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Transcript of Scientific Review
“UseofBacteriophagesintreating
bacterialinfections”
LucindaJ.CamidgeBScBiomedical
Sciences
SchoolofBiological,Biomedicaland
EnvironmentalSciences
UseofBacteriophagesintreatingbacterialinfections
Introduction
Antibioticresistancehasbecomeamajorworldwideproblemandisthreateningtheabilitytotreat
evensimplebacterialinfections(CDC2013).Forexample,in2012,therewerearound450,000new
casesofmultidrug-resistanttuberculosisresultinginlongertreatmentandincreasingthepotential
ofresistantbacteria(WHO2014).Whenantibioticswerefirstdiscoveredmostbacteriadidnotshow
signsofresistanceandiftheydiditwaseasytofindanotherantibiotictheyweresensitiveto(Davies
&Davies2003).Asresistancehasincreased,thediscoveryofnewantibioticshasdecreasedresulting
inaminimalamountofeffectiveantibiotics;thiscombinedwithcompaniesnotwantingtoinvest,
becauseoftheirpoorprofitmargin,meansnewtreatmentsforbacterialinfectionsareneededas
soonaspossible(WHO2011).
Antibioticresistanceoccurswhenapopulationofbacteriacontainasmallpercentageoforganisms
thatareresistanttoanantibiotic.Theantibiotictreatmentisgivenandkillsthemajorityofbacteria,
butleavestheresistantones.Thesearethenlefttoproliferateandcancausefurtherinfectionthat
cannotbetreatedwiththeoriginalantibioticused(CDC2013).
Forantibioticstoworktheymustdothreethings:enterthecell,normallybychannelsinthe
bacterialcellmembranesknownasporins;accumulatetoaminimumconcentration;actontheir
target.Resistancemechanismsthereforetargetthese,forexample:decreasingcellwall
permeability;usingeffluxpumpsonthecellwalltopumpoutsubstancesthataretoxictothecell
includingsomeantibiotics;changinganaminoacidsequenceontheantibiotic’starget,suchascell
ribosomesorproteins,sothatthedrugcannolongerbindandcarryoutitsaction.Bacteriacanalso
useenzymaticinactivationwhereanexistingenzymeinthecellismodifiedtoreactspecificallywith
theantibiotic.Onemajorexampleofthisisβ-lactamaseswhichbreakdowntheβ-lactamringin
antibioticssuchaspenicillins(Goering.etal.2012).
Consequently,alternativestotraditionalantibioticsmustbefoundandonesuchexampleistheuse
ofbacteriophages.Thesearevirusesthatinfectbacterialcellsandhelpmaintainagood,natural
microbialbalance.
Phagesareoneofthemostabundantorganismsonearthwitharound6000discoveredsofar.This
abundancemakesthechanceofdeclinelowerthanthatofantibiotics,whichonlyhaveahandfulof
classesandoverthepastfewdecadesdiscoveryofnewoneshasrapidlydeclined(Moore2015;
Davies2012;Wittebole.etal.2014).Resistancetoantibioticsoccursnottoindividualones,butto
thewholeclass.Bacteriophagesnormallyonlyinfectoneparticularstrainofbacteriumwhichmeans
that,unlikeantibiotics,theycauseminimaldisruptiontonormalfloraanddecreasethesideeffects
associatedwithsomeantibiotics,suchasCandidainfections(Birge,EA.2000).Candidainfections
oftenoccurwhenantibioticshaveremovednormalflora,allowinguncontrolledgrowthoftheyeast.
Researchalsosuggeststhatphagetherapymaybelessexpensivethanantibiotictreatment
(Międzybrodzkietal.2007).Asdescribedfurtheron,thenatureofdifferentphagelifecycles
suggeststheycouldbeusedasacompletealternativetoantibioticsorusedinconjunctionwith
them,bymakingbacteriamoresusceptibletoantibiotictreatment.
Structure
Thefirsttherapeuticphagestobediscoveredandthemostabundantcontaindouble-strandedDNA
andtendtohaveasimilarstructureforexample,theT-evenfamily(Milleretal.2003).Phagescan
alsobepresentwithsingle-strandedDNAordoubleorsinglestrandedRNAgenomes.
Figure1belowshowsthetypicalstructuresofcommonphages.TheT4phageisoneofthemost
widelystudiedphagesandinfectsEscherichiacolicells.Itconsistsofanelongatedicosahedralhead,
containingtheviralDNA,attachedtoahelicaltube,knownasthecomplextail,whichisthen
attachedtoasheath,connectingtheneckandcollartogetherwithacomplexendplate.Long,
jointedtailfibresarethenattachedtothis.Thesehelpthephageadheretothebacterialcellsurface
Figure1.Stucturesofcommonbacteriophages(Madigan,M.etal.2009).
Fromtoptobottom:ExamplesofsingleanddoublestrandedRNAbacteriophages;singlestrandedDNAbacteriophages,microviridaeleft,inoviridaeright;doublestrandedDNAphages.
(Madigan,M.etal2009).Otherphagesdifferinshape,normallydependingonwhatformofgenetic
materialtheycarry.Asshowninfigure1,doublestrandedDNA(dsDNA)phages,liketheT4,have
thecharacteristichead,bodyandtailfibres.
Single-strandedDNAbacteriophagescanbesplitintotwogroups;themicroviridaewhichhavean
icosahedralheadstructurewithnobody,ortailfibresandtheinoviridae.Thesehaveflexible,
filamentousphageswhichcarryacircular,positivesenseDNAgenomeandahelicalarrayof
thousandsofcopiesofamajorcapsidprotein(Brentlingeretal.2002;Kingetal2011).RNAphages,
bothsingleanddoublestranded,haveasimilarstructuretothatofthemicroviridae.
Phageswhichinfectdifferentstrainsofthesamespeciesareoftensimilar:mostTphagesinfectE.
colistrainsandthereforehavesimilarstructuresasshowninfigure1.(Madigan,M.etal2009).
Lifecyclesofbacteriophagesandhowthesecanbeappliedtophagetherapy
Bacteriophageshavetwolifecyclesdemonstratedinfigure2.Onthelefthandsideisthelyticcycle,
whichdirectlykillsbacterialcells,andontherighthandsideisthelysogeniccycle,whichintegrates
phageDNAintothehostbacterialgenome(Sahani&Gakkhar2014).Somephages,suchasT4,are
Figure2.LifecyclesofBacteriophages(Reece,J.etal2011)
Diagramshowsthelyticandlysogeniclifecyclesofabacteriophageandhowtotwointertwine.However,somephagescanonlycarryoutoneofthecycles.
knownas“virulentmode”virusesastheycanonlyperformthelyticcycle.However,asseenbelow,
itispossibleforthelifecyclestointerlinkandforsomephagestocarryoutbothlyticandlysogenic.
Theseareknownas“temperatemode”viruses.Whetherornotaphagecancarryoutjustone
lifecycleorbothdependsuponwhichgenesarepresentintheirgenome.
Virulentmodebacteriophages
Anexampleofacommon,virulentmodephagewouldbethedouble-strandedDNAT4phage.Inan
infectionofthiskindtherearefourmainstages;adsorptionontothehostcellmembrane,
penetrationofcellmembrane,intracellularreplicationofthevirusandlysisofthehostcellwith
releaseoftheprogeny(Adams1959).
Adsorptionoccurswhentipsofthebacteriophagetailsbindtospecificreceptorsonthecellsurface.
Thesereceptorsvarydependingoncellwallcomposition,soaredifferentforgram-positiveand
gram-negativebacterialcells,andalsovaryfordifferentphagegroups.Theycanbeproteinssuchas
porins,lipopolysaccharides,orteichoicacidreceptors(Rakhubaetal.2010).Adsorptionisarandom
eventandsoinfluencedbyfactorswhichincreaseinteractionsbetweenhostandviralcellsuchas
concentrationortemperature(Adams1959).
Therearethoughttobetwostagesofadsorption,reversibleandirreversible.Duringreversible
bindingthetailfibresbindandunbindto‘walk’acrossthemembraneuntilanidealsiteislocatedfor
irreversiblebinding(Birge2000).Irreversiblebindingisinfluencedbyfactorssuchashost
electrochemicalmembranepotentialandATP,itisonlyafterthisthatpenetrationcanoccur.This
involvesthetailfibrescontractingtoallowcontactofthemainbodyofthephagewiththehostcell
membrane.TheviralDNAorRNAisthenejectedfromtheheadintothehostcell(Rakhubaetal.
2010).
Onceinsertedtranscriptionandtranslationofearlyproteinsoccursresultinginthetakeoverofthe
bacterialcelltoonlyproducephageDNA.InT4phageaprominentearlyproteinisNddwhich
causesthebreakdownofthehostnucleoidandinterruptsthehostDNAreplicationsystemby
bindingtoDNAandcausingobstaclesonreplicationforks.Thereisnoeffectongeneexpressionor
degradationofhostDNAsothehostcellcanstillsurvive,butisforcedtoproducephageDNA
insteadofitsown(Bouetetal1998).Lateproteinsarethenproduced,havingmainlystructural
properties,althoughthelysozymeenzymeisalsomadehere.Assemblyofphageprogenythen
beginsandlysozymeenzymeattacksthehostcellwall,releasingtheprogenyintothesurrounding
environmenttoinfectnewcells(Birge2000).Althoughthiskillsthebacterialcellsitalsoreleasesby-
productssuchasendotoxinsofgram-negativebacteria,whichareamaincauseofsepticaemia
(Slopeketal.1983;Horadagodaetal.2001)
AstudybyWang,N.etal.(2011)concludedthatrecombinantT4lysozyme(T4L)couldbeusedto
inhibitgrowthofsomegram-positiveandgram-negativebacterialcells,howeverthecomparative
controlofhenegg-whitelysozymestillhadahigherenzymaticactivity.Oneofthereasonsforthis
wasthoughttobetheformationofdisulphidebondsintherecombinantT4Lresultinginalossof
enzymaticactivity.Iffurtherinvestigationswerecarriedouttofindawaytopreventthis,
recombinantT4Lcouldbeusedasanantimicrobialtherapyonitsownasopposedtousingthefull
T4phage.Isolatingandrecombiningenzymesisawidelyusedmoleculartechniqueinmany
laboratoriessothiscouldproveeasierthanisolatingwholephagesfortreatment,whichisnotas
common,especiallyinthewesternworld.T4phageisspecifictoE.colibacteriaandsocouldonlybe
usedagainsttheseinfectionsintreatment;T4Lhowever,hasprovedeffectiveinlysingawiderrange
ofbacteriainthestudybyWang,N,etal.(2011)thereforecouldbeofmoreuse.
Temperatemodephages
Thegenetic-networkoftemperatemodephages,suchaslambda,consistsofanumberofpositive
andnegativefeedbackloopsenablingthephagetoswitchbetweenlyticandlysogeniclifecyclesby
respondingtoenvironmentalsignals(Kobileretal2005).Theyenterthecellinthesamewayvirulent
phagesdoandundergothelyticcycle.Ifconditionsinsidethehostcellarefavourablegenesthat
carryoutthelyticcyclearerepressedandlysogeniccyclegenesareupregulated.Thephagethen
integratesitsgenomeintothebacterialchromosomeandrestsinprophageform,beingreplicated
withbacterialDNAasthehostcelldividesthereforepassingontobacterialdaughtercells,until
conditionsbecomeunfavourablewhereitresortsbacktolyticmode(refertofigure2).
InlambdaphageQandCllproteinarecotranscribed,butcontroloppositepathways(lyticand
lysogenicrespectively).IfconditionsinthecellarefavourableCllisupregulatedwhichinturncauses
adownregulationofQ.ProteinsIntandClarethentranscribed,Intintegratingthephagegenome
intothebacterialchromosomeandClrepressingphagepromotors,stoppingthelyticcyclefrom
happening(Kobileretal2005).
Ifconditionsinsidethecellbecomeunfavourable,forexamplebacterialDNAisdamagedandthe
hostSOSsystemisactivated,theClrepressoriscleavedandactivated,repressingClandQ
expressionisupregulatedcausingtheprophagetoreleaseitselffromthebacterialDNAandundergo
thelyticcycle(Atsumi&Little2005).Thisisthoughttobeamethodofself-defenceandsurvival
mechanismforthephage(Zhuetal.2004).
AsthelysogeniccycleincorporatesthephageDNAintothehostDNA,replicatingandgettingpassed
downtohostprogeny,anyresistancegenestheywerecarryinggettranscribedandpasseddown
too.Hence,phagescanactasefficientvectorsfortheacquisitionanddisseminationofresistance
genes(Marti2014).Researchontheprevalenceofantibioticresistancegenesinbothbacterialand
phageDNAinwatersamplesfromtheenvironmentshowedphagescarryingmultipleresistance
genes(Marti2014).Theseincludedβ-lactamasegenesassociatedwithwidespreadresistanceto
antibioticssuchaspenicillin.
Previousexperimentshaveshowntoxinssuchastheshiga-liketoxinonenterohemorrhagicE.coli
andcholeratoxinarebothcarriedbyphages,henceworkafterthismainlyfocussedonvirulent
modephages(O’Brien1989;Waldor1996)Incontrast,someviralgenes,ifreplicated,cansuppress
certainmetabolicpathwayshinderingthebacteriaandmakingthemmoresusceptibletoantibiotics
(Lu&Collins2009).
Historyofbacteriophagesandearlyexamplesofphagetherapy
Bacteriophageswerefirst“officially”discoveredandnamedbyFrenchmicrobiologist,Felix
d’Herelle,in1910;however,thereisevidencetosuggesttheyhadbeennotedbyotherspreviously
suchasBritishbacteriologistErnestHankin(Hankin1896).D’Heurellemanagedtofirstisolate
bacteriophagesin1916andusethemintherapysuccessfullyforthefirsttimein1919totreatsevere
dysenteryinFrenchhospital,HôpitaldesEnfants-Malades(Summers1999).Hecontinuedhiswork
onphagesinhislaboratoryinPariswithfivephagepreparationsbeingmarketedbyL’Orealincluding
Bacté-coli-phageandBacté-staphy-phageforE.coliandStaphylococcusaureus.D’Herellebelieved
selectionandpurificationofbacteriophageswaskeytotheirefficacyintreatment.
Phagepotencycanvarygreatly,withsomephagesbeingmuchbetteratattackingcertainstrainsof
bacteriathanothers.Ifaphagewithalowerpotencyisgiven,itcanmeaneradicatingthebacteria
takesmuchlonger,which,inturngivesthebacteriamorechanceofdevelopingphageresistance
genesandgivingthepatientapoorerprognosis(d’Herelle1931).Therefored’Herellebelieved
selectionmusttakeplacetoensurethephagewiththehighestpotencyisadministered.
Purificationofphageswasalsoimportanttod’Herelletoensurenobacteriawerepresentinphage
preparations.Ifpresent,thesemayenhancethepatient’ssymptomsorcauseaseparateinfection,
especiallyasmanyphagetherapiesweregivenintravenously,sobacteriawouldhavethechanceto
disseminateall-roundthebody.Healsonotedformanyphagestheirpotencyfortreatmentinvivo
decreasedwithage,despitenochangeinpotencyinvitro.Withthisinmind,ifphagesweretobe
producedcommerciallyforlargescaletreatmenttheywouldhavetobeisolatedasnearto
administeringtimeaspossibleanddelaysinthatisolation,suchasfindingthephagewiththe
highestpotencyfortheinfection,coulddecreaseefficacy(d’Herelle1931).
Fromthe1930’sonward,thediscoveryofantibioticsmeantthatphagetherapytookabackseatin
thewesternworld.Despitethis,institutionssuchastheELIAVAInstituteinGeorgiaandtheHirtzfeld
InstituteofImmunologyandExperimentalTherapies(HIIET)inPolandstillcarriedoutextensive
research.
Otherstudieswentontolookatthebestwaystopreservebacteriophagesfortreatment.One
experimentstoredphagesfor2yearsatbothroomtemperatureand4°Candprocessedthemin
differentways:brothlysates,glycerol,driedandfreezedried.Resultsshowedbrothpreparations
storedat4°Ctobethemostpotent,howeverallpreparationsdeclinedwithtime(Clark1962).
Furtherinvestigationhasnowleadtostocksbeingstoredinamixtureofconditions,dependingon
thephage,withvaryingtemperaturesandglycerolmedias.
1980’sexperimentsfromSmithandHugginsshowedphagetherapytobemoreeffectivethan
antibiotictreatmentininvivoanimalexperimentsagainstvariousE.colistrainsthatcancause
infectioninbothmanandanimals(Smith&Huggins1982;Smith&Huggins1983).Thefirst
experimentoccurredinmiceandshowedtimingofadministeringphagestobekey;thesoonerafter
theonsetofbacterialinfectiontheyweregiven,themoreeffectivetheywere.Thephageefficacy
wascomparedtonumerousantibioticsincludingtetracyclineandampicillin.Thedeathrateofmice
treatedwithantibioticswasmuchhigherthanthosetreatedwithphages.Nodeathsoccurredin
micetreatedwithphageconcentrationsabove3x103viableparticles,whereasmultipledeaths
occurredinallmicetreatedwithantibiotics(Smith&Huggins1982).
Thesecondexperimenttestedtheefficacyofphagesincalves,pigletsandlambsagainstvarious
enteropathicE.coli.Incalves,ifphageswereadministeredbeforetheonsetofdiarrhoea,theywere
protectedagainstthebacterialinfection,againhighlightingtheimportanceoftimingof
administration.Afterfaecalanalysis,calvesthatdidnotsurviveshowedtohavehighlevelsof
mutantE.colihence,thephagesusedwouldnothavehadashigherpotency(Smith&Huggins
1983).Thislinksinwithd’Herelle’spointearlieraboutphageselectionandspecificity.
Inpigletsandlambsmixturesofphagesweregiven.Thishadanamelioratingeffectonthediseasein
both.Infaecalanalysisfewermutantstrainswerefoundwhichcorrelateswiththeirlowerdeath
ratethancalves.Bacteriophagesareusedtodayinveterinarymedicineandfoodsciencetotreat
livestockagainstinfectionssuchasCampylobacterinpoultry,which,ifdigestedcancausefood
poisoninginhumans(Tiwarietal.2014;FSA2015).
Between1981and1986theHirtzfeldInstituteofImmunologyandExperimentalTherapies(HIIET)
usedbacteriophagetherapyon550patientsandfoundtreatmenttobesuccessfulinover90%of
cases.7%ofcasessawonlytransientimprovementwhichcouldhavebeenduetolackofphage
specificityduetothepresenceofmutantstrainslikeinSmith&Huggins(1983)experiment.518of
thecasestreatedhadbeenpreviouslytreatedwithantibiotics;however,duetoresistancethis
primarytreatmenthadbeenineffectivesophagetherapywasofferedasanalternative(Slopeketal.
1987).Phagetherapyprovedtoworkirrespectiveofageandsexofpatientandtypeofinfection
(monoandpolyinfections).
Followingthis,during1987-2000HIIETtreatedafurther1307patientsusingphagetherapywith
varyinginfections,normallywithmulti-drugresistantbacteria,whereantibioticshadfailed.S.aureus
causedthemajorityofinfections,butE.coli,Pseudomonas,KlebsiellaandProteuswerealsopresent.
Therapywasgiveninavarietyofwaysincludingoralsolution,earandnosedropsandtopicalcreams
forskininfections.Thesewereadministered10ml3timesaday.Patient’sagesrangedfrom4weeks
to86yearsandaltogetherhadafullrecoveryrateof85.9%,markedimprovementof10.2%leaving
only3.8%withnoeffect(Weber-Dabrowska2000).
Treatmentmethods
Phagetherapyisadministeredeitherasasingletreatmentmethodor,morecommonly,in
conjunctionwithantibiotics.Treatmentcanbewithbacteriophagescarryingouteitherlifecycle.
Lyticmodephagescanbeusedwithorwithoutantibioticstoeithereradicatethebacteria
completelyorbreakupabacterialcolony,allowingeaseofaccessforantibiotics.Thesecanalsobe
engineeredtobecomenon-lytic,decreasingthebacterialby-productsthatcouldcausesepticaemia.
Thelysogeniccyclecanbemanipulatedtoproducegeneswhichhinderthebacteriaandmakethem
moresusceptibletoantibiotics.
Lyticphagetreatment
Rhoadsetal.(2009)usedbacteriophagesasthesoletreatmentofinfectionsinvenouslegulcersin
theformofaphagecocktailWPP-201,whichcontainseightlyticphagesagainst:Pseudomonas
aeruginosa,S.aureusandE.coli,threebacteriaknowntohavehighinstancesofantibioticresistance
andcauseproblemsintreatment(CDC2013).Lyticphageswereusedtopreventthepotential
spreadofresistancebytransferofgenesfromphagetobacteria.AfurthermeasureoffullDNA
sequencingonallphageswasalsodonetomakesurenoresistancegenesortoxin-encodinggenes
werepresent.
TheresultsoftheWPP-201studyshowednoincreaseinadverseeffectsorhealingtimebetween
patientstreatedwiththephagecocktailandthoseonstandardtreatment.Thediscussionofthis
trial,includingsuggestionsforfuturework,focussedontheimportanceofthephagecocktail’s
specifityandtailoringittoeachpatientandorganisms.Ifresistancegenestoanyofthesephages
weretobeacquiredthephagecanbesubstitutedforanothereasilyasphagesaresoabundant.This
hasalreadybeenputinplaceinthefoodindustryforphagecocktailsinfoodsafetyhowever,aswith
thistrialandmanyothers,furtherclinicalstudiesareneeded(Rhoads,D.etal2009).
Anotherstudyin2009carriedoutaphaseIIdouble-blindplacebotrialfortheefficacyofphage
preparation,Biophage-PA,containing6separatelyticphagesalltargetingPseudomonasaeruginosa
strains.P.aeruginosaistheprimarycauseofotitisandalmostallstrainsnowcarryresistancegenes.
Samplesfrompatientswhosufferedlongterm(rangeof2-58years)chronicotitisweretakenand
testedforsensitivitytoeachofthephagespresentinthepreparation.Iftheyshowednosensitivity
toanyofthephagesthepatientcouldnotgofurtherinthetrial.Overall86.2%ofisolateswere
sensitivetoatleastone.Patientswerethenseparatedintoplaceboandtestgroupsand
administeredasingledoseof0.2mLofeitherBiophage-PAorplaceboliquidfromaspindleneedle
intotheear(Wrightetal.2009).
Patientscamebackforcheck-upsondays7,21and42aftertreatment,92%ofthetestgroup
showedsomeimprovementwith25%havingcompleterecovery.Resultsalsoshowedasignificant
80%decreaseinP.aeruginosacountinthetestgroupandonly10%decreaseinthecontrolgiving
evidenceforthehighefficacyofthislyticphagepreparationinthetreatmentofotitis(Wrightetal.
2009).
A2011casereportusinglyticphageswithantibioticsdetailssuccessfultreatmentofanelderly
patientsufferingfromUTIwithpersistentP.aeruginosainfectionfollowingsurgerytoremovea
malignanttumour.Thepatienthadpreviouslybeentreatedwithfourdifferentantibiotics
(Gentamicin,Ceftazidime,Ciproflozin,Meropenem)overthecourseoftwoyearshowever,resistant
strainsofthebacteriakeptreappearing.Aphagecocktailwaschosenfromaprexistinglibraryin
Georgiaandtwoantibiotics(Colistindays6-10andMeropenemday6-30)weregiventhenafterday
sixofphagetreatment.Fromdayeightonwards,i.e2daysofcombinedtherapyand8daysofphage
therapy,thebacterialviablecountfelltozero.Forsixmonthsafterwardsurinewasroutinelytested
andremainedsterile.Thepatientwasreadmittedtohospitaloneyearlaterforotherreasonsand
urinewasagainsterile(Khawaldeh,A.etal2011).Theviablecountinthisinstancefellfrom3.00
x106to3.00x105overthe5dayperiodthatphagetherapywasusedasthesoletreatmentandonly
fellto0aftertheantibioticsweregiven,thereforeitisarguablethatphagetherapyonitsown,in
thisinstance,wouldhavebeenineffective.However,previoustreatmentwithMeropenemhadbeen
ineffective,whichsuggeststhatthephagemayhavebeenabletotargettheresistancemechanism
andallowtheantibiotictoenterthebacterialcell,accumulateandactonthetarget.Itisarguable
thattheuseofthesecondantibioticColistinalonemayhavebeeneffective,sincethiswasnotused
inthefirstinstance,sothetoxicityithadonthebacteriainvivoisunknown.
Phagetherapyalonedidnotseemtohaveasignificantimpactonthebacteriaandantibiotic
treatmentpreviouslyhadfailed.However,acombinationofbothofthesewassuccessfulgiving
evidenceforthesynergisticactionofthetwowhenusedintreatment.
Nonlyticphagetreatment
Duetothelyticcycleofphagescausingthereleaseofharmfulby-productssuchasendotoxins,a
prominentcauseofsepticaemiafoundingram-negativecellwalls,scientistshavelookedinto
engineeringlyticmodephagestostillkillthebacteria,butkeepthebacterialcellstructureintact.
AstudyinViennareplacedanexportproteininP.aeruginosaphagePf3topreventphagereplication
andthereleaseofthephagefromthecell,thusthereleaseofendotoxins.Resultsfrominvivomice
experimentsshowedtherecombinantPf3(Pf3R)phagetobesuccessfulinkeepingthemajorityof
thecellsintactcomparedwiththelyticPt1phagethatwasusedasacontrol.
Pt1phageonlyhada20%survivalrateafterthe7dayincubationperiodfollowingadministration
whereasPf3Rhadasurvivalrateofover70%.Thiswasthoughttobeduetothemuchlower
endotoxinlevelreleaseandinflammatoryresponsepresentinmicegivenPf3RthanonesgivenPt1
(Hagensetal.2004).
Asmentionedpreviously,somescientistsarehesitanttousetemperatemodephagesintreatment
astheyarenaturalvectorsforresistancegenesandvirulencefactors,suchasthebotulinumtoxin
(Boyd2012).
Temperatemodephagescanbeengineeredtochangetheirgeneexpressiontoaffectbacterial
metabolicprocessesmakingtreatmentwithantibioticsmoresuccessful.Theycanexpressgenesthat
hinderthebacteriaorrepresstheirresistancemechanismsmakingiteasierforantibioticstoattack.
Onestudythattookadvantageofthisusedwild-typebacteriophageM13,afilamentousE.coli
phage;andengineeredM13mp18,acircularformofDNAderivedfromM13(Neb2015).Thiswas
usedtoover-expresslexA3protein,whichrepressedtheSOSDNArepairnetworkinE.coliandin
doingso,enhancedtheactionofvariousquinoloneantibiotics.
Allinvitroexperimentsandfurtherinvivomiceexperimentsonwild-typeandresistantbacteria
showedtheuseofM13phageenhancedantibioticactivity,butuseofmodifiedM13mp18phage
enhancedactivityevenmore(Lu,T.Collins,J.2009).
M13phageisoftenusedasatoolfortheframeworkfornanostructuresduetoitsmanyinsertion
sitesmakingiteasilyengineered,M13mp18isreadilyavailableasaproductfromNewEngland
Biolabs.Despitethisitisnotyetusedinclinicalpractise;furtherinvivoanimalandhumantrialsstill
needtobeconductedtoseehowtomanipulatethephageexactlytotargetspecificstrainsof
bacteria.Forexample,iftheDspgenedescribedbelowisinsertedintothegenomeM13couldbe
usedtotargetbacteriathathaveformedbiofilmcolonies.
Treatmenttargetingbiofilms
Rhoadsetal.(2009)alsonotedtheuseofbacteriophagesintargetingbiofilms-surface-associated
biofilmcommunitiesencasedinextracellularpolymericsubstances(EPS).Theseareessentialand
protectivestateforbacteriaassociatedwithpersistentinfectionandantibioticresistance(Fuxetal.
2004).Bacteriawithinbiofilmshaveachangedphenotypepreventingantibioticsfromreachingtheir
targetascellsgointoasloworstationarygrowthknownaspersistercells.Thechanceofresistance
isincreasedduetosomanycellsbeinginclosecontactallowingeaseforhorizontalgenetransfer
(Hoibyetal.2009).
Phages,unlikeantibiotics,caninfectpersistercellsandremaindormantuntiltheybecomeactive
wherethephagethendestroysthemuponreplicationusingthehostsactiveproteins;ordisperse
theprotectivematrixtoallowantibioticsthroughviamechanismslikeenzymaticdegradationas
citedearlierbyLu&Collins(2007).T4phageinE.coliinfectionshasbeenshowntohavean
influenceonbiofilmmorphology(Corbinetal.2001)
OnetrialmanipulatedT7phageto,firstly,multiplyrapidlyonceinsidethecellsand,secondly,
expressDispersinB(DspB)uponinfectionreleasingitintotheextracellularenvironmentduringcell
lysis.DspBisanenzymewhichcatalysesthehydrolysisofN-acetyl-D-glucosaminesfoundinthe
biofilmEPSbycleavingterminalmonosaccarideresidues(Ramasubbuetal.2005).Itisfound
naturallyinsomebacteriaandusedasamechanismtoreleasenearbycellsandallowthemtostart
newcolonies.
Ramasubbuetal.(2005)usedanE.colistrainknowntoformstrongbiofilmsandcontainmany
biofilm-promotingfactorstomakesurethat,ifthephageworked,ithadastrongefficacy.Inthe
experimentstheengineeredphageprovedeffectiveindispersingthebiofilmsshowingthiscouldbe
usedinaclinicalsetting.Oftenmedicaldevicessuchascathetersgrowbiofilmscausingdifficultyin
treatment,especiallywiththeincreasedchanceofresistancespreadinginbiofilmsandthe
resistancetoantibioticspersistercellsshownaturallyincomparisontoplanktonicbacteria(Pradeep
Kumaretal.2013).Usingbacteriophagestodispersebiofilmscouldbeaverypositivedevelopment.
Removingbiofilmsformedonexternalsurfaceswithphageshasalreadybeeninvestigated.Zhang
andHu(2012)usedRNAphagestoattackbiofilmsformedbyP.aeruginosa.Evenwithhigh
concentrationsofthephagetherewasstillevidenceofgrowthinthebiofilm.Theythenuseda
combinationofphagesandchlorinewhichonitsownagainhadlittleeffectonthebiofilm.Thetwo
togethershowedsignificantimprovementoneradicatingtheplaque(Zhang&Hu2012).Chlorine,
howeveristoxictohumansasitreactswithwaterinandoutsidethebodycausingseverepoisoning
(MedlinePlus2015).Hence,thismethodoferadicatingbiofilmscouldbeusedoncathetersifthey
wereremovedfromthebody,althoughextracarewouldhavetobetakentoensurethedevicewas
thenclearedofchlorine,orinsteadresearchintofindinganalternativelesstoxic.
CurrentThinking
ManycurrentmethodsofphageresearchtargetresistantbacteriasuchasP.aeruginosaandS.
aureustotrytofightthegrowingincreaseinresistance(Ampliphi2015;PhageTherapyCenter
2015).Alotofresearchisalsoaimedatisolatingspecificenzymes,suchasT4lysozyme,orproteins
ofphagestoeaseproductionandachievemorespecificresultsthancanbeachievedbymakingthe
entirephage(Borysowskietal.2006;Wangetal.2011).
In2006onestudylookedintophageendolysins,asthesetargetthepeptidoglycaninbacterialcell
walls.Duetotheirtarget,peptidoglycan,endolysinscanattackthebacterialcellregardlessofits
defencemechanisms.Thereisalowchanceofbacteriadevelopingresistancetothemsince,inorder
todoso,thewholebacterialcellwallstructurewouldhavetobealteredoranextraprotectivelayer
abovethiswouldhavetobeformedsomehow.Normallyendolysinsarereleasedfromphagesinside
thecelltocausecelllysisandreleaseofphageprogeny;however,theyarealsoeffectivewhen
appliedexternallytothecell(Borysowskietal.2006).
Currentresearchalsolooksintophagecocktails;beingabletousetheseagainstpolyinfectionsand
targetanybacteriathatdifferslightlyfromtheoriginal.
OnephagecocktailalreadygoingthroughhumanclinicaltrialsistheBFC-1againstS.aureusand
P.aeruginosatobeusedasasyringesprayonburnwoundinfections(Merabishvili,M.etal.
2009).ThereportbytheEliavaInstituedetailstheselectionprocessforthethreephagesisolated,
againstP.aeruginosaandS.aureus,thenthequalitycontrolstepsinvolvedafterselection.
AfollowupreportexplainshowtheBFC-1,althoughapprovedbyBelgianMedicalEthicsCommittee,
hasnotyetbeenfullyapprovedfortreatment.Itishowever,usedasasalvagetherapyatQueen
AstridMilitaryHospital,appliedtopicallytoburnwoundinfections,especiallyfrommulti-drug
resistantbacteria.Thisisduetoevaluationreportsclaimingphagesshouldundergopharmaceutical
testsandclinicaltrialswhichtakeyearstofinish.Thereportputsforwardtheargumentthatphages
aretherapeuticagents,notdrugs,andtreatingthemthesameasstabledrugswouldnotpermit
theirflexibilityintreatment.Phagesarebiologicalagentswhichcanvary;thisgivestheadvantageof
beingabletotargetbacterialstrainsthathavegainedmutationsfromtheoriginalbacteria(Roseet
al.2014).Duetothespecifiticyofphagesifbacterialstrainsvariedslightly,theoriginalphagepicked
outforusewouldbeineffective.Thisreporthighlightstheneedforincreasedknowledgeonphages
andhowtheyworkinclinicalusetofastforwardtheiracceptanceofbeingavalidtherapyfor
treatingbacterialinfections.
PhagesusedintheBFC-1trialweretakenfromexistinglibrariesinGeorgiaandRussia.Sharingof
knowledgebetweentheseinstitutionsandthewesternworldcouldhelptopromotephagesasa
treatmentmorereadily.AlreadyEliavahaveworkedwithAustraliancompanyAmpliphi(detailed
below)whohaveconnectionsintheUK(Khawaldehetal.2011;Ampliphi2015).
TheBFC-1studyusedlyticmodephages.Oneimportantstephighlightedinthequalitycontrol
processwastheremovalofanytemperatemodephagesthatmayoriginatefromthewild-type.This
wasforthesamereasonsascitedbyRhoadsetal.(2009).Itseemsalthoughothershaveworkedon
engineeringlysogenicphagesandbeensuccessful;muchmoreresearchneedstobedonetoallay
thefearofintroducingfurtherresistanceorvirulencefactorgenes.
Themostobviousreasonsthatbacteriophagesarenotyetusedintreatmentconcernlackof
knowledgeandprotocolsonisolationandpurificationofphages.TheBFC-1reportcouldbeusedas
atemplateinthefuturetreatmentofburninfections,whicharethoughttoaccountforaround75%
ofdeathsfollowingthermalinjuryandarethemostcommonoriginofsepsis,oftenwithmultidrug
resistantbacteria(Altoparlark,U.etal2004).
Limitations
Aspreviouslymentionedthelysogeniccyclecanintroduceantibioticresistanceandpathogenicityby
insertingitsgenomeintobacterialcellswhichisthenreplicatedwithbacteria.Asamanyphagesare
stillnotfullyunderstoodandtheirgenomeshavenotbeensequenced,thereposesariskofchoosing
aphagefortreatmentandthenintroducingresistancegenesintobacteria,suchasCTX-M(β-
lactamaseclassAenzymes)(Martietal.2014).Sequencingofgenomeshasdisplayedevidencethat
someoftheseareencodedforbyphage-likeelements(Oliveretal.2005;Falgenhaueretal.2014)
Lyticmodephages,althoughseeminglymoreeffectivethanlysogenicphagesintreatment,could
causephageresistanceinthesamewayasantibioticresistancearose.Iflyticphagesareusedto
treatapopulationofbacteriacontainingasmallpercentagethathaveacquiredphageresistance
genes,themajorityofbacteriawillbedestroyed,howevertheresistantonesremain.Theseareleft
toproliferateandpassdowntheresistancegenestotheirprogenycellsorviahorizontalgene
transmissiontootherbacterialspecies(Lu&Collins2007;Labrieetal.2010).Thus,iftreatmentwith
lyticphageswastogoaheaditwouldhavetobeusedcarefullyandmonitoredforanymechanisms
whichseemedtoshowresistance.BacteriasuchasS.aureusandgram-negativeslikeE.colihave
beenveryadeptatgainingandsharingantibioticresistancegeneshence,monitoringofphage
resistancewouldbeoftheutmostimportancewithsuchspecies.
Celllysisbylyticphagescanalsoreleasetoxicby-productsofthebacterialcellsincludingendotoxins
thatmaybecauseofsymptomsandnottheactualbacterialcell.Endotoxinsarealsoamaincuase
forsepticaemia(Slopeketal.1983;Horadagodaetal.2001).
Isolatinganyviruscanbedifficultasahostcellisrequiredforgrowth.Phagetherapyprovesan
extrachallengeasphagesarestrain-specific.Nevertheless,asreferredtoearlier,therearepre-
existingstudiesandexampleswhichcanbeusedasprotocolforisolation.
Intermsofphageproduction,itisimportanttohaveashortproductiontotreatmenttimescaleas
phagepreparationsdecreaseinefficacyovertime.IncountriessuchasGeorgiaandPolandto
addressthistheyhavea“surmeasure”approachwherebyphagepreparationsaremadeonsitein
hospitalsortherapycentresatlowcostandtakingonlydaystoweeks.Unfortunately,thisisnot
viableinmostofthewesternworldduetolicensinglawsmeaningthat“PrêtáPorter”approachof
productionbypharmaceuticalcompaniesisrequired.Thiscantakeyearsandcomeswithhighcost
withmanyclinicaltrialsonhumansandanimalsneeded.Duetothetimingdifficulty,phage
preparationsthataremademaybeuselesswhenthefinalproductismade(Pirnay,JP.etal2010).
Amainadvantageofphagetherapyisthepossibilityofcreatingcustomcocktailsforindividual
patientsandspecifyingthemforwhicheverstrainsofbacteriatheyareinfectedwith,havingthe“sur
measure”approachwouldsuitthisperfectlywithhospitalstaffabletomakeupacocktailandtreat
thepatientquickly,especiallyifitisatraumaorburnpatientwheretreatmentneedstobequickto
preventsepsis.
Atthemoment,themainlimitationwithphagetherapyinthewestisthelackoflargescale
randomizedtrials;however,trialsthatareavailablefromvariousinstitutionswhousephage
therapy,suchasHIEETandELIAVA,shownosignificantadverseeffectsandtrialsinanimalsalso
seemtobesuccessful(Wittebole,X.2014).
CompaniesstartingupsuchasFixed-PhageinScotlandandAmpliphiinAustraliaarelookingto
overcometheseandhavephagetherapyasastrongalternativetoantibiotics.Ampliphicurrently
havetwophageproductsinpreclinicalstagesandafurthertwoindevelopmental;allagainst
bacteriacommonlyassociatedwithresistanceanddifficultyintreatmentsuchasP.aeruginosa,
S.aureusandC.difficile(AmpliPhi2015).
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