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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 – [email protected]
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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.
