Our data showing the use of our Anthrax bacteriophages entitled "The Enemy of My Enemy if My Friend: Anthrax Specific Bacteriophages." that was presented at the 15th Medical Biodefense Conference in Munich (April 26th - 29th 2016)

1. RW AB1 LC1H911 3B6
Plaque appearance Clear Turbid Clear Clear
Capsid Diameter (nm) 62 ±6 70 ±5 77 ±6 123 ±6
Tail Length (nm) 197 ±20 344 ±32 406 ±47 449 ±40
Classification SiphoviridaeSiphoviridaeSiphoviridaeMyoviridae
The Enemy of My Enemy is My Friend: Anthrax Specific Bacteriophages
Ashley D. Otter1,2, James Blaxland2 and Les Baillie2.
1 Royal Veterinary College, Dept. of Pathology and Pathogen Biology, London.
2 Cardiff University School of Pharmacy and Pharmaceutical Sciences, Cardiff.
Corresponding author: Ashley D. Otter – aotter@rvc.ac.uk
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Introduction
• Anthrax is a bacterial zoonotic disease caused by Bacillus anthracis, a disease usually associated with cattle but can also infect humans, a
characteristic which terrorists could exploit.
• Anthrax spore contaminated land is extremely difficult to decontaminate and currently requires the application of toxic, harsh chemicals
such as formaldehyde.
• Bacteriophages are viruses which infect bacteria, are usually species specific and represent one of the commonest life forms on the planet
• We wish to exploit this specificity and employ B. anthracis specific bacteriophages as part of an environmentally friendly decontamination
strategy.
• In this poster we summarize our efforts to isolate B. anthracis specific bacteriophages from Welsh soil and demonstrate their ability, when
combined with spore germinants, to eliminate the pathogen.
Discussion and Conclusion
• We isolated B. anthracis lytic phages from soil in Wales
with no previous history of B. anthracis contamination
• The LC1H911 phage infected a gamma phage resistant
variant of B. anthracis suggesting a possible different
receptor site to the other phages
• In preliminary studies, a phage cocktail in conjunction
with a mixture of germinants reduced bacterial numbers
• Further work is required to exploit the potential of this
approach as an environmentally friendly
decontamination strategy including optimisation of
phage cocktail mixture, germinant concentration and
method of application.
Methods
Phage isolation: Phages were isolated from soil taken from various locations across Wales using the following method:
1. A total of 25 g of topsoil was taken from each site and it’s coordinates recorded
2. Soil was stored in a sterile 50 ml falcon tube for transport to the lab and either used immediately or frozen at -20°C.
3. 25 g of soil was added to 25 ml ofsterile TSB followed by 10 ml mid-log phase B. anthracis Sterne 34F2.
4. Samples were vortexed and then decanted into a culture flask and incubated at 37°Cfor 24 hours
5. Soil slurries were then centrifuged for 20 mins at 5,000 x g to separate potential phage from bacteria and soil particles.
6. Solutions were tested against lawns of B. anthracis strains Sterne & the Sterne variantSdT12 as well as B. cereus 4342
and incubated overnight. Any plaques that were seen were picked off and purified further for 3 rounds.
Electron microscopy: Using a Zeiss SIGMA field emission gun (FEG) electron microscope (Brighton Uni), equipped with a
scanning transmission electron microscopy (STEM) detector (FEG-STEM), samples stained with 2%phosphotungstic acid at pH
7.4 were visualised using parameters of 3.2 – 3.5 mm working distance, 20 µm aperture and 20 kV accelerating voltage.
Host range: A standardized solution of ~1x108 CFU/ml for each Bacillus strain was spread on TSA and allowed to dry. Each
phage (a minimum solution of 1x108 PFU/ml) was then spotted onto the overlays. Host range was performed in triplicates.
@asherichia / @cornishman100 / @lesbaillie1
Results
• A total of 12 Bacillus phages were isolated, of which, 4 could infect multiple B. anthracis strains. Isolation locations are found in Figure 5.
• On the basis of EM morphology all phages classified under the Siphoviridae family of phages, whilst 3B6 is Myoviridae(Figure 2).
• Phage 3B6 infected the majority of Bacillus spp - a total of 47 strains out of a total of 58 (full data not shown).
• LC1H911 showed high specificity for B. anthracis, even LSU463, a Ɣ phage resistant strain. No other phages tested were able to infect
LSU463 (Table 1 and Figure 3).
• 3B6 was the largest phages isolated, whereas the smallest was RW (Table 2).
• A phage cocktail (AB1, RW and LC1H911) in a germinant solution reduced the anthrax TVC count by ~2 log following 5 days incubation at
37°C with shaking (Figure 4)
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
Spot test
Overlay showing
phage plaques
Figure 1: Schematic diagram of isolating anthrax phages from Welsh soil
Table 2: Phage characteristics.
Table 1: B. anthracis host range.
LC1H911
AB1
3B6Gamma
RW
100 nm
Figure 2: EM images of phages, bar shown is 100 nm.
Figure 4: Phage ’cocktails’ (RW, AB1 and LC1H911) were
tested in conjunction with germinants Alanine and Inosine over
a 5 day period to test the ability of phages with germinants to
reduce the total count of B. anthracis spores and vegetative
cells. Test conditions – 37ºC with shaking at 250 RPM, one
addition of phage and germinant mixture. BHI and PBS
controls had both germinants but no presence of phage.
Total viablecountsafter addition of phage cocktails and
germinants against B. anthracis spores
Anthrax
cluster
Figure 3: MLST treeof Bacillus combined with phage infection data. Figure 5: Phage isolation locations.