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Please complete the form and return to Bailliel@cardiff.ac.uk along with evidence of the duration of the secondment (e.g. copies of flight tickets) and a short written report outlining your findings whilst at the host University.

Researcher details:

First Name: Rhodri

Last Name: Williams

Address: 54 Glynne Street, Canton, Cardiff

Post code: CF11 9NS

Contact Number: 07792 942444

Email Address: williamsro1990@gmail.com

Gender: Male

Date of Birth: 25/09/1990

Nationality: British/Welsh

Passport Number: 208910506

Details of Secondment:

Home University: Welsh School of Pharmacy and Pharmaceutical SciencesCardiff University – Professor Les BaillieRedwood Building, King Edward VII Avenue,Cardiff CF10 3XF, Wales, UK

Host University: G. Eliava Institute of Bacteriophages, Microbiology and VirologyAddress - 3, Gotua street, Tbilisi 0160, Georgia

Dates of secondment: 01/02/2016 - 01/03/2016

Description of activities and results

During my visit to the G. Eliava Institute of Bacteriophages, Microbiology and Virology (01/02/16 – 01/03/16) I was hosted by the laboratory of Dr. Mzia Kutateladze. The research group is primarily interested in developing the understanding, and applications, of bacteriophages as antibacterial agents. Although the researchers conduct studies with a vast array of bacterial species, during my visit I was exclusively concerned with the effects of bacteriophages against Bacillus anthracis. This was due to our research group at Cardiff possessing a shared interest in the decontamination of this particular spore-forming pathogen. B.anthracis spores are highly durable, which enable them to resist degradation from heat sources well above those typically tolerated by bacterial cells. Spores are also immune to many biocidal compounds and are able to persist in soil microcosms for many years until being reintroduced to favourable conditions, which initiates germination and causes B.anthracis cells to restart metabolic processes and become infectious. This can pose a major health risk to many mammalian species (including humans) in areas where B.anthracis is endemic.

Our research group in Cardiff is interested in developing optimised germination formulations. The aim is to induce B.anthracis spores to germinate and increase the susceptibility of the cells to physical and chemical degradation. Alternatively, germination also causes vegetative cells to become prone to infection by specific bacteriophages, which require hosts to be metabolically active. It would be of both medical and agricultural significance if bacteriophages could be adapted and optimised for deployment as biological decontaminants. This would likely reduce the risk of infection to both humans and livestock and would also eliminate the time that farmland is rendered unusable after treatment with harsh sporicidal compounds. By combining both our research regarding the germination of spores and developing bacteriophages as decontaminants, it may be possible to develop an ecologically-friendly, non-toxic protocol for wide-area decontamination of B.anthracis.

Although we have isolated and studied bacteriophages in our research at Cardiff, the staff at the G. Eliava Institute work exclusively with bacteriophages and there were some contrasts in our methods. For example, they used different propagation and enumeration methods (using a soft agar overlay) which proved to be more efficient in terms of producing higher bacteriophage titres over similar time scales. The researchers at the G. Eliava Institute also used an overnight method of establishing the quality of bacteriophages against different bacterial strains via serial dilution. The molecular techniques used, which I will briefly detail below, were also methods that I had not previously performed.

The experiments conducted during my secondment at the G. Eliava Institute involved the genetic identification of 5 bacteriophage strains (N8, N12big, N12small, N15big, N15small – big and small were labels derived from plaque morphology) isolated from Turkish soil contaminated with B.anthracis. The strains were tested against a B.anthracis strain library for optimal host specificity. We then propagated each bacteriophage sample until a final titre of at least 1x109 PFU/mL was achieved. At this titre an ample yield of bacteriophage DNA was collected by initially lysing bacteriophage samples and then utilising a phenol-

chloroform extraction method. The bacteriophage DNA samples were subjected to analysis using basic gel electrophoresis (Southern blot) and pulse-field gel electrophoresis to establish both purity and DNA concentration of each sample. DNA samples were then treated with individual restriction enzymes overnight and gel electrophoresis repeated to separate the resulting fragments.

We concluded after analysis that bacteriophage N8 was genetically distinct from the other bacteriophages analysed in this project, although it had a similar genome size to N15. Each of the bacteriophages examined was genetically divergent from gamma bacteriophage. There was no apparent difference between N12big and N12small, which suggests that they are closely related (if not identical) genetically but also that the bacteriophage produces polymorphic plaques. The results obtained for N15big and N15small were similar to the results described for N12 bacteriophages, with no significant differences in band fragmentation or migration between the two N15 samples.

As a result of my informative secondment I have learned multiple techniques involving the propagation, enumeration and identification of bacteriophages. This knowledge will augment our research at Cardiff University concerning the use of bacteriophages as environmental, bacterial decontaminants. I would like to thank Dr. Mzia Kutateladze and her colleagues for their hospitality during my time in Tbilisi and Prof. Les Baillie for the chance of studying at the G. Eliava Institute.

Boarding passes

Methods and results

Southern blot of lysed, washed, bacteriophage samples to establish relative DNA content. This experiment provides data which can be used to adjust the starting volume of sample used in subsequent restriction digest analyses in order to standardise DNA abundance and, subsequently, band density upon visualisation.

An example of one of multiple gel electrophoresis experiments performed with restriction digest samples using a different restriction enzyme each time to generate a more accurate fragment profile between samples. By comparing the fragment profile of each sample when digested with each restriction enzyme it is possible to identify whether they are genetically similar or not.

This image was captured after Pulse Field Gel Electrophoresis (PFGE) was utilised on complete, undigested bacteriophage DNA samples. Although similar to a Southern blot, PFGE alternates the direction of the current applied and therefore provides greater resolution of DNA bands over a longer time. From this image it can be concluded that N8 (lane 1) N15big and N15small (lanes 4 and 5) have genomes which are of similar size and may be closely related if not genetically identical. A fainter second band can also be seen in lane 5, which indicates a potential contamination with DNA from another sample.