chakvetadze_NCDC_BACT_2015_20151020-2

Modern Turkish approaches to soils ‘decontamination from anthrax’ agent with attention to Ukraine. Biosafety and Biosecurity 2015. Ukraine paper

 

 

Milestone 7 Report

Romuald Gryko – Military Institute of Hygiene and Epidemiology – Poland

Milestone 7:  A germanant/phage lysin decontamination formulation suitable for field trials- (WiHE)- progress report April 2015

Colleagues at the Military Institute of Hygiene and Epidemiology  in Poland (WiHE) are working to identify bacteriophages which contain lysins capable to targeting the cell wall of B.anthracis.   They  first isolated bacteriophages from environmental samples with activity against B.anthracis 34F2.

Environmental isolation method

In brief, 20 gram of  environmental samples (agricultural soils, sewages) was added to 40ml of Tryptose Soy Infusion  supplemented with 5 mM MgSO4  (TSIMg).   The suspension was then inoculated with  100 µl of a  24 h. culture of B. anthracis 34F2  and incubation   for 24 hr at 370C. Following incubation  the sample was treated with chloroform and centrifuged at 4000rpm for 30 min (2952 x g).

To determine if any phages were present  sample supernatant was dropped onto the surface of a  Tryptose Soy Infusion Agar  plate supplemented with 5 mM MgSO4  (TSAMg)  in which the  upper layer of agar had been inoculated  with  B. anthracis 34F2.

Following overnight incubation at 370C clear zones of inhibition  were  harvested from the agar and  placed in 8 ml of TSIMg.  Following agitation the solution was treated with chloroform and  centrifuged after which the  supernatants were diluted and  plated onto a  bilayer TSAMg  as described above. Following overnight incubation clear plaques were again harvested and the process repeated on a further four occasions. The host range of the resulting phages and there lysins were determined using the methods  described below.

Method  to determine the bacterial host range

A 5 ml of suspension of  test cells of Bacillus spp. strains  in 0,7% TSAMg was laid over the surface of 15ml of TSAMg. Once the agar had solidified tested phage lysates were dropped (about 25 µl) on the surface of plates and  were incubated for 24 h in 370C and zones of lysis were noted. The gamma and  Fah phages were included as positive  controls.

Screening method  to determine of phage produced  lysins

A 5 ml  1% agarose overlay contained heat inactivated cultures of the test bacteria.  This was  achieved by  harvesting the growth from 50ml of a 24 hr culture of the test bacteria  at 370C and resuspending it in 5ml of water. This was then mixed with 10 ml of  1.5% agarose and boiled for 15 mins.  The agarose was then overlaid onto  TSAMg and after solidifing 6 mm diameter holes were cut and the bottom of each hole was sealed with a drop molten TSAMg.  For each hole 25 µl of 24h liquid culture of B. anthracis 34F2 (host strain for all phages) were dropped. After 3 h of incubation in 370C lysates of tested phages (25 µl) in the rate 1.8 – 3.2 x 106pfu were dropped to individual holes. Dishes were incubated for 24 – 48 h in 370C and zones of brightening around holes were noted. The gamma and  Fah phages were included as positive  controls.

In the case of poorly visible of the zones  plates were dipped on 15 min in the water containing 1 – 5 µg/ml of active  chlorine. 

Results

An example of the results achieved using this method can be seen in figure 1 and a summary of all of the results is presented in table 1.  As expected the control phages, gamma and Fah  were only able to infect the B.anthracis isolates include in this study. They also  produced lysins which  were specific to B.anthracis.    While the majority  of environmentally isolated bacteriophages were highly specific for B.anthracis  there were exception such B.cereus ATCC 23261 and B.sp. Ba 813. We saw a different picture for the lysins and observed that a number of phages produced lysins with activity against B.anthracis  other members of the Bacillus test panel.

figure 1

 

Figure 1. A example of the activity of phage lysates on the lysis of dead cells of B.thuringiensis T7-030 and Bacillus sp. B.a. 813. #7.  K1- is a negative control in that it only contains  B.anthracis  34F2 and no phage

Table 1. The activity of  15 environmentally isolates bacteriophages and two control bacteriophages (gamma and Fah)  propagated in B.anthracis  4342 against living and dead cells of test bacteria.  A= activity of phages against living bacteria, B= activity of lysins against dead cells.

Strains   Bacteriophages
 

F3

 

F4 F7 F8 F9 F12 F13 F14 F15 F16 F17 F18 F19 F20 F22 Gamma Fah  
   B. anthracis

   1584

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   211

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   SL 1809

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   Sterne 34F2

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. thuringiensis

   ATCC 33679

A  
B  
   B. thuringiensis

   ATCC 35646

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. thuringiensis

   ATCC 10792 T

A  
B +

 

+ + + + + + + + _- _- nd Nd nd nd  
   B. thuringiensis

   T7 – 128

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. thuringiensis

   T7 – 019

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 19637

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   UW 85

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   F 17202

A  
B + + + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 14579 T

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   F 16959

   A  
B +

 

+ + + + + + + + nd Nd nd nd  
   B. cereus

   F – 1728 S

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 23261

A + + + +  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. mycoides

  ATCC 21929

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. subtilis

   ATCC 6633

A  
B +

 

+ + + + + + + + + + nd Nd nd nd  
   B. sp. Ba 813

   15 (11614-2)

 

A nd Nd nd nd  
B + + + + + + + + + + + nd Nd nd nd  
   B.sp. Ba 813 #

   6 (I/2)

A + + + + nd Nd nd nd  
B +

 

+ + + + + + + + + + nd Nd nd nd  

To further understand the nature of the environmental bacteriophages three isolates, F9, F15 and F17 were selected for further characterization by electron microscopy, PFGE and restriction enzyme digestion. Analysis of the  images  of F15 and F17  (figures 2 and 3) revealed that these phages belonged to the Siphoviride family. The morphology of F9 has yet to be determined.

                        Figure 2. An electron Microscopy image of the F15  bacteriophage

Measurement  of the phage revealed a head  size of 36.9-38nm and a tail of  172 nm.

                     Figure 3. An electron Microscopy image of the F17  bacteriophage

Measurement  of the phage revealed a head size of 43.3-41nm and a tail of 154.35 nm.

To further characterize these phages their  DNA  was extracted and  subjected to  pulse field gel electrophoresis (PFGE) and to restriction enzyme digestion.

Pulsed field gel electrophoresis (PFGE)

Following dialysis a 50 µl  suspension of phage was mixed with 50 µl of 2 % (w/v) plug agarose (CleanCut, Bio-Rad) and dispended into a plug mold were it was left to solidified.  The plug was then  removed from the  mold,  suspended in phage lysis buffer (50mM EDTA, 50mM Tris pH 8.0, 1% w/v SDS and 1mg/ml of proteinase K) and incubated overnight at 54 ºC, with shaking.  The digestion buffer was then decanted, and the samples were washed three times using TE buffer (10mM Tris, 1 mM EDTA, pH 8)  after which it was incubated in TE buffer (4 ºC, 1 h).   The plug was  then placed into  a well cut into a 1% Pulsed Field Certified agarose (Bio-Rad)  made using  0.5 % TBE. Lambda Ladder PFG Markers (New England Biolab) were used as the MW standards. The samples were electrophoresed using a CHEFDRII System (Bio-Rad) at 6Vcm/1 with pulse ramps from 1 to 25 s  for 20 h at 14 ºC in 0.5 % TBE buffer.   Following electrophoresis, nucleic acids were stained with ethidium bromide (1 µg/ml) for 30 min [Clokie MRJ, Kropinski AM (2009) Bacteriophages: Methods and Protocols.Volume 2: Molecular and Applied Aspects. Humana Press, Totowa, NJ.]

eee

Figure 4. The DNA extracted from the phages F9, F15 and F17 were subjected to pulse field gel electrophoresis (PFGE)

Isolation of the phage DNA for restriction digestion

Following incubation the phage infected culture was treated with chloroform    and then centrifugation at 25 000 rpm ( max 115900 x g) for 2 hours at 4 ºC in  Sorvall T-890 rotor the   phage pellet was  suspended in SM buffer and  sodium dodecyl sulfate (SDS) (final concentration of 0.5 %) and proteinase K (final concentration 50 µg/ml) were  then added. The  suspension was then incubated at 55 °C for 1 hour.  An equal volume of phenol-chloroform (1:1) was then added to remove the proteinaceous material. This  extraction was repeated twice, and the DNA was precipitated according to the standard procedures [Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]   Restriction enzyme digestions of the phage DNA were carried out according to the instructions provided by the suppliers. Restriction endonucleases tested for digestibility of the F9,F15 and F17 phages  DNA were: EcoRV, HaeIII and Bsp. After the enzymatic digestion electrophoresis of the samples in 0.4 % (w/v) agarose with addition of ethidium bromide (1 µg/ml) was performed.

As can be seen in Figure 5 the restriction digestion profiles on all three phages differed suggesting  that they were unrelated phages.

Picture5.png

 

AEDnet FP7 project management meeting, NCDC Tbilisi, Georgia, 22nd April

Present
Les Baillie (CU)
Mzia (EIG)
Lile (NCDC)
Mitat Sahin (KAU)

Agenda items
1. Missed secondments from 2014
2. Moving secondments from one institution to another
3. Secondment reports
4. Project related presentations
5. Progress on project deliverables
6. Project workshop in Tbilisi, Sept 2015
7. Project website
8. Any other business.

Notes
1. Missed secondments from 2014
During the first year of the project we only completed 16 of the proposed 50 secondments. We need to reschedule the missed secondments over the remaining period of the project and send a revise a copy of the plan to our EU project officer. Lile and Mzia will send there plans to reschedule the secondments from there institutions which were not completed.

2. Moving secondments from one institution to another.

1. A request was made by Lile to investigate the possibility of moving secondment from NCDC to KAU (3 months in year 1 and 1 month in year 2) to another EU partner institution within the project. Les will contact the EU project officer to determine if this is possible as it will involve changes to individual budgets.

2. There was also a request to determine if a secondment from UHOH to NCDC planned for year 1 which could not be completed by UHOH could be reallocated to NCDC. Les will contact the EU project officer to determine if this is possible.

3. Jason Farlow has not been able to resolve his position at ISU and as consequence it has been proposed that we reassign all of the secondment that will be going to ISU to NCDC. Les will contact the EU project officer. We will also have to reassign any secondments which we planned to take place from ISU.

4. The group from IZPSB are concerned about sending there start to Ukraine and have request that there secondments be shifted from the Ukrainian institution to the Intitution in Georgia. I will contact the EU project officer to see if this is possible.

5. The group in IZSPB would like to break a one month secondment to Georgia into two blocks of 15 days and to send two researchers to Georgia at the same time, both for 15 days. The total funding for this secondment would add up to 1 month as budgeted. I will approach the EU project officer to determine if this is possible.

3.Secondment reports- At the completion of the secondment the secondee must write a short report of there visit describing the new skills and/or knowledge that they have gained from the visit. Please include pictures if possible. Send the report to Cardiff. Please also send copies of airline tickets as confirmation of the secondment dates. Attached is an example of a recent secondment report for a visit to IZSPB. Thesae reports will be posted on the website.

4.Project related presentations- Please send details of any presentations/ publication which have arisen from the project to Cardiff so that they can be included in the progress report to the EU and posted on the website. Also for future presentations please ensure that the Marie Curie and EU logos are included.

5. Progress on project deliverables for 2015. We have the following deliverables scheduled for the end of 2015. The lead institution for each activity is shown in brackets.
i) Training to characterise phages from field experiments (EIG)

ii) Training in the cloning and expression of B.anthracis specific lysins (WiHE)

iii) Identification and optimization of site specific spore germinants (CU)

iv) Full characterization anthrax spore contaminated sites in Georgia (NCDC), Turkey (KAU) and Ukraine (NSC IECVM)

v) A germinant/ phage decontamination formulation suitable for field trials- (CU)

vi) A germinant/phage lysin decontamination formulation suitable for field trials- (WiHE)

We are making good progress in all of these areas and we call review progress during our meeting in Tbilisi in Sept 2015.
6. Project workshop to be held in Tbilisi towards the end of Sept 2015, date and details to be finalised. – To reduce organizational costs this will be a combined meeting of two anthrax related projects both of which involve partners in Tbilisi.- Outline programme is attached- we need to agree presentations for the AEDnet session. The suggestion is for each institutional lead to give an overview as to there activities and then for individual young researchers who have been on secondment to give a brief presentation of their experience an the knowledge they have gained.

7. Project website.- https://aednetproject.wordpress.com/
Project deliverables, reports and papers arising from the project will be posted on the website as they are generated. Please check the partner institution section and confirm that you are happy with the text which as been posted, if not please send us new text.

7. Any other business
The next meeting will be held in Tbilisi in Sept before or after the workshop on the 29th Sept 2015