Ashley Otter - Research Poster - 15th Medical Biodefense Conference, Munich

RW AB1 LC1H911 3B6

Plaque appearance Clear Turbid Clear Clear

Capsid D iameter (nm) 62±6 70±5 77±6 123±6

TailLength (nm) 197±20 344±32 406±47 449±40

Class ification 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• Anthraxisabacterialzoonoticdiseasecausedby Bacillusanthracis,adiseaseusuallyassociatedwithcattlebutcanalsoinfecthumans,a

characteristicwhichterroristscouldexploit.• Anthraxsporecontaminatedlandisextremelydifficulttodecontaminateandcurrentlyrequirestheapplicationoftoxic,harshchemicals

suchasformaldehyde.• Bacteriophagesareviruseswhichinfectbacteria,areusuallyspeciesspecificandrepresentoneofthecommonestlifeformsontheplanet• WewishtoexploitthisspecificityandemployB.anthracis specificbacteriophagesaspartofanenvironmentallyfriendlydecontamination

strategy.• InthisposterwesummarizeoureffortstoisolateB.anthracis specificbacteriophagesfromWelshsoilanddemonstratetheirability,when

combinedwithsporegerminants,toeliminatethepathogen.

DiscussionandConclusion• WeisolatedB.anthracis lyticphages fromsoilinWales

withnoprevioushistoryofB.anthracis contamination• TheLC1H911phageinfectedagammaphageresistant

variantofB.anthracis suggestingapossibledifferentreceptorsitetotheotherphages

• Inpreliminarystudies,aphagecocktailinconjunctionwithamixtureofgerminants reducedbacterialnumbers

• Furtherworkisrequiredtoexploitthepotentialofthisapproachasanenvironmentallyfriendlydecontaminationstrategyincludingoptimisationofphagecocktailmixture,germinantconcentrationandmethodofapplication.

MethodsPhage 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 recorded2. 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 of sterile 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°C for 24 hours5. 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 variant SdT12 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• Atotalof12Bacillusphageswereisolated,ofwhich,4couldinfectmultipleB.anthracisstrains.IsolationlocationsarefoundinFigure5.• OnthebasisofEMmorphologyallphagesclassifiedundertheSiphoviridae familyofphages,whilst3B6isMyoviridae (Figure2).• Phage3B6infectedthemajorityofBacillusspp - atotalof47strainsoutofatotalof58(fulldatanotshown).• LC1H911showedhighspecificityforB.anthracis,evenLSU463,aƔ phageresistantstrain.Nootherphagestestedwereabletoinfect

LSU463(Table1andFigure3).• 3B6wasthelargestphagesisolated,whereasthesmallestwasRW(Table2).• Aphagecocktail(AB1,RWandLC1H911)inagerminantsolutionreducedtheanthraxTVCcountby~2logfollowing5daysincubationat

37°Cwithshaking(Figure 4)

Soilf romsamplesite

25gsoil

Bacteriaandphageseparated

Vortex

+25mlTSB+10mlB.anthracis

Centrifuge

20mins.5,000xg

37°C18–24hours

Filter

Phagesolution

Spottest

Overlay showingphageplaques

Figure1:SchematicdiagramofisolatinganthraxphagesfromWelshsoil

Table 2: Phage characteristics.

Table 1: B. anthracis host range.

LC1H911

AB1

3B6Gamma

RW

100nm

Figure 2: EM images of phages, bar shown is 100 nm.

Figure 4: Phage ’cocktails’ (RW, AB1 and LC1H911) weretested in conjunction with germinants Alanine and Inosine overa 5 day period to test the ability of phages with germinants toreduce the total count of B. anthracis spores and vegetativecells. Test conditions – 37ºC with shaking at 250 RPM, oneaddition of phage and germinant mixture. BHI and PBScontrols had both germinants but no presence of phage.

Total viable counts after addition of phage cocktails and germinants against B. anthracis spores

Anthraxcluster

Figure 3: MLST treeof Bacillus combined with phage infection data. Figure 5: Phage isolation locations.