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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