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PIRSES-GA-2013-612309 AEDnet Project Mid-Term Report December 2014

ANNEX 1: MID-TERM REPORT

Marie Curie Actionsseventh framework programme

Marie Curie Actions – International Fellowships

Project n°: _PIRSES-GA-2013-612309________
Project Acronym: ___AEDnet_________
Project Full Name: __Anthrax Environmental Decontamination Network___
Marie Curie Actions
IRSES Mid-term Report
Period covered: from ___01.01.14_________ to ____31.12.16____
Period number: _______1_____
Start date of project: _____1st Jan 2014_______
Project coordinator name: ___Prof Les Baillie_________
Project coordinator organisation name: __Cardiff University UK__________
Date of preparation: ____24th Dec 2014________
Date of submission (SESAM): ___24th Dec 2014_________
Duration: ____12 months________
Version: _____1______

1. GENERAL PROGRESS OF THE PROJECT
Please indicate if the project:
a) has fully achieved its objectives and technical goals for the period;
b) has achieved most of its objectives and technical goals for the period with relatively minor deviations;
c) has failed to achieve critical objectives and/or is not at all on schedule.

If you answered b) or c) please include a detailed description of the modifications in the report
While the project has achieved all of its deliverables, we have not completed all of the planned secondments. On reflection, front loading the distribution of the secondments to the first 12 months (50 months in total) of the project was unrealistic. We were not helped by the fact that it took several months to complete the contractual arrangements between the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. Finally the on going problems in Ukraine meant that only 5 of the planned 13 secondments from that institution took place.
It is our intention to re-arrange the missed secondments so that they are completed during the lifetime of the project. A summary of the planned and completed secondments is shown in the attached GNATT chart.

Qualitative indicators of progress and success in line with workplan and milestones (description of progress towards the milestones and deliverables)
The project has met all but one of its 6 deliverables within the 12 month period and the 6th deliverable, the Month 12 meeting of the Project Management Group will be completed early in the new year.

D1.1, Month 12: A standardised method for the isolation of B.anthracis specific bacteriophages- (see attached).

D4.1, Month 3: Meeting of the Project Management Group
Held at the project meeting at the Anthrax workshop in Cardiff in March 2014. At this meeting, the contractual arrangements between the partners and Cardiff University were explained, as was the process by which funds would be transmitted to all the institutes. The reporting requirements were also explained. Due to the unrest in Ukraine, our colleagues from NSC IECVM were unable to attend the meeting in Cardiff. Information with transmitted to them via email.

We held a second informal project meeting in Tbilisi, Georgia in June 2104, to coincide with a congress on Chemical, Biological, Radiological, Nuclear and explosives (CBRNe) Science and Consequence Management which was attended by a number of the partners.

D4.2, Month 6: First Internal technical report- see attached

D4.3, Month 6: Establish project website- A project website was established and can be viewed at https://aednetproject.wordpress.com/ We are still waiting for all of the partners to provide information about their institutions which we will post on the site.
D4.4. Month 12: Internal technical report- see attached

D4.5. Month 12: Meeting of the Project Management Group- This meeting is yet to be held and will be hosted in the new year by Cardiff via Skype. The meeting will address the problem of identifying staff willing to go on secondment and will identify how the secondments which were not completed in the first 12 months are reassigned across the rest of the project period.

D4.6, Month 12: Preparation of mid- term review report to the EU Commission
Completed and submitted to EU- completed

2. PROJECT ACHIEVEMENTS
Scientific highlights and research achievements
1. With support from researchers from CU, KAU and EIG we developed A standardised method for the isolation of B.anthracis-specific bacteriophages

2. We have isolated novel B.anthracis-specific bacteriophages which will be assessed for their suitability as potential decontamination agents

3. As a consequence of the interaction between KAU and NCDC, and with support from CU, we have established a new project which started in Nov 2014 and is funded by the US Biological Engagement Program to genetically type the pathogenic strains of B.anthracis which cause anthrax in Georgia and Turkey. The project will reinforce the link formed as part of this EU funded project and will provide a foundation for future joint research efforts. The project aims to provide short-term training by WRAIR and NCDC Lugar Center collaborators to Turkish personnel in the areas of bacterial DNA extraction and molecular analysis of anthrax isolates.

Transfer of knowledge and Training activities (workshops…)
Secondment activities
WP1- Development and characterisation of phage-based decontamination agents: Nino Modebadze, Ketevan Sidamonidze from NCDC visited the group at CU for 1 month for training in the isolation and characterization of B.anthracis specific bacteriophages (task 1.2). They isolated phages form soil and observed the effect of different phages on a vaccine strain of B.anthracis. As part of this process, they employed a number of different methods which included enrichment from soil, propagation of phage in liquid broth, determination of bacteriophage titre and purification of bacteriophage.
Ms Aleksandra Nakonieczna, from WiHe visited the group at EIG for 2 months for training in the isolation and characterisation of B.anthracis-specific bacteriophages (task 1.1). During the visit she worked in a Molecular Biology laboratory led by Dr. Mzia Kutateladze. The aim of this exchange was to share and extend the knowledge about bacteriophages, including B. anthracis phages. During the secondment she took part in bacteriophage isolation from sewage samples, purification and propagation of bacteriophages, phage DNA isolation, DNA restriction analysis, PFGE analysis, phage characterization assays (adsorbtion rate, one step growth experiments, phage host range), phage adaptation according to the Appelman’s method, media preparation, and API testing.

WP2- Development of methods to enhance the activity and stability of sporicidal agents: Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve effective killing of the pathogen in soil.
Ekaterine Khmaladze, Mariam Zakalashv from NCDC visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). The were introduced to the method that shows the effect of germinants alone on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites (task 3.1). He visited a number of potential decontamination sites in Turkey with a view to identifying similar sites in Ukraine.

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory where they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

The following researchers Dr Anatoliy Paliy,Dr Olena Kolchyk, Dr Oleksandr Rula and Mr Mykola Kalashnyk from NSC IECVM visited IZSPB Italy for one month for training in the following topics: characterization of the genetic diversity of B. anthracis (genotyping by 15 loci Multi Locus VNTR Analysis -MLVA) isolates from environmental samples via G.A.B.R.I. technique (task 3.2) and training on safely working with B. anthracis in laboratory Biosafety Level 3. (task 3.5).

Dissemination of results (conferences, publications)
To date, we have not disseminated the results of our work outside of the network. It is our intention to disseminate our results and activities to the wider scientific community and to advertise our activities on the project website at https://aednetproject.wordpress.com/
3. PROJECT MANAGEMENT
Overview of the activities carried out by the partnership, identification of problems encountered and corrective action taken.
This project involves the co-ordination of 9 institutions, five based in EU member or associate states, three based on Georgia and one based in Ukraine. Co-ordination and control of such a disparate group is a challenge. The project coordinator has been greatly assisted by the efforts of a part time project administrator who in addition to coordinating details of secondments, is also responsible for managing the project website.

We held a kickoff meeting of the Project Management Group in Cardiff in march 2014. A second informal project meeting was held in Tbilisi, Georgia in June 2104 and was attended by representatives of the majority of the groups. Our 12 month meeting is yet to be held and will be hosted in the new year by Cardiff via Skype. The meeting will address the problem of identifying staff willing to go on secondment and will identify how the secondments which were not completed in the first 12 months will be reassigned across the rest of the project period.
4. ADDITIONAL INFORMATION
Please indicate any additional information, which may be considered useful to assess the work done during the reporting period. The socio-economic aspects of the project may be addressed in this section.
Ethical issues
As stated in the proposal, no exchange of viable strains of pathogens between laboratories and between countries has or will take place (monitoring safety issues).

Also there have been no anthrax cases in the environment of the research sites.

All of the research which has been undertaken as part of this project complies with the ethical standards and guidelines of the European Union.

There is no intension to transport samples containing B.anthracis from Georgia or the Ukraine.

Statements from members of the external, independent Advisory Board stating that the project has not created any concerns with regards to bioterrorism, misuse and dual use are attached.
Appendix A: D4.2. Month 6: First Internal technical report- delayed- will be same as phage report
Appendix B: D4.4. Month 12: Internal technical report- summary of project reports
Visits to date
Attachment: Deliverable 1.1.A standardised method for the isolation of B.anthracis-specific bacteriophages

Person in charge of the project for the beneficiary/consortium
Name: Les Baillie
Date 24th Dec 2014
Signature

Appendix A: D4.2. Month 6: First Internal technical report- delayed- will be same as phage report

For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. As a consequence, there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficient killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Appendix B: D4.4. Month 12: Internal technical report- summary of project reports visits to date

WP1- Development and characterisation of phage based decontamination agents

Isolation of B.anthracis specific phages using the Sterne vaccine strain of B.anthracis

1. The group at CU with help from colleagues from EIG and NCDC isolated novel B.anthracis specific phages from environmental samples and determine there biological activity. Electron microscopy images of the phages can be seen below.

micro 1micro 2micro 3
2. Ms Aleksandra Nakonieczna, from WiHe with support form the the group at EIG isolated and characterisation B.anthracis specific bacteriophages

Researchers from NCDC received training in the isolation and characterisation of B.anthracis specific bacteriophages (task 1.2) and as a consequence should be better equipped to isolate bacteriophages from there own B.anthracis contaminated sites.
WP2- Develop of methods to enhance the activity and stability of sporicidal agents

Researchers from Georgia have received training in how to set up and run soil microscosms. They were introduced to the method that shows the effect of germinants on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites with a view to identifying similar sites in Ukraine.

Researchers from KAU in Turkey visited the group at NCDC in Georgia for training in the use of molecular methods to characterize the genetic diversity of B.anthracis. Researchers from NSC IECVM in Ukriane underwent similar molecular characterization training at IZSPB Italy in the method of genotyping using 15 loci Multi Locus VNTR Analysis –MLVA.

Researchers from NCDC, EIG, KAU and NSC IECVM received training in how to operate safely 8in Biosafety level 2 and 3 facilities

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Deliverable 1 1 A standardised method for the isolation of B anthracis specific bacteriophages

Introduction: One of the aims of this project is to harmonise and standardize methods across the network so that results obtained from different laboratories can be compared. We have developed a B.anthracis specific bacteriophage isolation method for the recovery of phages from B.anthracis contaminated animal burial sites (WP1.1). The method shown below is a composite of the approaches developed by researchers from Kafkas University in Turkey (KAU), the Eliava Institute in Georgia (EIG) and Cardiff University in the UK (CU). Training in this combined method has already been provided to researchers from the National Center for Disease Research in Georgia.

Composite B.anthracis specific Bacteriophage isolation and production methods

Bacteriophage isolation from soil
Environmental phage isolations were performed by adding 25 g of soil to 25 ml of TSB followed by the addition of 10 ml of mid-log phase culture of the Sterne strain of Bacillus anthracis. The resulting suspension was mixed thoroughly and was incubated overnight at 37°C with shaking at 125 RPM.

Each incubated mixture was centrifuged at 5,000 x g for 20 mins and the supernatant fraction was filtered using syringe filter of pore sizes 0.45 μm and then 0.22 μm. Each filtrate was screened for the presence of bacteriophages against B. anthracis Sterne using the agar method described below.

To recover the phage a plug of agar which included the plaque was taken and
suspended in 500 µl phage buffer to which a small drop of chloroform was added to lyse any remaining bacterial cells. The suspension was vortexed and then left overnight at 4°C to allow any phage particles to diffuse out of the agar. The samples were centrifuged at 5,000 x g for 10 minutes and the supernatant fraction decanted into a sterile microcentrifuge tube.

This solution was then retested for the presence of phage using the agar method and if plaques were seen, the extraction was repeated a minimum of three times to ensure that the phage preparation was pure. If plaques were not seen after an initial round of purification and testing against B. anthracis, no further testing was performed.
Bacteriophage plaque morphology: The plaque morphology of each phage was determined using an agar overlay method. The phage preparation at a range of dilutions was added to overnight cultures of the propagation strain of bacteria in 5ml molten TSA agar. The agar was poured over the surface of a TSA plate and allowed to dry. The plate was then incubated overnight at 37°C for 18 – 24 hours and examined for the presence of plaques.

Bacteriophage propagation
Bacteriophage propagation in broth was performed by inoculating fresh TSB with 50 μl of an overnight culture of the bacterium spore and 50 μl bacteriophage which was then incubated at 37°C and 180 RPM overnight. Following overnight incubation Vortex mix. Add a drop of chloroform at a ratio of 1:1000 chloroform: broth and Vortex mix again. Leave the suspension for 5 minutes and then centrifuge at 5,000 x g for 20 minutes to pellet the bacterial debris. The supernatant is then filtered using a double stacked syringe filter (0.45um and 0.22um filter joined together). The resulting phage suspension was stored at 4°C.

Bacteriophage enumeration (drop count method to determine PFU/ml): A version of the Bacteriophage agar propagation method was used.
1. Prepare a fresh culture of the bacteria (inoculate fresh media with 1ml of overnight culture of bacteria – usually will result in exponentially growing bacteria after 6 hr)
2.Prepare 8 eppendorfs filled with 900 μl of sterile PBS/water
3.Serial dilute bacteriophage in the 8 eppendorfs ensuring vortexing between each dilution using 100 μl neat bacteriophage culture
4.Add 0.1ml of exponentially growing bacterial host to 4.9ml of 50% TSA (or TSB with 0.7% agar-agar): pour agar as an overlay onto set TSA plate. Prepare two agar plates in total, allow to dry and divide into four sections.
5.Drop 10 μl of each bacteriophage dilution in triplicates onto one of the four sections.
6.Allow the drops to dry on the plate, then incubate overnight.
7.Phage titre determined as above (multiply by dilution and amount spotted to determine PFU/ml).

Spore production methods
The spore production method is adapted from a method developed by Curtis Thorne (J. Virol. 2(7):657.1968) The method is as follows;
1 Prepare an overnight broth and Incubate broth at 37oC
2 overnight with shaking at 250RPM
3 Remove broths from incubator and centrifuge at 3800 x g for 10 min at 20oC
4 Discard the supernatant and re-suspend in 10 mL Sterile distilled water
(SDW)
5.Innoculate 1 mL of this suspension into 50 mL NBYS broth. Incubate at 28 oC for 3 days with shaking at 250 RPM
6.Following incubation transfer broth to centrifuge tubes and centrifuge at 3800 x g for 15 min at 4oC.
7.Discard supernatant and re-suspend in 30 mL SDW
8.Store in the fridge at 4oC for 3 days
9. Remove suspensions from the fridge and centrifuge at 4oC for 15 min at 3800 x g.
10. Discard the supernatant and re-suspend pellet in 30 mL SDW
11. Heat shock suspensions at 65oC for 30 min
12. A 1 mL aliquot of the suspension will then be removed and perform pre and post heat shock viable counts. Aliquots are also looked at under x100 magnification phase contrast to see phase bright numbers.

D4 4 Month 12- Internal technical report

Progress in the first six months
For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and there respective responsibilities. As a consequence there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardise soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficacy killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in Characterization of the genetic diversity of B.anthracis isolates (task 3.2) and – Safe working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Progress by month 12

WP1- Development and characterisation of phage based decontamination agents

Isolation of B.anthracis specific phages using the Sterne vaccine strain of B.anthracis

1. The group at CU with help from colleagues from EIG and NCDC isolated novel B.anthracis specific phages from environmental samples and determine there biological activity. Electron microscopy images of the phages can be seen below.

micro 1 micro 2 micro 3
2. Ms Aleksandra Nakonieczna, from WiHe with support form the the group at EIG isolated and characterisation B.anthracis specific bacteriophages

Researchers from NCDC received training in the isolation and characterisation of B.anthracis specific bacteriophages (task 1.2) and as a consequence should be better equipped to isolate bacteriophages from there own B.anthracis contaminated sites.
WP2- Develop of methods to enhance the activity and stability of sporicidal agents

Researchers from Georgia have received training in how to set up and run soil microscosms. They were introduced to the method that shows the effect of germinants on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites with a view to identifying similar sites in Ukraine.

Researchers from KAU in Turkey visited the group at NCDC in Georgia for training in the use of molecular methods to characterize the genetic diversity of B.anthracis. Researchers from NSC IECVM in Ukriane underwent similar molecular characterization training at IZSPB Italy in the method of genotyping using 15 loci Multi Locus VNTR Analysis –MLVA.

Researchers from NCDC, EIG, KAU and NSC IECVM received training in how to operate safely 8in Biosafety level 2 and 3 facilities

D4 2 Month 6- First Internal technical report

Progress in the first six months

For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. As a consequence, there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficient killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.