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APPLIEDANDENVIRONMENTALMICROBIOLOGY,July2008,p.
4149–4163Vol.
74,No.
130099-2240/08/$08.
000doi:10.
1128/AEM.
02371-07Copyright2008,AmericanSocietyforMicrobiology.
AllRightsReserved.
InSilicoandInVivoEvaluationofBacteriophageEF24C,aCandidateforTreatmentofEnterococcusfaecalisInfectionsJumpeiUchiyama,1,2MohammadRashel,2IyoTakemura,2HiroshiWakiguchi,1andShigenobuMatsuzaki2*DepartmentsofPediatrics1andMicrobiologyandInfection,2KochiMedicalSchool,Oko-cho,NankokuCity,Kochi783-8505,JapanReceived22October2007/Accepted1April2008Alongwiththeincreasingthreatofnosocomialinfectionsbyvancomycin-resistantEnterococcusfaecalis,bacteriophage(phage)therapyhasbeenexpectedasanalternativetherapyagainstinfectiousdisease.
Al-thoughgenomeinformationandproofofapplicabilityareprerequisitesforamoderntherapeuticphage,E.
faecalisphagehasnotbeenanalyzedintermsoftheseaspects.
Previously,wereportedanovelvirulentphage,EF24C,anditsbiologyindicateditstherapeuticpotentialagainstE.
faecalisinfection.
Inthisstudy,theEF24CgenomewasanalyzedandtheinvivotherapeuticapplicabilityofEF24Cwasalsobrieyassessed.
Itscompletegenome(142,072bp)waspredictedtohave221openreadingframes(ORFs)andvetRNAgenes.
InourfunctionalanalysisoftheORFsbyuseofapublicdatabase,noproteinsundesirableinphagetherapy,suchaspathogenicandintegration-relatedproteins,werepredicted.
Thenoncompetitivedirectionsofrepli-cationandtranscriptionandthehost-adaptedtranslationofthephagewerededucedbioinformatically.
ItsgenomicfeaturesindicatedthatEF24CisamemberoftheSPO1-likephagegenusandespeciallythatithasacloserelationshiptotheListeriaphageP100,whichisauthorizedforprophylacticuse.
Thus,thesebioin-formaticsanalysesrationalizedthetherapeuticeligibilityofEF24C.
Moreover,theinvivotherapeuticpotentialofEF24C,whichwaseffectiveatalowconcentrationandwasnotaffectedbyhostsensitivitytothephage,wasprovenbyuseofsepsisBALB/cmousemodels.
Furthermore,nochangeinmouselethalitywasobservedundereithersingleorrepeatedphageexposures.
Althoughfurtherstudyisrequired,EF24CcanbeapromisingtherapeuticphageagainstE.
faecalisinfections.
Enterococcusspeciescanbefoundintheenvironmentandnormalmicrooraoflargemammals,andsomeofthesebac-terialspeciesoccasionallycauseavarietyofdiseases(30).
Theemergenceofvancomycin-resistantEnterococcus(VRE)dis-ruptseffectiveconventionalchemotherapyandcausesfatalinfectionsinnosocomialsettings.
AnincreaseinthenumberofVREcaseshasbeenreportedworldwide(5,9,14).
Amongtheenterococcalinfections,E.
faecalisisthemostclinicallyiso-latedspecies(19,30).
Bacteriophage(phage)therapyharnessesaliveprokaryoticvirusasabioagenttotargetanddestroydisease-causingbac-teria(7,15).
PhagetherapyhasalonghistoryofsuccessfuluseintheformerEasternbloccountries,whereasithasalmostnosuchhistoryintheWest(15,27).
Therecentincreaseinthenumberofmultiple-drug-resistantbacteriaincludingVREhasrenewedtheinterestoftheWesternscienticcommunityinphagetherapy(15,27).
However,becausethepastfailuresinphagetherapyresultedfromalackofscienticknowledgeofphagebiology,thistherapeuticapproachneedstobescientif-icallyrationalized(27).
Hence,eachtherapeuticphageneedstobewellcharacterized,likeotherapproveddrugs.
Genomeanalysisandproofofapplicabilityaresimpleandeffectivemethodsfortheprimaryevaluationofeachphage.
First,thephagegenomereectsbiologicalinformationsuchasmorphology(e.
g.
,drugformulation)andlifecycle(e.
g.
,prop-agationmechanismanddrugefcacy),enablingtheelucidationofphagedrugfeatures.
Inaddition,genomeanalysisallowsustoexaminethesafetyoftheapproachbydeterminingthepresenceorabsenceofundesirablegenessuchaspathogenicandintegration-relatedgenes(36,43).
Moreover,thelyticac-tivityandintrinsiceffectsofeachphageinvivoareusuallyunknown,sothatinvivotherapeuticeffectivenessandphagetoxicitymustalsobeexamined.
Unfortunately,notherapeuticphagewithsuchevaluationiscurrentlyavailableagainstE.
faecalisinfections.
Previously,EF24Cwasisolatedanditsbiologywaschar-acterizedbriey.
EF24C,whichwasclassiedinthefamilyMyoviridaemorphotypeA1,hasabroadhostspecicitywithstrongvirulenceagainstE.
faecalis,includingtheVREstrains(47).
ThemorphologyofEF24C,togetherwiththeN-termi-nalsequencesofitsstructuralproteins,impliesarelationshiptomembersoftheSPO1-likephagegenus(28,47).
Someofthesemembersareconsideredtobetherapeuticandprophy-lacticphagecandidates(e.
g.
,StaphylococcusphagesKand812andListeriaphageP100)(8,20,29,40).
Consequently,EF24Cwasproposedasaputativetherapeuticcandidate.
Inthepresentstudy,theEF24Cgenomewasanalyzedforthersttime.
Next,invivotherapeuticeffectivenesswasevaluatedusingseveresepsismousemodelsinfectedwithanE.
faecalisstrainhavingeitherhighorlowphagesensitivity.
Inaddition,phagetoxicitywasbrieyexaminedinvivo.
*Correspondingauthor.
Mailingaddress:DepartmentofMicrobiologyandInfection,KochiMedicalSchool,KohasuOko-cho,NankokuCity,Kochi783-8505,Japan.
Phone:81-88-880-2323.
Fax:81-88-880-2324.
E-mail:matuzaki@kochi-u.
ac.
jp.
Supplementalmaterialforthisarticlemaybefoundathttp://aem.
asm.
org/.
Publishedaheadofprinton2May2008.
4149onMarch11,2021byguesthttp://aem.
asm.
org/DownloadedfromMATERIALSANDMETHODSCulturemedia.
Bothtrypticsoybroth(TSB)andheartinfusionbrothwereobtainedfromBecton,DickinsonandCompany(Sparks,MD).
ConstituentsofculturemediausedwerepurchasedfromNacalaiTesque(Kyoto,Japan)unlessotherwisestated.
Heartinfusionbrothsupplementedwith20mMMgCl2and20mMCaCl2(HIMC)wasprepared.
Forthephageplaqueformationassay,TSB-basedsolidmediacontaining1.
5%and0.
5%agarwereusedforthelowerandupperlayers,respectively.
Bacterialstrains.
E.
faecalisstrainEF24wasemployedasahostforthephagepropagationandphageplaqueformationassay.
E.
faecalisstrainsEF14andVRE2wereemployedintheanimalexperiments.
Thesebacterialstrainsweredescribedpreviously(47).
Phagepurication.
Phagepuricationwascarriedoutasdescribedpreviously(47).
Briey,phagewaspropagatedwithhoststrainEF24in1literofTSBmedium.
Aftertheremovalofbacterialdebrisbycentrifugation(10,000g,4°C,10min)andsupplementationofthelysatewithpolyethyleneglycol6000(Sigma-AldrichCo.
,MI)andNaCl(nalconcentrationsof10%and0.
5M,respectively),phagewasprecipitatedbycentrifugation(10,000g,4°C,30min).
PhageprecipitatewastreatedwithDNaseI(typeII;Sigma-Aldrich)andRNaseA(typeIA;Sigma-Aldrich)(both50g/ml).
Finally,phagewassequentiallypuriedbyCsClstepgradientultracentrifugation(50,000g,4°C,2h)twice.
AnS80AT3rotorandaGXseriesHimacCS100GXmicroultracentrifuge(HitachiLtd.
,Tokyo,Japan)wereusedforultracentrifugation.
Afterthephagebandwascollected,thepuriedphagewastreateddifferentlyforeachexperimentalpur-pose.
Genomesequencing.
TheextractionofpuriedphageDNAwascarriedoutasdescribedpreviously(47).
Briey,thepuriedphagesuspensioncontainingCsClwasdilutedwithAAS(0.
1Mammoniumacetate,10mMNaCl,1mMCaCl2,1mMMgCl2,pH7.
2)fourtimes,andphagewaspelletedbyultracentrifugation(100,000g,4°C,1h).
ThephagepelletwasincubatedwithproteinaseK(TakaraBio,Kyoto,Japan),andphenolextractionandethanolprecipitationwereconducted.
Finally,thegenomeDNAwassolubilizedinwater.
ThephageDNAwasdigestedbyrestrictionendonucleaseHindIII(50ngDNA/Uin50l,37°C,4h)(TakaraBio)andthenelectrophoresedin0.
8%agarose.
Aftervisualizationwithethidiumbromide(1g/ml),theDNAfrag-mentswereexcisedandextractedfromthegel.
TheDNAfragmentswereclonedintopUC19vectorsandtransformedintocompetentEscherichiacoliDH5.
SequencingofeachclonedfragmentwasperformedbyPCR,usingtheBigDyeTerminatorv1.
1cyclesequencingkit(AppliedBiosystems,CA).
ThePCRprod-uctswerepuriedthroughaSephadexG-50column(Sigma-Aldrich)andwerethenanalyzedusinganABIPrism3100-Avantgeneticanalyzer(AppliedBio-systems).
Clonedfragmentsweresequencedbyprimerwalking.
TheregionsuncoveredbytheclonedfragmentswereampliedbyPCRwithprimersbasedontheclonedfragments,andthenthePCRproductswerepuriedbyLaboPassPCR(HokkaidoSystemScience,Sapporo,Japan)andsequencedbyprimerwalkingasdescribedabove.
Bothstrandsweresequenced,andthesequencedatawereconnectedusingtheGenetyx-MacATSQprogram,version4.
2.
1(GenetyxCo.
,Tokyo,Japan).
Thesequencecoverageredundancywasatleastdouble.
Genomeanalysis.
Thepotentialopenreadingframes(ORFs)thatpossiblyen-codethegeneproductswererstpredictedbythefollowinggenepredicationtools:GeneMarkVIORIN(http://opal.
biology.
gatech.
edu/GeneMark/)(3),FGENESB(http://www.
softberry.
com/berry.
phtml),andMicrobialGenomeAnnotationTools(http://www.
ncbi.
nlm.
nih.
gov/genomes/MICROBES/glimmer_3.
cgi)(12,46).
ATG,TTG,andGTGwereconsideredtobestartcodons,andTAA,TGA,andTAGwereconsideredtobestopcodons.
TheORFswerethendeterminedfromtheprogram-predictedORFsbasedonthecriteriaofalengthofmorethan72nucleotidesandamaximumlengthequivalenttothatoftheprogram-predictedORFswithstopcodonsatthesamelocations.
ToexaminethetherapeuticeligibilityofEF24Cstrictly,suchcriteriawerepurposelyusedtodeterminetheORFsinthisstudy.
Toincreasethepossibilityofidentifyingprotein-codingsequences,theribosomalbind-ingsite(RBS)sequenceofeachORFwasalsosubsequentlyinvestigated.
Moreover,tRNAgeneswerepredictedusingthetRNAscan-SEprogram(http://lowelab.
ucsc.
edu/tRNAscan-SE/)(34).
TheputativeproductsoftheORFswereanalyzedbyBLASTPattheNationalCenterforBiotechnologyInformation(http://www.
ncbi.
nlm.
nih.
gov/blast/Blast.
cgi)andbyInterProScanwithBlastProDom,FPrintScan,HMMPIR,HMMPfam,HMMSmart,HMMTigr,ProleScan,ScanRegExp,SuperFamily,HMMPanther,andGene3DattheEuropeanBioinformaticsInstitute(http://www.
ebi.
ac.
uk/InterProScan/)(38).
Transmembranedomainsandsignalpeptideswerealsopre-dictedbyTMHMM(http://www.
cbs.
dtu.
dk/services/TMHMM/)andSignalIP3.
0(http://www.
cbs.
dtu.
dk/services/SignalP/),respectively(2,31).
UsinganEvaluethresholdof0.
1forbothBLASTPandInterProScan,thefunctionsoftheputativegeneproductswerespecied.
Thefollowinggenomefeatureswerealsoanalyzedusinginsilicomolecularcloninggenomicsedition(InSilicoBiology,Inc.
,Yokohama,Japan):GCcon-tent,GCscanning,GCskew,cumulativeGCskew,andcodonusage.
Anunrootedphylogenictreewasconstructedbytheneighbor-joiningmethod,usingDNASISPro(HitachiSoftwareEngineeringCo.
,Ltd.
,Tokyo,Japan).
Bootstrapanalysiswasperformedbyresamplingthedatasets10,000times.
Bootstrapvaluesofgreaterthan95%wereconsideredtobestatisticallysignif-icantforthegrouping.
Thegeneorderwascomparedwiththemostrelatedphagegenome;thiscomparisonwasmanuallyperformedbyin-houseBLASTsearch,usinginsilicomolecularcloninggenomicsedition(InSilicoBiology,Inc.
).
AnEvalueoflessthan0.
1wasconsideredasindicativeofhomology.
ComparativedataforthefollowingwereretrievedfromtheGenBankdata-base:bacteriophageG1(accessionnumberAY954969),StaphylococcusphageK(accessionnumberAY176327),StaphylococcusphageTwort(accessionnumberAY954970),ListeriabacteriophageP100(accessionnumberDQ004855),Lacto-bacillusplantarumbacteriophageLP65(accessionnumberAY682195),andE.
faecalisV583(accessionnumberAE016830.
1).
Phagepreparationforanimalexperiments.
Forthepurposeofamouserescueexperiment,thepuriedphagesamplewascontinuouslydialyzedagainstSMC(salinewith20mMMgCl2and20mMCaCl2)(4°C,30min)andHIMC(4°C,30min).
Ontheotherhand,thepuriedphagesamplewasdialyzedagainstSMC(4°C,60min)fortherepeatedadministration.
Thetiters(PFU/ml)ofthephagewerethendeterminedbyinoculationintobacterialstrainEF24.
Thephagewasstoredat4°Cuntiluse.
Animalexperiments.
Allanimalexperimentswereconductedwiththeap-provaloftheAnimalExperimentCommitteeofKochiMedicalSchool.
Female6-to8-week-oldBALB/cmice(weighingupto18g)wereusedinthefollowingexperiments.
E.
faecaliscells(eitherstrainEF14orstrainVRE2)weregrownin300mlofTSBmediumat37°Cuntiltheearlystationaryphase(uptoca.
200Klettunits)andwerethencentrifuged(10,000g,4°C,10min).
Thecellpelletwaswashedwith300mlofsaline,centrifuged(10,000g,4°C,10min),andnallyresus-pendedin3mlofsaline.
Afterappropriatedilutionusingsaline,thebacterialconcentration(bacteria/ml)wasdeterminedbyturbidity(inKlettunits),mea-suredwithaKlett-Summersonphotoelectriccolorimeter(KlettMfg.
Co.
,NY).
EF14waspreparedtobeat1.
01010,2.
01010,5.
01010,1.
01011,2.
01011,and5.
01011bacteria/ml.
VRE2waspreparedtobeat5.
01010,1.
01010,2.
11010,5.
01010,1.
01011,and2.
11011bacteria/ml.
Saline(0.
2ml;control)orbacterialsuspensionatdifferentconcentrationswasinjectedintotheperitonealcavitiesof5micethroughtheleftsideoftheabdomen(intotal,70micewereused).
Thesurvivalratesforandactivitiesofalltestedanimalswereobservedfor7days.
Theminimumbacterialconcentrationshowing100%lethal-itywasdeterminedastheminimumlethalbacterialdosage.
Forthemouserescueexperimentbyphage,thephagewasdilutedinHIMCtothefollowingconcentrations(expressedinmultiplicitiesofinfection[MOI]):100,10,1,0.
1,0.
01,0.
001,and0.
0001.
Micewereinoculatedontheleftsideoftheabdomenwith0.
2mloftheminimumlethalbacterialdose(intotal,70mice).
About20minaftertheinoculationofthelethalbacterialdose,0.
2mlofphagesolutionatdifferentconcentrations(inMOI),HIMC,orsalinewasadministeredtovemiceontherightsideoftheabdomen.
Thedataonthesurvivalratesofthemicewereanalyzedstatisticallywithatwo-tailedFisher'sexacttest.
More-over,tomeasuretheintrinsiceffectsofphagealoneandbuffers,0.
5-mlaliquotsofHIMC,saline,orphagesuspension(total,1.
01012PFU)alonewasadmin-isteredintotheabdominalcavitiesof5mice(atotalof15mice).
Thesurvivalratesforandactivitiesofalltestedanimalswereobservedfor7days.
Toexaminetheeffectsofrepeatedphageexposure,0.
5mlofanSMCphagesuspension(intotal,3.
51010PFU)orSMCalonewasintraperitoneallyadministeredseventimesat4-dayintervalsintotheabdominalcavitiesof10mice(atotalof20mice).
Themousesurvivalratewasrecordedfor2monthsfromtheinitialphageadministration.
Nucleotidesequenceaccessionnumber.
ThegenomedataofphageEF24CwasdepositedtoGenBank(accessionnumberAP009390).
RESULTSANDDISCUSSIONGeneralgenomedescription.
TheEF24Cgenomewasde-terminedtobe142,072bp.
Also,thegenomesequencewascircularlypermutedbecausenodeniteterminalendswereidentiedbygenomicsequencing.
TheGCcontentwas35.
7%.
4150UCHIYAMAETAL.
APPL.
ENVIRON.
MICROBIOL.
onMarch11,2021byguesthttp://aem.
asm.
org/DownloadedfromTwohundredtwenty-oneORFsandvetRNAgenesweredetermined.
WhenwecheckedforthepresenceofanRBSupstreamfromeachORF,mostORFsseemedtohaveatyp-icalRBS(seethesupplementalmaterial).
Inthefollowingtext,ORFsconsideredtohaveafunctionalputativeproductweredenedasputativegenesandaredesignatedusingan"orf"prex.
Theputativegene(i.
e.
,orf)productislikewiseshownusing"Orf.
"Bioinformaticanalysisrevealedthat45.
2%(100/221)oftheputativeORFproductswereassumedtohaveeitherproteinfunctionaldomainsorsimilaritytootherphagegeneproductsorboth.
Overall,20.
8%(46/221)oftheORFswerededucedtoencodefunctionalproteins.
Nogenescodingforsite-specicintegrase,toxinandantibioticresistancegenes,orotherpatho-genicfactorswerepredicted.
ABLASTPsearchonthehypo-theticalORF-encodedproteinsshowedsimilaritieswiththegeneproductsoftheotherlargevirulentphages,includingStaphylococcusphageK,G1,Twort,LactobacillusphageLP65,andListeriaphageP100.
Thegenomesofthesephagesarewellcharacterized(8,10,32,39).
ThegenomemapofEF24CisshowninFig.
1.
TheannotationofthegenomeisalsoshowninTable1.
TheN-terminalaminoacidsequencesoftheproductsoftheorf16,orf23,orf24,orf40,orf69,andorf219genespreciselycor-respondedtothoseofsixvirionproteins(42,62,16,26,21,and12.
6kDa,respectively)ofEF24Casreportedinourpreviouspaper(47).
OnlyOrf16andOrf23werebioinformaticallyspec-iedasmajorcapsidprotein(MCP)andtailsheathprotein,respectively;theotherswereunknown.
ExceptforthatofOrf219,theN-terminalendswereconsideredtobeprocessed.
Duringmorphogenesis,theN-terminal20aminoacidresiduesoftheMCP(Orf16)wereconsideredtoberemoved,whichwasassumedtobemediatedbytheputativeproheadprotease(Orf14).
Theotherstructuralproteins(Orf23,Orf24,Orf40,andOrf69)wereassumedtobesimilarlydigestedbetweenmethionineandalanine.
Ingeneral,functionallyrelevantgenesareclusteredasamoduleinphage(6,11).
Threemoduleswerespeculatedtorangefrom1bpto71kbpinEF24C,accordingtogenomeannotationandstructuralproteinidentication(Fig.
1).
Thelargestructuralmoduleseemedtobeassociatedwithheadandtailcomponents(1bpto46kbp).
Thereplication-associatedgeneswereclusteredasaDNAreplicationmodule(46kbpto68kbp).
Thesmallstructuralmoduleincludedatleastorf68andorf69(69kbpto71kbp)duetoanimmunoglobulin(Ig)-likedomainspeciedonOrf68(anIg-likedomainistypicallyfoundinavirionproteinofphage)andtheidenticationofOrf69asastructuralproteinfromtheproteomicanalysis(16,17).
Inthefunctionallyuncategorizedregion,someputativegenesassociatedwithdenovosynthesisofnucleicacidprecur-sorswerespeculated.
TheseincludedthegenesforOrf103FIG.
1.
GenomemapofphageEF24C.
ArrowsindicateputativeORFsandtRNAgenes,alongwiththeirorientations.
Functionallyassignedgenesaredifferentlycolored(blue,structuralgene;red,lysisgene;green,DNA-associatedgene;violet,tRNAgene).
Speculatedmodulesareenclosedbyboxes(black,structuralmodule;pink,DNAreplicationmodule).
,geneforstructuralprotein.
VOL.
74,2008THERAPEUTICPHAGEAGAINSTE.
FAECALISINFECTIONS4151onMarch11,2021byguesthttp://aem.
asm.
org/DownloadedfromTABLE1.
FeaturesofphageEF24CgeneproductsandtheirfunctionalassignmentsORFPositionStrandaLength(nt)bSize(aa)cMolmass(kDa)dpIePutativefunctionalassignmentNCBIdatabasesearchresultProteindomainsearchresultPredictedTMHfandsignalpeptideStartEndSimilarities/homologiestogeneproductsofphagesandbacteriaToolBitsEvaluePredicteddomainToolEvalueTMHHMgSignalPh11143832708910.
19.
8224046822799210.
39.
9ORF133(StaphylococcusphageTwort)BLASTP64.
39.
00E-113692111442314015.
94.
4gp3(ListeriabacteriophageP100)BLASTP73.
23.
00E-1241114148236912213.
89.
6LargeterminaseLargeterminase(Bacillussubtilisphage1102phil-3)BLASTP1602.
00E-385176732721,50650156.
76.
3Largeterminasegp5(ListeriabacteriophageP100)BLASTP6338.
00E-180Phageterminaselargesubunit(GpA)RPSBLAST4.
00E-0863372416078926229.
67.
6gp6(ListeriabacteriophageP100)BLASTP1114.
00E-2374265498171723827.
54.
7gp8(ListeriabacteriophageP100)BLASTP1021.
00E-2084971531534511412.
86.
6gp9(ListeriabacteriophageP100)BLASTP350.
9295409627887028931.
47.
0Endolysin-associatedproteinN-Acetylmuramyl-L-alanineamidase,negativeregulatorofAmpC,AmpD(RubrobacterxylanophilusDSM9941)BLASTP70.
59.
00E-11N-Acetylmuramoyl-L-alanineamidase,family2RPSBLAST4.
00E-12106445708363921223.
14.
4Endolysin-associatedproteinLysMdomainprotein(EnterococcusfaecalisV583)BLASTP1591.
00E-37LysMdomainRPSBLAST6.
00E-09Y117227757134511413.
55.
9gp13(ListeriabacteriophageP100)BLASTP76.
63.
00E-1312758693101,72557464.
95.
6Portalproteingp14(ListeriaBLASTP6440PhageportalproteinRPSBLAST6.
00E-08bacteriophageP100)139344943087283.
19.
31494171020879226329.
65.
2Proheadproteasegp15(ListeriabacteriophageP100)BLASTP2812.
00E-74PeptidaseU35HMMPfam8.
10E-0615102151114192730834.
84.
3IgA-specicmetalloendopeptidase(EC3.
4.
24.
13)type1precursor-Haemophilusinuenzae(strainHK613)BLASTP47.
87.
00E-041611282126761,39546451.
25.
2MCPCps(ListeriabacteriophageP100)BLASTP674011712763130442829310.
36.
7gp18(ListeriabacteriophageP100)BLASTP29.
33.
418130571395690029934.
04.
9gp19(ListeriabacteriophageP100)BLASTP3797.
00E-10419139761484587028932.
65.
9gp20(ListeriabacteriophageP100)BLASTP2645.
00E-6920148381546162420723.
710.
5gp21(ListeriabacteriophageP100)BLASTP1835.
00E-4521154651631084628131.
84.
6ORF6(ListeriabacteriophageA511)BLASTP2391.
00E-61221631016543234779.
09.
3ORF185(StaphylococcusphageTwort)BLASTP66.
63.
00E-102316547182561,71056962.
04.
9TailsheathproteinTsh(ListeriabacteriophageP100)BLASTP69704152UCHIYAMAETAL.
APPL.
ENVIRON.
MICROBIOL.
onMarch11,2021byguesthttp://aem.
asm.
org/Downloadedfrom24183171873942314015.
55.
4StructuralproteinORF8(ListeriaBLASTP2054.
00E-52bacteriophageA511)25188631969983727832.
19.
8261971319859147485.
85.
027199952046847415718.
34.
9gp26(ListeriabacteriophageP100)BLASTP1286.
00E-2928205362111157619122.
44.
4RNApolymarasegp27(ListeriabacteriophageP100)BLASTP90.
93.
00E-17MitochondrialDNA-directedRNApolymeraseRPSBLAST0.
0062921156248003,6451214129.
48.
3Taillysingp28(ListeriabacteriophageP100)BLASTP4002.
00E-109SLTdomainproteinsRPSBLAST7.
00E-093024839280243,1861061118.
15.
2Taillysingp29(ListeriabacteriophageP100)BLASTP5484.
00E-154NlpC/P60familyRPSBLAST0.
000002PeptidasefamilyM23/M37RPSBLAST0.
00023128108335855,4781825203.
04.
7Tailbergp30(ListeriabacteriophageP100)BLASTP2773.
00E-73Galactose-bindingdomain-likeSuperFamily0.
00021LuxS/MPP-likemetallohydrolaseSuperFamily0.
0123233685361172,43381089.
49.
0MinorstructuralProphageLambdaSal,BLASTP70.
15.
00E-10LipocalinProleScanproteinminorstructuralprotein,putative(Streptococcusagalactiae2603V/R)33361113683672624127.
94.
4343686837014147485.
74.
935371513784069022925.
59.
1gp31(ListeriabacteriophageP100)BLASTP2203.
00E-5636378443838053717820.
24.
6gp32(ListeriabacteriophageP100)BLASTP1463.
00E-3437383673907170523426.
34.
6Baseplategp33(ListeriabacteriophageP100)BLASTP2434.
00E-633839087401391,05335039.
65.
2Structuralproteingp34(ListeriabacteriophageP100)BLASTP4091.
00E-112UncharacterizedhomologofRPSBLAST0.
000033940158415581,40146653.
14.
7gp35(ListeriabacteriophageP100)BLASTP3223.
00E-86phageMuproteingp4740416644220954618120.
35.
9Structuralproteingp36(ListeriabacteriophageP100)BLASTP1842.
00E-454142224456883,4651154128.
84.
9Adsorption-associatedtailproteingp37(ListeriabacteriophageP100)BLASTP14640Sialidases(neuraminidases)SuperFamily0.
0037424576545971207687.
85.
7Putativetransposase(WolinellasuccinogenesDSM1740)BLASTP32.
75.
44346226479981,77359067.
26.
4Helicasegp42(ListeriabacteriophageP100)BLASTP7060HelicasesuperfamilyC-terminaldomainRPSBLAST2.
00E-114448026496541,62954262.
98.
4Transcriptionalregulatorgp43(ListeriabacteriophageP100)BLASTP3117.
00E-83PredictedtranscriptionalregulatorRPSBLAST0.
0024549687511591,47349055.
44.
8Helicasegp44(ListeriabacteriophageP100)BLASTP4231.
00E-116DnaBhelicaseCterminaldomainRPSBLAST4.
00E-134651159522141,05635139.
85.
5Exonucleasegp45(ListeriabacteriophageP100)BLASTP2591.
00E-67SbcD,DNArepairexonucleaseRPSBLAST2.
00E-164752330542221,89363071.
25.
2ExonucleasePutativeexonuclease(StaphylococcusphageK)BLASTP3746.
00E-102SbcCDandotherMre11/Rad50(MR)complexesRPSBLAST6.
00E-0948542315489666622125.
94.
9ORF065(StaphylococcusphageTwort)BLASTP49.
71.
00E-044954897559551,05935240.
47.
1Primasegp49(ListeriaBLASTP3361.
00E-90DnaG,DNAprimase(bacterialtype)RPSBLAST3.
00E-13bacteriophageP100)50559725660463321024.
25.
151566305751488529432.
76.
0gp50(ListeriabacteriophageP100)BLASTP1507.
00E-35ContinuedonfollowingpageVOL.
74,2008THERAPEUTICPHAGEAGAINSTE.
FAECALISINFECTIONS4153onMarch11,2021byguesthttp://aem.
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org/DownloadedfromTABLE1—ContinuedORFPositionStrandaLength(nt)bSize(aa)cMolmass(kDa)dpIePutativefunctionalassignmentNCBIdatabasesearchresultProteindomainsearchresultPredictedTMHfandsignalpeptideStartEndSimilarities/homologiestogeneproductsofphagesandbacteriaToolBitsEvaluePredicteddomainToolEvalueTMHHMgSignalPh525751757747231769.
09.
0dUTPasegp51(ListeriaBLASTP32.
36.
5dUTPaseRPSBLAST0.
0008bacteriophageP100)53577495805730910212.
18.
654580445835531210312.
24.
6Phosphotransferase/aniontransportproteinPhosphotransferase/aniontransportproteinSuperFamily0.
01455583485871937212314.
36.
2gp53(ListeriabacteriophageP100)BLASTP46.
24.
00E-0456587395940766922225.
75.
0ResolvaseHypotheticalproteinBLASTP1728.
00E-42HollidayjunctionRPSBLAST0.
001KgORF78(Staphylo-coccusphageK)resolvase,archaealtype57594095971430610111.
35.
458597156019448015918.
56.
3Hypotheticalproteinphil2p15(Staphylococcusaureusphagephi12)BLASTP73.
63.
00E-1259602876108179526431.
57.
0gp66(ListeriabacteriophageP100)BLASTP2404.
00E-6260610746138531210311.
99.
7IntegrationhostfactorPutativeintegrationhostfactor(StaphylococcusphageK)BLASTP727.
00E-12Integrationhostfactor(IHF)andHURPSBLAST0.
000261614756454630721023119.
06.
1DNApolymerasePutativeDNApoly-merase(Staphylo-coccusphageK)BLASTP7910DNApolymerasefamilyARPSBLAST7.
00E-6762646496519154318021.
55.
2gp70(ListeriabacteriophageP100)BLASTP1241.
00E-276365246665351,29042948.
24.
9gp71(ListeriabacteriophageP100)BLASTP1256.
00E-276466620678671,24841546.
25.
7RecAgp72(ListeriabacteriophageP100)BLASTP4345.
00E-120RecARPSBLAST7.
00E-3765679216830738712814.
78.
6gp73(ListeriabacteriophageP100)BLASTP72.
46.
00E-1266683006891461520423.
76.
2Sigmafactorgp74(ListeriabacteriophageP100)BLASTP1734.
00E-426768975692502769110.
26.
2HolinHolin-likeproteinsimilartoORFofbacteriophageBK5-T(BidobacteriumlongumNCC2705)BLASTP43.
50.
002HolinRPSBLAST0.
0003268692987026096332035.
24.
5Ig-likeproteinIg-like,group2(FlavobacteriumjohnsoniaeUW101)BLASTP891.
00E-17BacterialIg-likedomain(group2)RPSBLAST3.
00E-0769702817072444414716.
64.
5Structuralproteingp77(ListeriaBLASTP1133.
00E-24bacteriophageP100)70708307113530610112.
15.
271711327208595431735.
95.
7gp79(ListeriabacteriophageP100)BLASTP1384.
00E-317272139734221,28442748.
85.
7gp80(ListeriabacteriophageP100)BLASTP4346.
00E-120Metallophospho-esteraseHMMPfam0.
0009673734347380837512414.
29.
6gp81(ListeriabacteriophageP100)BLASTP43.
50.
003374738477446762120623.
25.
7gp82(ListeriabacteriophageP100)BLASTP1134.
00E-2475744677520774124628.
39.
3gp84(ListeriabacteriophageP100)BLASTP2873.
00E-764154UCHIYAMAETAL.
APPL.
ENVIRON.
MICROBIOL.
onMarch11,2021byguesthttp://aem.
asm.
org/Downloadedfrom76751977570350716819.
110.
3gp85(ListeriaBLASTP942.
00E-18bacteriophageP100)77757177606735111612.
63.
878760927694985828531.
54.
9ORF036(bacteriophageBLASTP55.
82.
00E-06G1)79770547789984628132.
44.
8NucleotidyltransferaseSuperFamily0.
00168077892795201,62954262.
77.
0ORF137(Lactobacillusplantarumbacterio-phageLP65)BLASTP75.
56.
00E-12YC53_LISIN_Q925W4BlastProDom7.
00E-1381798398052869022926.
34.
9gp96(Listeriabacterio-phageP100)BLASTP2035.
00E-5182805398100646815518.
24.
8gp97(Listeriabacterio-phageP100)BLASTP90.
92.
00E-17838110683385228075986.
54.
6HypotheticalproteinBLASTP71.
61.
00E-10KgORF105(Staph-ylococcusphageK)848344583651207687.
56.
52Y85836708417650716818.
65.
32868424984353105343.
93.
4878435584525171566.
74.
58884515847872739010.
34.
4898489585146252839.
69.
2TranscriptionalDNA-bindingproteinBLASTP46.
63.
00E-04Helix-turn-helixXRE-RPSBLAST4.
00E-06regulator(Pseudomonassyringaepv.
phaseolicolafamily-likeproteins1448A)9085159854462889511.
44.
591854498623778926229.
96.
79286320873931,07435738.
34.
3LPXTG_anchorHMMTigr0.
0047193875198782130310011.
510.
394878238812530310011.
74.
09588128884212949710.
69.
52968841888597180596.
84.
197886108896335411713.
74.
198889908936737812514.
38.
92998936089590231768.
85.
22100895948996837512414.
24.
71018996590192228758.
84.
71029021090398189627.
34.
9103903959099460019923.
05.
0CytidinedeaminaseCytidinedeaminaseRPSBLAST0.
0006104910369187884328032.
24.
8105918919252063020923.
95.
01069259892840243809.
64.
81079285892998141465.
39.
01089301193205195647.
65.
2109932219386264221324.
25.
3110938749431744414716.
65.
2111944549490044714817.
04.
211295256955553009911.
28.
411395633959232919611.
85.
3114960069632932410712.
34.
91159642696566141465.
510.
111696625969182949711.
56.
91179702597231207687.
69.
221189731397555243809.
44.
21199763297877246819.
47.
8ORF160(LactobacillusBLASTP64.
31.
00E-09plantarumbacterio-phageLP65)1209794498180237789.
23.
81219823198437207687.
96.
31229845998626168556.
810.
1123986239871290293.
39.
8112498770990662979811.
38.
91259913299362231769.
04.
9126994819991243214316.
74.
3ContinuedonfollowingpageVOL.
74,2008THERAPEUTICPHAGEAGAINSTE.
FAECALISINFECTIONS4155onMarch11,2021byguesthttp://aem.
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org/DownloadedfromTABLE1—ContinuedORFPositionStrandaLength(nt)bSize(aa)cMolmass(kDa)dpIePutativefunctionalassignmentNCBIdatabasesearchresultProteindomainsearchresultPredictedTMHfandsignalpeptideStartEndSimilarities/homologiestogeneproductsofphagesandbacteriaToolBitsEvaluePredicteddomainToolEvalueTMHHMgSignalPh1279999610034635111612.
85.
412810037810080342614116.
43.
9129100797101009213708.
37.
9130101038101229192637.
44.
613110124210160136011914.
010.
0132101606101707102333.
88.
111331026211028752558410.
04.
2134102964103230267889.
84.
8135103328103495168556.
18.
7136103580103756177586.
74.
4137103838104011174576.
74.
3138104087104311225748.
44.
113910441410472230910211.
84.
914010479010510131210311.
89.
2141105193105351159525.
94.
9142105485105724240799.
04.
2143105805106050246819.
15.
714410617110686669623127.
04.
0MyosinheavychainRPSBLAST0.
006145106973107221249829.
84.
6146107292107486195647.
44.
9147107512107691180596.
99.
6148108021108281261869.
88.
7149108278108550273909.
95.
4215010855510889634211313.
05.
215110892910924031210311.
35.
03152109233109496264879.
94.
7215310949710980230610111.
96.
5154110021110212192637.
06.
5155110256110480225748.
33.
815611049411089840513415.
45.
0157111288111485198657.
54.
615811148511200051617119.
94.
9Proteingp51(ListeriaBLASTP58.
22.
00E-07DUF1642HMMPfam6.
10E-09monocytogenesstrain4bH7858)159112013112261249829.
35.
116011227411279852517420.
45.
3HypotheticalproteinEfaeDRAFT_2183(EnterococcusfaeciumDO)BLASTP729.
00E-12DUF1642HMMPfam2.
70E-3016111279911319139313015.
75.
0HypotheticalproteinEF1441(EnterococcusfaecalisV583)BLASTP1094.
00E-2316211318411354336011914.
04.
5HypotheticalproteinEF1441(EnterococcusfaecalisV583)BLASTP42.
70.
005163113547113717171566.
43.
8HypotheticalproteinSdys1_01002815(Shigelladysenteriae1012)BLASTP42.
70.
00516411370511424754318020.
86.
4HypotheticalproteinEF2118(EnterococcusfaecalisV583)BLASTP97.
82.
00E-1916511425011471446515418.
28.
7HypotheticalproteinBLASTP65.
56.
00E-10EF0326(EnterococcusfaecalisV583)4156UCHIYAMAETAL.
APPL.
ENVIRON.
MICROBIOL.
onMarch11,2021byguesthttp://aem.
asm.
org/Downloadedfrom166114711114815105344.
14.
316711492811529336612113.
95.
5168115290115469180596.
83.
416911548311600151917220.
09.
3gp158(ListeriabacteriophageP100)BLASTP1172.
00E26ConservedhypotheticalproteinCHP02464TIGRFAM3.
40E58COG3236RPSBLAST2.
00E2617011600211647547415718.
16.
417111647211717970823526.
25.
317211720811764844114616.
95.
0173117698117886189627.
65.
117411788311834446215316.
93.
9ABCtransporterProleScan175118358118534177586.
73.
8176118531118728198657.
84.
817711872511916243814517.
59.
7117811925511999574124628.
04.
9Serinethreonine-proteinphosphatasegp135(ListeriabacteriophageP100)BLASTP1482.
00E-34Serum/threonine-specicproteinphosphataseandbis(5-nucleosyl)-tetraphosphataseFPrintScan1.
10E0517911999212047148015918.
19.
441801204751207262528310.
39.
818112072312126554318020.
88.
0PhosphoesterasePhosphoesterase(putative)(LactobacillusreuteriJCM1112)BLASTP1203.
00E26Phosphoesterase/phosphohydrolaseRPSBLAST4.
00E1418212134512169535111613.
76.
42183121692121808117384.
23.
8184121822122034213708.
04.
518512203412211481262.
85.
6118612211512252841413716.
04.
918712253312291638412714.
64.
518812298512387589129634.
45.
1189123877124065189627.
55.
6190124156124329174576.
69.
61Y19112431912466334511413.
45.
0192124663124917255849.
64.
81931248981251522558410.
05.
119412516312549833611112.
94.
519512547312591043814517.
25.
319612592612638746215317.
54.
9PutativeTNP-liketransposableelement[Oryzasativa(japonicacultivar-group)]BLASTP36.
20.
45197126380126634255849.
58.
9198126635126853219728.
24.
519912684112727243214316.
74.
5200127275127505231768.
73.
920112750212793343214316.
54.
8202127937128089153505.
79.
820312808912903694831536.
05.
7ThymidylatesynthaseThymidylatesynthase(Bacillusmojavensis)BLASTP1991.
00E49ThymidylatesynthaseRPSBLAST3.
00E5020412900612909896313.
610.
320512908912947538712814.
99.
0HypotheticalproteinF116p23(PseudomonasaeruginosaphageF116)BLASTP83.
23.
00E15206129566129715150495.
78.
01Y20712971513057285828531.
18.
4AntiproliferativeproteinPhage-likeprotein(BacilluslicheniformisATCC14580)BLASTP1774.
00E43Band7domainofotillin(reggie)-likeproteinRPSBLAST1.
00E131Y20813073313171698432737.
34.
6RibonucleotidereductaseUnknown(bacteriophageSPBc2)BLASTP2538.
00E66RibonucleotidereductaseR2/betasubunit(RNRR2)RPSBLAST2.
00E63ContinuedonfollowingpageVOL.
74,2008THERAPEUTICPHAGEAGAINSTE.
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75.
1RibonucleotidereductaseRibonucleotide-diphosphatereductasealphasubunit(LactobacillusplantarumWCFS1)BLASTP9260Class1ribonucleotidereductase(RNR)RPSBLAST4.
00E119210133882134124243809.
05.
7RibonucleotidereductaseRibonucleotidereductase,NrdH-redoxin(Lactobacillussakeisubsp.
sakei23K)BLASTP68.
69.
00E11NrdH-redoxin(NrdH)familyRPSBLAST5.
00E122111342511345172678810.
04.
921213452313482830610111.
99.
0213134919135149231768.
99.
1TranscriptionalregulatorORF187(bacteriophageG1)BLASTP59.
35.
00E08Helix-helixXRE-family-likeproteinsRPSBLAST2.
00E08214135213135437225748.
49.
8gp166(ListeriabacteriophageP100)BLASTP32.
74.
9215135519136610109236341.
75.
0AAAproteinfamilyHypotheticalproteinRBTH_07188(Bacillusthuringiensisserovarisraelensis)BLASTP3482.
00E94ATPasefamilyassociatedwithvariouscellularactivities(AAA)RPSBLAST1.
00E07tRNA-Met13698113705474AnticodonCAU2161373701376362678810.
04.
8tRNA-Leu13767713776185AnticodonUAGtRNA-Arg13848313855674AnticodonUCU21713869813876972232.
69.
7tRNA-Trp1391571392287223AnticodonCCAtRNA-Asp13947813955376AnticodonGUC218140224140358135445.
08.
921914102814136633911212.
64.
8Structuralproteingp171(ListeriabacteriophageP100)BLASTP37.
70.
14STATSUPERFAMILY0.
007222014143014177434511413.
34.
9gp172(ListeriabacteriophageP100)BLASTP92.
46.
00E181221141809142069261869.
97.
73aOrientationsoftheORFsintheEF24Cgenome.
and,rightwardandleftwardorientations,respectively,ofORFsinFig.
2.
bSizesofORFs,includingstopcodons,innucleotides(nt).
cSizesofORFproducts,inaminoacids(aa).
dPredictedmolecularmassesofORFproducts.
ePredictedpI.
fTMH,transmembranehelix.
gTMHHM,thepredictiontooloftransmembranehelicesinproteins;thenumbersoftransmembranehelicesareshowninthecolumn.
hSignalP,thepredictiontoolforsignalpeptideinproteins.
"Y"indicatesthepresenceofasignalpeptide.
4158UCHIYAMAETAL.
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MICROBIOL.
onMarch11,2021byguesthttp://aem.
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LikeT4phage,EF24Cmayproduceitsownmod-iedbasefromhostDNAbreakdown(22,37).
Atthenalstageofthelatentperiod,progenyphageswithinabacterialcellwerereleasedbythedegradationofthecellwall(25,27).
Thiscellwalllysisistypicallyinducedbytwophage-encodedproteinscalledholinandendolysin(25,27).
Atthelateperiodofinfection,holinformsaholeinthecellmem-brane,andendolysinpassesthroughtheholeanddestroysthepeptidoglycanstructure(25,27).
InthephageEF24Cge-nome,theputativegenesforendolysin(Orf9andOrf10)andholin(Orf67)arethoughttobedistantlypositioned,asoccursinphageT4(37).
orf9andorf10arethoughttobelocatedinonestructuralmodule,andorf67isthoughttobelocatedinverycloseproximitytoanotherstructuralmodule.
Thus,thesegenesarepossiblyexpressedlateintheperiodofinfection(37).
Consequently,Orf9,Orf10,andOrf67mayfunctionasaholin-endolysinsystem.
PhylogeneticanalysesofEF24CwithintheSPO1-likephages.
TheEF24Cgenomecontainsapproximately142kbp,theGCcontentofwhichis35.
7%,asdescribedabove.
Thegenomeiscircularlypermuted,andDNApolymeraseA(Orf61)hasbeententativelyidentied.
Theseattributes,to-getherwiththemorphologicalandbiologicalfeaturesofEF24C,suggestthatEF24CisamemberoftheSPO1-likephagegenus(28).
Therefore,thephylogenicrelationshipsofEF24CtotheotherSPO1-likephageswereexamined.
AphylogenictreebasedontheMCPisfrequentlyusedinphagephylogenicanalysis(1,26).
TheMCPsofthefollowingphageswereobtainedfromtheirgenomesequences:Staphy-lococcusphageK,G1,Twort,LactobacillusphageLP65,andListeriaphageP100.
TheMCP-basedphylogenicanalysisshowedthatEF24CismostcloselyrelatedtoListeriaphageP100amongthesephages(Fig.
2A).
Next,thegeneorganiza-tionoftheEF24CgenomewasalsocomparedwiththatofListeriaphageP100.
Genomesyntenywasobserved,particu-larlyonthepredictedstructuralandDNAreplicationmodulesinEF24C(ca.
70%ofthegenome)(Fig.
2B),whereasthegenesontheotherregionofEF24C(theremainingca.
30%ofthegenome)arenotonlyfunctionallyunknownbutalsodissimilartoanygenesofListeriaphageP100.
ByconsideringthedifferenceinbacterialspeciesandphylogenicrelationbasedonMCP,EF24CandListeriaphageP100wereconsid-eredtohaveevolveddivergentlyfromthesamevirusorigin(44).
Noncompetitivenaturebetweenreplicationandtranscrip-tiondirections.
Theoriginofreplication(ori)andreplicationterminus(ter)canbededucedbyGCskewandcumulativeGCskewanalyses(23,24,45).
ThecumulativeGCskewanalysisforFig.
3showsthepredictedoriandter,whicharethelowest(ca.
1-bp)andhighest(108-kbp)regions,respectively.
Al-thoughtheprocessofEF24Cgenomereplicationremainsunknown,itseemstoreplicateinamannerslightlydifferentfromthatofanotherlargephage,T4ofE.
coli.
TheEF24Cgenomeseemstohaveonlyoneori,whereasphageT4hasmultipleoris(37).
Moreover,thespeculatedreplicationdirec-tion(oritoter)matchedwiththedirectionoftranscription(directionofgenes).
Accordingtothismutualcorrespondence,bimolecularcollisionsduringreplicationandtranscriptioncanbeavoided(13,18).
Hence,EF24Cisassumedtomultiplywithoutsuchmutualinterferencebetweenreplicationandtranscription.
Host-adaptedtranslation.
HostandphagecodonusagecomparisonsandphagetRNAanalysisimpliedefcienttrans-lation.
ThecodonusagesofEF24CandE.
faecalisV583wereFIG.
2.
PhylogeneticanalysesofEF24C.
(A)PhylogenetictreebasedonMCPs.
(B)ComparisonofthegeneorderbetweenEnterococcusphageEF24CandListeriaphageP100.
TheORFofEF24CisconnectedtothatofP100byablacklinewheretheEvaluefromthein-houseBLASTsearchislessthan0.
1.
VOL.
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4A),whichcanindicateoverallefcienttranslationinthehost.
Moreover,thecodonusageofEF24CexceededthatofthehostonvepredictedtRNAswhosegeneswerecarriedbythephage(Fig.
4B).
Thus,wecaninferthatEF24CsuppliesspecictRNAsonitsownincaseoftRNAdeciency.
E.
faecalissepsismousemodel.
Sepsismousemodelsaretypicallyusedforthepreliminaryassessmentofphagetherapyagainstnosocomialbacteria(4,35,48–50).
Toexaminethera-peuticeffectivenessandtheeffectofhostsensitivitydifferenceonthephageinvivo,E.
faecalissepsismousemodelsusingBALB/cmiceweresetupusingtwostrains,EF14andVRE2.
Inthepreviousstudy,EF14hadphagesensitivityabout32timesgreaterthanthatofVRE2(efciencyofplating[EOP]againstEF14,1;EOPagainstVRE2,0.
032)(47).
Afterintra-peritonealbacterialinoculationatdifferentconcentrationstomice,theminimumlethalbacterialdosagesofEF14andVRE2weredeterminedtobe1.
01010and4.
2109bac-teria,respectively;thesedosagesresultedin100%lethalitywithin2days.
Thesebacterialdosageswereusedforthefol-lowingexperimentstoassessmouserescuebyphage.
Inbothcases,inoculationoftheminimumlethalbacterialdosagesseemedtoinduceseveresepsiscomplications.
Astimeelapsedafterbacterialinoculation,anincreaseinthenumberofbacteriainthebloodwasalsoobserved(bacteremia).
Inaddition,anincreasingfrequencyofunusualchangesinthemicewasobserved,suchasdecreaseinactivity,lowbodytem-perature,shivering,bloodclotting,andhyperventilation.
Theseabnormalconditionswereconsideredtobetypicalfeaturesofseveresepsis,althoughhistologicalanalyseswerenotper-formed(21,41).
EF24Ctherapeuticeffectivenessinvivo.
Noabnormalmousebehaviororalteredsurvivalratewasobservedfollowingtheadministrationofsaline,HIMC,orphagealone(1.
01012PFU).
Thus,thephagerescueexperimentswereconsideredtobeconductedwithoutbias.
TheinvivotherapeuticeffectivenessofEF24Cwasthenexamined.
Aftertheinoculationoftheminimumlethalbacte-rialdosage,HIMCorEF24CatdifferentMOIof10,1,0.
1,0.
01,0.
001,and0.
0001wasadministered.
Figure5showstheresultsofmouserescueexperimentsusingEF24C.
Thedose-dependenteffectivenesswasobservedtobelessthanMOIof0.
01and0.
1intheEF14andVRE2mousesepsismodels,respectively.
Comparedwiththecontrol(HIMCorsalinetreatment),theEF24CtreatmentwassignicantlyeffectiveforbothEF14-infectedmiceatMOIof10,1,0.
1,and0.
01(P0.
01)andVRE2-infectedmiceatMOIof10,1,and0.
1(P0.
01)or0.
01(P0.
05).
Accordingtotheseresults,EF24CcanefcientlyrescuemiceinfectedwithbothEF14andVRE2atanMOIof0.
01.
Undertheseexperimentalconditions,thetherapeuticefcacyofEF24Cdidnotseemtobeaffectedbythesensitivityofthehosttothephage.
TheEF24Cburstsizeinvitrowasreportedasca.
100inapreviouswork(47).
Underinvivoconditions,thephagemaynotinfectthebacteriaorpropagateasefcientlyasitdoesunderinvitroconditions.
Therefore,theseresultssuggestthattheefcientpropagationofEF24Cledtoitssignicanttherapeuticeffectivenessinvivo.
EligibilityofEF24Casapotentialtherapeuticphage.
EF24Cisconsideredtobeeligibleasatherapeuticphageforthefollowingreasons.
First,asdemonstratedinapreviousstudy,EF24ChasbroadhostspecicityandstrongvirulenceagainstE.
faecalisstrains.
Second,undesirablegenesforphagetherapysuchasintegration-relatedandpathogenic(e.
g.
,toxinandantibioticresistance,etc.
)geneshavenotyetbeenidenti-ed.
Third,wecaninferfromthefollowingfeaturesthatthebiologicalnatureofEF24Cisappropriateforatherapeuticphage:itsdenovonucleicacidsynthesisfromhostDNAbreak-down,itsholin-endolysinsystem,itshost-adaptedtranslation,andthenoncompetitivenatureofitstranscriptionandrepli-cation.
Fourth,itsgenomicfeatures,togetherwithitsmorphol-ogy,allowEF24CtobecategorizedintheSPO1-likephagegenus,somemembersofwhich,includingphagesKandP100,areusedorareunderconsiderationfortherapyorprophylaxis.
PhylogeneticandgenomesyntenyanalysesrevealedacloserelationshipbetweenEF24CandtheListeriaphageP100,whichhasbeenapprovedforprophylacticuseandhasbeencommercialized(EBIFoodSafety[http://www.
ebifoodsafety.
com/])(8,42).
Thus,EF24Ccanbeencompassedinthegeneraltherapeuticphagegroup.
Fifth,accidentalhomologousrecombinationisalsonotlikelyintheuseofEF24C,becausesimilaritiesbetweentheEF24CgenomeandthehostE.
fae-calisV583genomewererestrictedtotRNAgenesandafewgenesforhypotheticalproteins,asfortheothertherapeuticphagesKandP100(datanotshown).
Thisanimalexperimentshowedthatasinglelow-dosageadministrationofEF24Ccaneffectivelytreatsepsisinmicewithouttheeffectofhostsensi-tivitytothephageobservedinvitro(i.
e.
,EOPdifferencebe-tweenstrainsEF14andVRE2).
Thephagebacteriolyticactionisbelievedtobetheprimarymechanismofmouserescueeffects.
However,EF24Cefcacymaybealteredindifferentmousestrainsandanimals.
Forexample,somepossiblephage-inactivatingfactors,suchasphage-neutralizingantibodyandliverorbileacids,mayalterphageefcacy,andthephagemaynotefcientlyreachthefocusofinfection.
Therefore,furtherstudyisrequired(i.
e.
,ofthepharmacokineticsandpharmaco-FIG.
3.
GC-associatedgenomeanalysis.
IntheGCscanning,thelowregioninGCisindicatedbythebar.
InthecumulativeGCskew,theoriginofreplication(blackarrow)andthechangingpointofgenedirection/terminationofDNAreplication(whitearrow)areindicated.
4160UCHIYAMAETAL.
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Safetyissuesareofgreatconcerninphagetherapy.
Surpris-ingly,nosignicantsideeffectsofphagetherapyhavebeenreportedtodateintheEast(36,43).
Inthisexperiment,phageadministrationdidnotcauseanylethalityormousebehaviorchangebothinthemouserescueexperimentandinthead-ministrationofahigh-concentrationdosageofphagealone.
InFIG.
4.
OptimizationofEF24CcodonusagetoitshostcodonusageanditspossibletRNAfunction.
(A)ComparisonofcodonusagebetweenEF24CandE.
faecalisV583.
(B)LocationoftRNAgenes(top),predictedsecondarystructuresoftRNAs(middle),andphageandhostcodonusagecomparisonsonthetRNAanticodons(bottom).
Inthebarchart,codonsforEF24CtRNAsareindicatedinboldface.
VOL.
74,2008THERAPEUTICPHAGEAGAINSTE.
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However,differentphageshavedifferentmolecularfeatures,anddifferentmousestrainshavedifferentlevelsofimmunity,sothesafetyofeachphagemustbeexaminedinthefuture(25,33,36).
Inthisstudy,EF24Cwasinvestigatedprimarilyasatherapeuticphage.
Althoughfurtherdevelop-mentofthisphageandothermethodsisstillnecessarytoaddresssomeremainingproblems,EF24CisapromisingtherapeuticphageagainstE.
faecalisinfections.
ACKNOWLEDGMENTSWethankHiromiKataoka(ClinicalLaboratoryCenters,KochiMedicalSchool)foraccesstoDNASISProandToshimitsuUchiyama(TohoUniversity,Tokyo,Japan)forhelpfulscienticadvice.
ThisstudywassupportedbyTheSpecialResearchProjectofGreenScience,KochiUniversity.
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