Ashley Otter Cardiff University School of Pharmacy and - - PowerPoint PPT Presentation

Isolation and characterisation of bacteriophages that infect Bacillus anthracis: The etiological agent of Anthrax

Ashley Otter

Cardiff University School of Pharmacy and Pharmaceutical Sciences, King Charles VII Avenue, Cardiff. Supervisor: Professor Les Baillie.

18/10/2016

Introduction

• Phages are the most abundant entity – 1031.
• Phages exist as lytic and lysogenic.
• B. anthracis is cause of zoonotic disease Anthrax.
• 3 types of disease:

– Cutaneous, Inhalational and Gastrointestinal (+ injection).

• Produces three toxins:

– Protective antigen, Lethal Factor and Edema Factor.

• Considered bioterrorism threat:

– Biowarfare agent – Category 3/A organism. – Considerable research after 2001 ‘Amerithrax’ attacks.

• Clinically detected using diagnostic phage

(γ phage) as well as other methods.

• Most recent UK case – March 2013

– Fatal infection in injecting heroin user

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Aims and Objectives

1) To isolate bacteriophages from surrounding areas of Cardiff and parts of Wales. 2) Characterise the bacteriophages. 3) Determine the host range of newly isolated bacteriophages. 4) Analyse the genomes of each bacteriophage using RFLP procedures and compare with other known bacteriophages. 5) Analyse proteins of each isolated bacteriophage via SDS- PAGE and compare with known bacteriophages. 6) Sequence the genomes of our newly isolated bacteriophages. 7) To study horizontal gene transmission.

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Bacteriophage isolation from soil

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Soil from sample site

25g soil Bacteria and phage separated Vortex + 25 ml TSB + 10 ml B. anthracis Centrifuge 20 mins. 5,000 x g 37°C 18 – 24 hours Filter Phage solution

Bacteriophage isolation from soil

18/10/2016 Vortex + 25 ml TSB + 10 ml B. anthracis Centrifuge 20 mins. 5,000 x g 37°C 18 – 24 hours

Bacteriophage isolation from soil

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Soil from sample site

25g soil Bacteria and phage separated Vortex + 25 ml TSB + 10 ml B. anthracis Centrifuge 20 mins. 5,000 x g 37°C 18 – 24 hours Filter Phage solution

Bacteriophage isolation from soil

18/10/2016 Vortex + 25 ml TSB + 10 ml B. anthracis Centrifuge 20 mins. 5,000 x g 37°C 18 – 24 hours

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Isolated bacteriophages

• Isolated on B. anthracis Sterne 34F2:

RW, AB1, LC1H911, GLJ5A2, GLJ7A2 and NMAB11.

• Isolated on B. cereus ATCC 4342:

BP8, BP10, TW511A, TW8, LH1 and LH3.

Results

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Results

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PlcR – Sastalla et al. (2010)

Results

I II III IV

Conclusions

• Specificity
• LH1 and LH3 – Same?
• LC1H911 – Host range
• pX01 – Abshire et al. (2005)

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Phage Number of Bacillus isolates phage can infect Number of different Bacillus species phage can infect

Species phage is capable of infecting (Bacillus…)

RW

7 4

anthracis, cereus, mycoides & thuringiensis.

AB1

9 6

anthracis, cereus, subtilis, megaterium, pseudomycoides & thuringiensis.

BP8

10 3

anthracis, cereus & thuringiensis.

BP10

33 6

anthracis, cereus, mycoides, pseudomycoides, weihenstephanensis and thuringiensis.

LH1

40 6

anthracis, cereus, mycoides, pseudomycoides, weihenstephanensis & thuringiensis.

LH3

40 6

anthracis, cereus, mycoides, pseudomycoides, weihenstephanensis & thuringiensis.

TW511A

6 4

anthracis, cereus, mycoides & thuringiensis.

TW8

4 3

anthracis, mycoides & thuringiensis.

LC1H911

5 2

anthracis & mycoides.

3B6

50 8

anthracis, cereus, subtilis, licheniformis, megaterium, mycoides, weihenstephanensis & thuringiensis.

Gamma

12 4

anthracis, cereus, mycoides & thuringiensis.

Conclusions

• Specificity
• LH1 and LH3 – Same?
• LC1H911 – Host range
• PlcR
• pX01 – Abshire et al. (2005)

18/10/2016 Species Isolate AB1 RW LC1H911 Gamma BP8 BP10 LH1 LH3 TW511A TW8

• B. anthracis

34F2

+ + + +

• ΔGerH
• +

+

• +

+ +

• SdT

+ + + +

• +

+ +

• SdT12

+ + + + + + + + + +

Future work

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• Testing against virulent B. anthracis
• Electron microscopy – October 2014
• RFLP/Sequencing
• MOD (DSTL) project:
• “Feasibility study into the use of bacteriophages against B. anthracis spores”
• DSTL contract: DSTLX1000095774
• SfAM

Acknowledgements

I would like thank all members of Lab 1.11 (Dr. Bettina Schelke, Dr. Callum Cooper, Dr. Tina Joshi, Chris Hatton, James Blaxland and Jennifer Hawkins) as well as everyone in Lab 1.49 (Dr. Mark Legget, Harsha Siani, Dr. Maru Saavedra, Dr. Anna Costanlo, Rebecca Wesgate and Leonam Goncalves) for all their help with lab work and helping me have an awesome time. I’d also like to thank my PTY supervisor, Prof. Les Baillie, for all his help and guidance with experiments and data analysis throughout, Dr. Beatrix Fahnert for her pastoral and academic support as my personal tutor as well as the Society for Applied Microbiology for their funding in my project. I also thank Professor Baillie, Dr. Cooper and Professor P. Keim for use of their photos in my presentation.

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References

Abshire, T. G., Brown, J. E. and Ezzell, J. W. (2005). Production and validation of the use of gamma phage for identification of Bacillus anthracis. Journal of clinical microbiology 43:4780-4788. Sastalla, I., Maltese, L. M., Pomerantseva, O. M., Pomerantsev, A. P., Keane-Myers, A. and Leppla, S. H. (2010). Activation of the latent PlcR regulon in Bacillus anthracis. Microbiology 156:2982-2993.