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Methods in Molecular Biology 1333 Jan Michiels Maarten Fauvart Editors
Bacterial Persistence Methods and Protocols M ETHODSIN M OLECULAR B IOLOGY SeriesEditor
JohnM.Walker
SchoolofLifeandMedicalSciences
UniversityofHertfordshire Hatfield,Hertfordshire,UK
Forfurthervolumes: http://www.springer.com/series/7651
BacterialPersistence MethodsandProtocols Editedby JanMichielsandMaartenFauvart
DepartmentofMicrobialandMolecularSystems, KULeuven-UniversityofLeuven, Heverlee,Belgium
Editors JanMichiels DepartmentofMicrobialandMolecularSystems
KULeuven-UniversityofLeuven
Heverlee,Belgium
MaartenFauvart
DepartmentofMicrobialandMolecularSystems
KULeuven-UniversityofLeuven Heverlee,Belgium
ISSN1064-3745ISSN1940-6029(electronic) MethodsinMolecularBiology
ISBN978-1-4939-2853-8ISBN978-1-4939-2854-5(eBook) DOI10.1007/978-1-4939-2854-5
LibraryofCongressControlNumber:2015945939
SpringerNewYorkHeidelbergDordrechtLondon # SpringerScience+BusinessMediaNewYork2016
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Preface Antibiotictreatmentoftenfailstoclearchronicinfections,evenintheabsenceofclinically detectableresistance.Thisislargelyduetotheso-calledpersistercellsthatcansurvive exposuretohighconcentrationsofbactericidalantibiotics.Itisgenerallyacceptedthat persistersareresponsiblefortherelapseofinfectionsbynotoriouspathogenssuchas Mycobacteriumtuberculosis, Staphylococcusaureus, Pseudomonasaeruginosa, Salmonella Typhimurium,and Candidaalbicans.Persisterstypicallymakeuponlyasmallpartofa cellpopulation.Theyresultfromatemporaryswitchtoastatethatisinsensitivetokillingby antibiotics.Theirrareandtransientnaturehaslonghamperedtheexperimentalstudyof persisters.Thisvolumebringstogetherthemostrespectedresearchersinthefieldofbacterial persistence.Itpresentsacomprehensivecollectionofmethodsthathavebeeninstrumental toourcurrentunderstandingofthetopicandwilllikelyremainsoforyearstocome.
Heverlee,BelgiumJanMichiels MaartenFauvart
Preface.. ..................................................................v
Contributors ...............................................................ix
PART
IINTRODUCTION 1AHistoricalPerspectiveonBacterialPersistence ..........................3 NatalieVerstraeten,WouterKnapen,MaartenFauvart, andJanMichiels
PART IIQUANTIFICATIONOF PERSISTENCE 2Persisters:MethodsforIsolationandIdentifyingContributing Factors—AReview. ...................................................17 SarahE.Rowe,BrianP.Conlon,IrisKeren,andKimLewis
3AGeneralMethodforMeasuringPersisterLevels in Escherichiacoli Cultures. ............................................29 NiiloKaldalu,ArviJo˜ers,HenriIngelman,andTanelTenson
4OptimizedMethodforMeasuringPersistencein Escherichiacoli withImprovedReproducibility ..........................................43 F.GoormaghtighandL.VanMelderen
5AMicroplate-BasedSystemasInVitroModel ofBiofilmGrowthandQuantification ...................................53 IlseVandecandelaere,HeleenVanAcker,andTomCoenye
6ProtocolforDeterminationofthePersisterSubpopulation in CandidaAlbicans Biofilms ............................................67 KatrijnDeBrucker,KaatDeCremer,BrunoP.A.Cammue, andKarinThevissen
PART IIISINGLE CELL ANALYSISOF PERSISTER CELLS 7QuantitativeMeasurementsofTypeIandTypeII PersistersUsingScanLag... ............................................75 IritLevin-ReismanandNathalieQ.Balaban
8AnalyzingPersisterPhysiologywithFluorescence-Activated CellSorting ..........................................................83 MehmetA.Orman,TheresaC.Henry,ChristinaJ.DeCoste, andMarkP.Brynildsen
9Single-CellDetectionandCollectionofPersisterBacteria inaDirectlyAccessibleFemtoliterDropletArray ..........................101 RyotaIino,ShouichiSakakihara,YoshimiMatsumoto, andKunihikoNishino
PART IVIDENTIFICATIONOF PERSISTER MUTANTSAND GENES
10AWhole-Cell-BasedHigh-ThroughputScreeningMethod toIdentifyMoleculesTargeting PseudomonasAeruginosa PersisterCells .........................................................113
VeerleLiebens,ValerieDefraine,andMaartenFauvart
11FunctionalAnalysisoftheRoleofToxin–Antitoxin(TA)Loci inBacterialPersistence .................................................121
AaronT.ButtandRichardW.Titball
12ExperimentalEvolutionof Escherichiacoli PersisterLevels UsingCyclicAntibioticTreatments.. ....................................131
BramVandenBergh,JoranE.Michiels,andJanMichiels
PART VCELLULARAND ANIMAL MODEL SYSTEMS FOR STUDYING PERSISTENCE
13InVitroModelsfortheStudyoftheIntracellularActivity ofAntibiotics .........................................................147
JulienM.Buyck,SandrineLemaire,CristinaSeral, AhalieyahAnantharajah,Fre ´ de ´ ricPeyrusson, PaulM.Tulkens,andFranc¸oiseVanBambeke
14AMurineModelfor Escherichiacoli UrinaryTractInfection. ..............159
ThomasJ.HannanandDavidA.Hunstad
15AnalysisofMacrophage-Induced Salmonella Persisters .....................177 RobertA.Fisher,AngelaM.Cheverton,andSophieHelaine
16PopulationDynamicsAnalysisofCiprofloxacin-Persistent S. TyphimuriumCellsinaMouseModelfor Salmonella Diarrhea ...........189 PatrickKaiser,RolandR.Regoes,andWolf-DietrichHardt
PART VIMATHEMATICAL MODELINGOF PERSISTENCE
17ComputationalMethodstoModelPersistence ............................207 AlexandraVandervelde,RemyLoris,JanDanckaert, andLendertGelens
Contributors AHALIEYAH ANANTHARAJAH Pharmacologiecellulaireetmole ´ culaire,LouvainDrug ResearchInstitute,Universite ´ catholiquedeLouvain,Brussels,Belgium
NATHALIE Q.BALABAN TheRacahInstituteofPhysics,TheHebrewUniversityofJerusalem, Jerusalem,Israel
MARK P.BRYNILDSEN DepartmentofChemicalandBiologicalEngineering,Princeton University,Princeton,NJ,USA;DepartmentofMolecularBiology,PrincetonUniversity, Princeton,NJ,USA
AARON T.BUTT DepartmentofInfectionandImmunity,MedicalSchool, UniversityofSheffield,Sheffield,UK
JULIEN M.BUYCK Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium;FocalAreaInfection Biology,Biozentrum,UniversityofBasel,Basel,Switzerland
BRUNO P.A.CAMMUE CentreofMicrobialandPlantGenetics(CMPG), KULeuven–UniversityofLeuven,Leuven,Belgium; DepartmentofPlantSystemsBiology,VIB,Ghent,Belgium
ANGELA M.CHEVERTON SectionofMicrobiology,MedicalResearchCouncilCentre forMolecularBacteriologyandInfection,ImperialCollegeLondon,London,UK
TOM COENYE LaboratoryofPharmaceuticalMicrobiology,GhentUniversity, Ghent,Belgium
BRIAN P.CONLON AntimicrobialDiscoveryCenter,DepartmentofBiology,Northeastern University,Boston,MA,USA
JAN DANCKAERT AppliedPhysicsResearchGroup(APHY),VrijeUniversiteitBrussel, Brussels,Belgium
KATRIJN DE BRUCKER CentreofMicrobialandPlantGenetics(CMPG),KULeuven–UniversityofLeuven,Leuven,Belgium
KAAT DE CREMER CentreofMicrobialandPlantGenetics(CMPG),KULeuven–UniversityofLeuven,Leuven,Belgium;DepartmentofPlantSystemsBiology,VIB, Ghent,Belgium
CHRISTINA J.DECOSTE DepartmentofMolecularBiology,PrincetonUniversity, Princeton,NJ,USA
VALERIE DEFRAINE CentreofMicrobialandPlantGenetics(CMPG), DepartmentofMicrobialandMolecularSystems,KULeuven–UniversityofLeuven, Leuven,Belgium
MAARTEN FAUVART CentreofMicrobialandPlantGenetics(CMPG), DepartmentofMicrobialandMolecularSystems,KULeuven–UniversityofLeuven, Leuven,Belgium
ROBERT A.FISHER SectionofMicrobiology,MedicalResearchCouncilCentreforMolecular BacteriologyandInfection,ImperialCollegeLondon,London,UK
LENDERT GELENS AppliedPhysicsResearchGroup(APHY),VrijeUniversiteitBrussel, Brussels,Belgium;DepartmentofChemicalandSystemsBiology,StanfordUniversity SchoolofMedicine,Stanford,CA,USA
F.GOORMAGHTIGH LaboratoiredeGe ´ ne ´ tiqueetPhysiologieBacte ´ rienne,IBMM,Faculte ´ desSciences,Universite ´ LibredeBruxelles(ULB),Gosselies,Belgium
THOMAS J.HANNAN DepartmentofPathologyandImmunology,WashingtonUniversity SchoolofMedicine,St.Louis,MI,USA
WOLF-DIETRICH HARDT InstituteofMicrobiology,Eidgeno¨ssischeTechnischeHochschule ETH,Zurich,Switzerland
SOPHIE HELAINE SectionofMicrobiology,MedicalResearchCouncilCentreforMolecular BacteriologyandInfection,ImperialCollegeLondon,London,UK
THERESA C.HENRY DepartmentofMolecularBiology,PrincetonUniversity,Princeton, NJ,USA;RutgersRobertWoodJohnsonMedicalSchool,Piscataway,NJ,USA
DAVID A.HUNSTAD DepartmentofPediatricsandMolecularMicrobiology,Washington UniversitySchoolofMedicine,St.Louis,MI,USA
RYOTA IINO OkazakiInstituteforIntegrativeBioscience,InstituteforMolecularscience, NationalInstitutesofNaturalScience,Aichi,Japan;DepartmentofFunctional MolecularScience,SchoolofPhysicalScience,TheGraduateUniversityofAdvanced Studies(SOKENDAI),Kanagawa,Japan
HENRI INGELMAN InstituteofTechnology,UniversityofTartu,Tartu,Estonia
ARVI JO ˜ ERS InstituteofTechnology,UniversityofTartu,Tartu,Estonia
PATRICK KAISER InstituteofMicrobiology,Eidgeno¨ssischeTechnischeHochschuleETH, Zurich,Switzerland
NIILO KALDALU InstituteofTechnology,UniversityofTartu,Tartu,Estonia
IRIS KEREN AntimicrobialDiscoveryCenter,DepartmentofBiology,Northeastern University,Boston,MA,USA
WOUTER KNAPEN CentreofMicrobialandPlantGenetics(CMPG), DepartmentofMicrobialandMolecularSystems,KULeuven–UniversityofLeuven, Leuven,Belgium
SANDRINE LEMAIRE Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium;GSKBiologicals, Rixensart,Belgium
IRIT LEVIN-REISMAN TheRacahInstituteofPhysics,TheHebrewUniversityofJerusalem, Jerusalem,Israel
KIM LEWIS AntimicrobialDiscoveryCenter,DepartmentofBiology,Northeastern University,Boston,MA,USA
VEERLE LIEBENS CentreofMicrobialandPlantGenetics(CMPG), DepartmentofMicrobialandMolecularSystems,KULeuven–UniversityofLeuven, Leuven,Belgium
REMY LORIS StructuralBiologyResearchCenter,VIB,Brussels,Belgium;Structural BiologyBrussels,DepartmentofBiotechnology(DBIT),VrijeUniversiteitBrussel,Brussels, Belgium
YOSHIMI MATSUMOTO LaboratoryofMicrobiologyandInfectiousDiseases, InstituteofScientificandIndustrialResearch,OsakaUniversity,Osaka,Japan
JAN MICHIELS CentreofMicrobialandPlantGenetics(CMPG),DepartmentofMicrobial andMolecularSystems,KULeuven–UniversityofLeuven,Leuven,Belgium
JORAN E.MICHIELS CentreofMicrobialandPlantGenetics(CMPG),KULeuven–UniversityofLeuven,Leuven,Belgium
KUNIHIKO NISHINO LaboratoryofMicrobiologyandInfectiousDiseases, InstituteofScientificandIndustrialResearch,OsakaUniversity,Osaka,Japan
MEHMET A.ORMAN DepartmentofChemicalandBiologicalEngineering,Princeton University,Princeton,NJ,USA
FRE ´ DE ´ RIC PEYRUSSON Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium
ROLAND R.REGOES InstituteofIntegrativeBiology,Eidgeno¨ssischeTechnischeHochschule ETH,Zurich,Switzerland
SARAH E.ROWE AntimicrobialDiscoveryCenter,DepartmentofBiology,Northeastern University,Boston,MA,USA
SHOUICHI SAKAKIHARA TechnicalDivision,InstituteofScientificandIndustrialResearch, OsakaUniversity,Osaka,Japan
CRISTINA SERAL Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium;Departmentof Microbiology,HospitalClı ´ nicoUniversitarioLozanoBlesa,Zaragoza,Spain
TANEL TENSON InstituteofTechnology,UniversityofTartu,Tartu,Estonia
KARIN THEVISSEN CentreofMicrobialandPlantGenetics(CMPG),KULeuven–UniversityofLeuven,Leuven,Belgium
RICHARD W.TITBALL Biosciences,CollegeofLifeandEnvironmentalSciences,University ofExeter,Exeter,UK
PAUL M.TULKENS Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium
HELEEN VAN ACKER LaboratoryofPharmaceuticalMicrobiology,GhentUniversity,Ghent, Belgium
FRANC¸OISE VAN BAMBEKE Pharmacologiecellulaireetmole ´ culaire,LouvainDrugResearch Institute,Universite ´ catholiquedeLouvain,Brussels,Belgium
BRAM VANDEN BERGH CentreofMicrobialandPlantGenetics(CMPG),KULeuven–UniversityofLeuven,Leuven,Belgium
L.VAN MELDEREN LaboratoiredeGe ´ ne ´ tiqueetPhysiologieBacte ´ rienne,IBMM,Faculte ´ desSciences,Universite ´ LibredeBruxelles(ULB),Gosselies,Belgium
ILSE VANDECANDELAERE LaboratoryofPharmaceuticalMicrobiology,GhentUniversity, Ghent,Belgium
ALEXANDRA VANDERVELDE StructuralBiologyResearchCenter,VIB,Brussels,Belgium; StructuralBiologyBrussels,DepartmentofBiotechnology(DBIT),VrijeUniversiteit Brussel,Brussels,Belgium
NATALIE VERSTRAETEN CentreofMicrobialandPlantGenetics(CMPG), DepartmentofMicrobialandMolecularSystems,KULeuven–UniversityofLeuven, Leuven,Belgium
Introduction
AHistoricalPerspectiveonBacterialPersistence NatalieVerstraeten,WouterKnapen,MaartenFauvart, andJanMichiels Abstract
Bactericidalantibioticsquicklykillthemajorityofabacterialpopulation.However,asmallfractionofcells typicallysurvivethroughenteringtheso-calledpersisterstate.Persistercellsareincreasinglybeingviewedas amajorcauseoftherecurrenceofchronicinfectiousdiseaseandcouldbeanimportantfactorinthe emergenceofantibioticresistance.Thephenomenonofpersistencewasfirstdescribedinthe1940s,but remainedpoorlyunderstoodfordecadesafterwards.Onlyrecently,aseriesofbreakthroughdiscoverieshas startedtoshedlightonpersisterphysiologyandthemolecularandgeneticunderpinningsofpersister formation.Wehereprovideanoverviewofthekeystudiesthathavepavedthewayforthecurrentboomin persistenceresearch,withaspecialfocusonthetechnologicalandmethodologicaladvancesthathave enabledthisprogress.
Keywords: Persisters,Persistence,Antibiotictolerance,Dormancy,Antibiotics,Review
1TheEarlyDays Thefirstreportonthesurvivalofasmallfractionofstreptococci cellsfollowingtreatmentwithpenicillindatesfrom1942[1]. Twoyearslater,JosephBiggerestablishedthatadditionofpenicillintostaphylococcidoesnotresultincompletesterilizationofall cellsinaclonalpopulation.Oneoutofamillioncellssurvivedeven prolongedtreatmentwithantibiotics.Heappropriatelynamedthe survivingcellspersisters[2].Morerecently,itwasshownthatin mostbacterialspecies,themajorityofcellsareefficientlykilledby relativelylowconcentrationsofbactericidalantibiotics.However, killingshowsabiphasicpatternandbeyondacertainthreshold, furtherincreasingtheconcentrationoftheantibacterialdoesnot resultincompleteclearingoftheculture(Fig. 1)[3].
For40yearsfollowingitsdiscovery,thepersistencephenomenonwaslargelyneglected,atleastbymoleculargeneticists. Thiswaspartlyduetothefactthattheclinicalrelevanceofpersister
JanMichielsandMaartenFauvart(eds.), BacterialPersistence:MethodsandProtocols, MethodsinMolecularBiology,vol.1333,DOI10.1007/978-1-4939-2854-5_1, © SpringerScience+BusinessMediaNewYork2016
Fig.1 Illustrationofpersistence:Themajorityofcellsinabacterialcultureareefficientlykilledbyrelatively lowconcentrationsofantibiotics.However,beyondacertainthreshold,akillingplateauisobservedasonly persistercellsremainviable.Whenregrowninfreshmedium,thesurvivingcellsgenerateapopulationas sensitivetotheantibioticastheoriginalpopulation
cellswasnotclear.Incontrast,thethreatposedbyinherited antibioticresistancewasgenerallyrecognized,addingincentives toresistanceresearch.Theproblemwascompoundedbytechnical challengesthatinevitablyaccompanythestudyofatransient phenotypethatisassociatedwithonlyaverysmallfractionofcells. Abreakthroughdiscoverycameintheearly1980s,from researchcarriedoutbyHarrisMoyedduringasabbaticalleavein thelabofAlexanderTomasz[4].Mutagenesisof Escherichiacoli populationswithethylmethanesulfonate(EMS)ledtothe identificationofthreehighlypersistent(hip)mutantsexhibiting 10–10,000-foldincreasedpersisterfractionsuponincubation withpenicillin[4, 5].Moyed’spioneeringworkledtotheidentificationoftwomutantshitinthe hipA locusthatupuntilnow remainsthebeststudiedpersistergene[6–10].Furthermore, becauseoftheirincreasedpersisterfraction, hipA mutantshave frequentlybeenusedasatoolinpersistenceresearch.Crucially andforthefirsttime, hipA mutantsenabledthedirectobservation ofpersistercells.Usingacombinationofmicrofluidicsandlivecell microscopy,NathalieBalabanrecordedhowpersisterssurvived killingbyantibioticsthroughdormancyandsubsequentresuscitation[11].Inaddition,the hipBA locusisarepresentativeforother toxin–antitoxin(TA)locithatarenowintensivelystudiedinrelationtopersistence.TAmodulesconsistofastabletoxin,typically targetingessentialcellularfunctions,andanunstableantitoxin,
whichcounteractstheactivityofitscognatetoxin[12, 13].TA systemswereoriginallyidentifiedonplasmids,wheretheyplaya roleinplasmidmaintenance;yetasignificantnumberofTAlociare chromosomallyencodedandthesehavebeenimplicatedinpersistence[14].ExamplesincludeRelE[6],MqsR[15–17],TisB[18, 19],MazF[20],andYafQ[21].Interestingly,withthenotable exceptionof Salmonella persistersresidingwithinmacrophage vacuoles[22],deletionofasingletoxingenerallydoesnotaffect persistence.ThiscanpartlybeexplainedbyredundancyofTA systemsinmostbacteria.DeletionofmultipleTAsystems,onthe otherhand,causesadecreasein E.coli persistence[23].
2TheRiseofPersistenceResearch Followingthediscoveryof hipA,persistenceasafieldofstudy steadilygainedattention.Thiswaspartlyduetotheacknowledgementofitsclinicalsignificance(summarizedby[24]).In1944, Biggeralreadyalludedtotheroleofpersistersintheresuscitationof chronicinfections[2].Decadeslater,KimLewispostulatedthat persistersmightcontributetotherecalcitranceofbiofilminfections [25, 26].Thisisofparticularinterestasbiofilmsareknownto withstandantibiotictreatment,therebycausingchronicinfections [27].Subsequently,mathematicalmodelingdemonstratedthat persistencecouldextendthedurationofantibiotictreatment, therebycausingtreatmentfailureandpromotingtheemergence ofresistance[28].Finally,twostudieshaveunambiguouslydemonstratedthatprolongedantimicrobialtherapyselectsforhighpersistencestrainsof Candidaalbicans duringcandidiasisand of Pseudomonasaeruginosa duringcysticfibrosislunginfections [29, 30].Inaddition,theroleofpersistercellsinthedevelopment ofresistanceisbecomingincreasinglyclear[31].Apartfrom providingincentivestofurtherintensifypersistenceresearch, thesefindingsalsopromotedthesearchforanti-persistertherapies. Atpresent,severalstrategieshavebeendescribed,buttheirinvivo effectivenessremainstobeinvestigated.Examplesincludethe useofresonantactivation[32],electrochemicalcurrents[33], cadaverine[34],metabolites[35, 36],antimicrobialpeptides [37],brominatedfuranones[38–41],andactivatedClpP[42] (summarizedby[43]).
Apartfromincreasedinterestduetotheclinicalimportanceof persistence,thedevelopmentofnoveltechniquesalsocausedpersistenceresearchtoboom.Anoverviewofthesenoveltechniquesis providedbelow.
2.1Screening Approaches Overtheyears,severalscreeningprocedureshavebeendeveloped thatledtotheidentificationofpersistergenes.Inafirstapproach,a non-redundant E.coli knockoutlibrarywasscreenedformutants
2.2Single-Cell Studies withalteredpersistence[44].Persistercellsofindividualmutants werequantifiedbytreatingastationary-phaseculturewithofloxacinandplatingthesurvivingcellsonagarmediumcontaining amdinocillin.Asthenumberofspontaneousamdinocillin-resistant mutantsisafractionoftheoriginalnumberofcells,thisobviates theneedfordilutionstepsandgreatlyreducesthelaborioustaskof screeningseveralthousandsofstrains.
Asecondscreeningapproachemployeda P.aeruginosa plasposonknockoutlibrary.Individualmutantsweregrownuntilstationaryphaseandtreatedwitheitherofloxacintokillnon-persistercells orwater,thelatterservingasacontrol.Subsequently,sampleswere dilutedandincubatedinanautomatedplatereader(BioscreenC, OyGrowthCurvesAbLtd),allowingtheopticaldensityof200 samplestobemeasuredsimultaneouslyasafunctionoftime. Giventhelinearrelationshipbetweenthenumberofcellsinan inoculumandthelagphase,thisallowedfortheselectionof mutantsdisplayingalteredpersisterlevels[45].
Bothscreeningsledtotheidentificationofanumberof interestingpersistergenesincludingsomeglobalregulators.In addition,notasinglemutantlackingpersisterswasidentified.Asa generalconclusion,thesescreeningsthereforeprovidedevidence pointingtothemultiplicityofpersisterformationmechanisms.
Inafinalapproach,arandomoverexpressionlibrarywas generatedin E.coli.Cellsfromtherecombinantlibrarywerepooled andlogarithmicallygrowingculturesoflibrarycloneswereexposed tomultipleroundsofexposuretoampicillin.Thisledtotheenrichmentofmutantswithincreasedprobabilityofpersisterformation andultimatelytotheidentificationof glpD asagenuinepersister gene[46].
Aspersistenceisaphenotypictraitexpressedinonlyasubfractionof apopulation,advancesinsingle-cellresearchsignifiedaneraofvast newpossibilities.FirstusedbytheBalabangroup[11],transparent microfluidicdevicesprovedinstrumentalformicroscopicexaminationofpersistercells[47–49].Thestrengthofthistechniqueliesin thepossibilitytomonitorindividualcellsforprolongedperiodsof timewhileadaptinggrowthconditions.Forexample,normal growthconditionscanbealternatedwithantibiotictreatmentsin ordertokillnon-persistercells.Thisallowstopinpointpersister cellssurvivingtreatment.Subsequently,thehistoryofpersistercells canbetracedbackthroughtherecordedimages.Severalstudies haveusedthistechniquetodemonstratepreexistingheterogeneity inbacterialpopulations[11],tocharacterizethedormantstateof singlepersistercells[47],tomonitorpersisterformationfollowing administrationofindole[48],andtocorrelatehighTAexpression tocessationofgrowth[49].
AlsodevelopedbytheBalabangroup,acolony-appearanceassay waselaboratedtoquantifysingle-cellpersisterlagphases[50].
2.3Transcriptomics
Experimentsdemonstratedthatathresholdconcentrationoftoxin moleculesisrequiredforinductionofpersistence.
Amajordrawbackofmicrofluidicdevices,ormorepreciselyof microscopy,isthelimitednumberofcellsthatcanbestudied simultaneously.Thiscanbecircumventedbyusingflowcytometry, allowingthousandsorevenmillionsofcellstobeevaluatedina high-throughputmanner.Ashortcomingofthistechniqueisthe inabilitytocontinuouslymonitorindividualcellsovertime.Nonetheless,flowcytometryhasbeensuccessfullyusedtostudythe kineticsofpersisterawakening[51].Inaddition,whileBigger postulatedthatpersistersareinadormant,nondividingstate[2], flowcytometryhasbeenusedtodemonstratethatdormancyisnot arequirementforentryintothepersisterstate[52].Finally,arecent studyperformedbytheHoldengroupshowedhowtocharacterize thedynamicsofintracellularbacterialreplicationatthesingle-cell level.Theyusedafluorescencedilutiontechniquetoquantifythe numberofreplicationcyclesofinternalized Salmonella [53].This showedtheexistenceofdifferent Salmonella subpopulationsin bonemarrow-derivedmacrophagesincludinganon-replicating butmetabolicallyactivesubpopulation,comprisingthepersister cells,possiblycapableofresuminggrowthandcausingrelapsing infections[22].Similarly,theBumanngroupexploitedaDsRed variantcalledTIMERbac,whichspontaneouslychangescolorfrom greentogreen/orangeovertime,asadynamicgrowthrate reportertoidentifypersistercellsinvivo[54].
Insightintoglobaltranscriptionalchangesinpersistercellscame fromseveralelegantstudiesbytheLewisgroup.Toenrichfor persisters,allthreeapproachesconvenientlyemployedthemetabolicinactivityofthesecells.Inthefirstreport,logarithmically growingpopulationsofthehigh-persistence E.coli mutant hipA7 [4]weretreatedwithampicillin,therebylysingnon-persistercells. IsolatedRNAwasenrichedformRNA,labeled,andhybridized to E.coli GeneChips[6].Similarly,geneexpressionprofilingof persisterswasperformedaftertreatinganexponentiallygrowing populationof Mycobacteriumtuberculosis with D-cycloserineand collectingsurvivingpersistercellsbycentrifugation.Transcriptome analysiswasperformedbymicroarrayhybridization[55].Thethird studyfollowedaslightlydifferentapproach.Itwasbasedonthe assumptionthatpersistersaredormantcellswithlowlevels ofproteinsynthesisandcorrespondinglowlevelsofrRNAtranscription. E.coli persistercellswereisolatedbylinkingthe rrnB promotertoageneencodinganunstablefluorescentprotein.Inso doing,persistercellsaredimascomparedtonormalcellsinthe population,whichallowsfortheisolationofpersistersusing fluorescence-activatedcellsorting(FACS).cDNAwasprepared frompurifiedRNAandhybridizedtospotted E.coli DNAmicroarrays[56].
2.4Experimental Evolution
Basedonthestudiescitedabove,stressresponsepathwaysas wellasTAlociwereshowntobehighlyexpressedinisolated persistercells.Ontheotherhand,biosyntheticfunctionsincluding energyproductionweredownregulated[6, 55, 56].
Theuseofexperimentalevolutionforelucidatingantibacterial resistancemechanismsisawidelyusedmethod.Arecentstudyby theBalabangroupusedthistechniqueforenrichingapopulation withpersistersbyrepeatedexposureofabacterialpopulationto highconcentrationsofantibiotics.Thisresultedinevolvedstrains showingveryhighpersisterfractionscausedbyfixedspecific geneticmutations.Theincreasedsurvivalappearedtobetheresult ofanadjustmentinthesingle-celllag-timedistribution,whichwas correlatedwiththeextentoftheantibioticexposureinterval[57]. TheyimplementedtheScanLagmethod,whichallowsthesimultaneousmeasurementoflagtimesofhundredsofcells[58].These findingsresultedinanewtheoryregardingpersistercellsandtheir abilitytoadapttohighdosesofdrugscalledtolerancebylag.
2.5Modeling
Apartfromthesewetlabtechniques,mathematicalmodelinghas providedinterestinginsights[28, 59–62](summarizedby[63]). Briefly,twomainstrategiescanbediscerned:thefirstonerelieson estimatingtheswitchingratesbetweenpersisterandnon-persister growthstatesandassumesthisprocesstotakeplacecontinuously andstochastically(e.g.,[11, 28, 59, 61, 64]).Thebalancebetween bothswitchingratesprovidesastraightforwardwaytomodela givenpersisterlevel,althoughignoringexactlywhatdetermines theswitchingrates.Thesecondstrategyfocusesonthemolecular mechanismsofpersisterformationbyTAsystems,withtheratioof (free)toxinoverantitoxinultimatelydetermining,atthesingle-cell level,thedecisiontoswitchtothepersisterstate(e.g.,[50, 60, 65, 66]).Acrucialfactorinthistypeofmodelsisthegenerationof phenotypicbistabilityatthepopulationlevel,typicallyrequiring noisygeneexpressionandnoiseamplificationthroughpositive feedbackmechanisms[67].Bothmodelingstrategieshavetheir merits,anduntilamoreintegratedapproachispresented,the choicebetweenbothwilldependonthegoalandspecificfocusof thestudyathand.
Mathematicalmodelingofpersistenceposesseveraladvantages. Experimentsthatarenotfeasibleinthelabcanbesimulatedto predicttheoutcome.Italsoallowstoexplainempiricallyobserved persisterlevelsintermsoftheparametersencompassedbythe model,andwhyvaryingsomeparametershasmoreimpacton persistencethanothers.Consequently,evolutionaryforcesthat shapepersisterlevelscanbeidentified,whichshouldhelptodevise strategiestoavoidhighpersisterlevelsemergingintheclinic.
3StateoftheArtandFuturePerspectives Recently,thefieldofmicrobialpersistenceresearchhasexploded,as evidencedbyahostofpublicationsintop-tierjournals[10, 22, 23, 35, 48–50, 54, 68–70].Currently,itisgenerallyacceptedthat persistercellsarepresentinabacterialpopulationprecedingantibiotictreatment[71].Itispostulatedthattheirformationresults fromnoisygeneexpression[72]aswasfirstsuggestedbyKim Lewis[6].However,overtheyears,severalstimulihavebeen showntoinducepersistence.Forexample,sub-inhibitoryconcentrationsoffluoroquinolonesareknowntoinducepersistencevia activationofthe tisAB/istR TAlocus[18, 19].Otherexamples includequorumsensingmolecules[73, 74],carbonsourcetransitions[70],andnutrientdeprivationleadingtoactivationofthe stringentresponse[75].AswasearlierdescribedforHipA[76], arecentmodelascribesTA-regulatedpersistencetostochastic fluctuationsincellularconcentrationsofthealarmone(p)ppGpp. High(p)ppGpplevelsactivateTAlocithrougharegulatorycascade requiringinorganicpolyphosphateandLonproteasetargeting proteintoxins[49].Foranelaboratediscussionontheroleof thesemechanismsinpersistence,thereaderisreferredtosome excellentreviewsonthetopic[3, 77–81].
Addingtothesignificanceofthesestudiesistherecentobservationofaphenotypicallydistinctsubpopulationoftransiently drug-tolerantpersistersincancercellpopulations.Thesecellsare heldresponsiblefor“fractionalkilling”uponchemotherapy[82]. Cell-to-cellvariationsinproteinlevelsweresuggestedtocontribute tothisphenomenoninwhicheachroundoftherapykillssomebut notallofthecellsinatumor[83].Thereisastrikinganalogy betweenbacterialandcancercell-derivedpersistenceasbothphenomenareflectatransientlyphenotypicheterogeneitycausingmultidrugtoleranceandrecurrenceofdiseasesymptomsuponremoval oftreatment[84].Addedinsightintobacterialpersistencemay thereforeimpactresearchareasfarbeyondinfectiousdisease.
Acknowledgements ResearchinthelabofJMissupportedbygrantsfromKULeuven ResearchCouncil(PF/10/010;IDO/09/010;IDO/13/008), IAP-BELSPO,FWO,andIWT.
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PartII QuantificationofPersistence Persisters:MethodsforIsolationandIdentifying ContributingFactors—AReview SarahE.Rowe,BrianP.Conlon,IrisKeren,andKimLewis
Abstract
Persistercellsarephenotypicvariantssurvivingalethaldoseofantibiotic,sufficienttokillthebulkofan exponentialphasepopulation.Inthischapterwesummarizecurrenttechniquestoisolatepersistersand discusslimitationsassociatedwithidentifyingmechanismsofpersisterformation.
Keywords: Hipmutant,Toxin-antitoxin(TA)modules,Antibiotic,Biofilm
1Introduction Persisterswerefirstdescribedin1944byJosephBigger,amedical doctorfromTrinityCollegeDublin[1].Biggerreportedthat additionofalethaldoseofpenicillintoapopulationof Staphylococcusaureus yieldedasmallsubpopulationofsurvivingcells.Upon reinoculation,thesecoloniesgrewintoaculturethatonceagain lysedinthepresenceofpenicillinandagainyieldedasubpopulation ofsurvivors.Biggerreferredtothesesurvivingcellsaspersisters[1].
Thesecellsarecontrarytoresistantcellsastheycannotgrowin thepresenceoftheantibiotic.Theproportionofpersistersina populationvariesdramaticallydependingongrowthphaseand environmentalconditions[2, 3].Specifically,persisternumbers aregreatlyincreasedinstationaryphaseandbiofilms[4, 5].
Persisterscanbeenumeratedbyaddingalethalconcentration ofabactericidalantibiotictoapopulationofcells.Thebulkofthe populationdiesrapidlyandthesurvivingpersistercellseitherdie muchmoreslowlyordonotdieduringtheexperiment(Fig. 1). Thepersisterfractioncanbeenumeratedatvarioustimeintervals byremovinganaliquot,washingwith1%NaCl,andplatingserial dilutionsforcolonycounting.Thesurvivingpersisterfraction emergesinaclearbiphasiccurveinresponsetotheantibiotic.
JanMichielsandMaartenFauvart(eds.), BacterialPersistence:MethodsandProtocols, MethodsinMolecularBiology,vol.1333,DOI10.1007/978-1-4939-2854-5_2, © SpringerScience+BusinessMediaNewYork2016
Drugsusceptiblecells Fig.1 Typicalbiphasickillcurveofabacterialpopulationinexponentialphaseofgrowth:Acultureof E.coli growntomid-exponentialphaseischallengedwith10 MICofabactericidalantibiotic(suchasciprofloxacin).Themajorityofcellsdierapidly.Thepersisterfractionisenumeratedbyremovinganaliquotofcellsafter 3and6hofexposuretotheantibiotic.Cellsarewashedwith1%NaClandserialdilutionsareplatedfor counting.Persistercellsdifferfromresistantcellsastheycantoleratetheantibioticbutcannotgrowinits presence
Thetimeperiodwherethepersisterfractionremainsconstantis referredtoasthe“persisterplateau”(Fig. 1).
Themajorityofresearchhasconcentratedonpersistersin Escherichiacoli butpersistershavebeenshowntobeproduced bymanyotherbacterialspeciesincluding Pseudomonasaeruginosa, Mycobacteriumtuberculosis, Salmonellaentericaserovartyphimurium, Staphylococcusaureus,and StreptococcusmutansBurkholderia cepacia andeveninthefungalpathogen Candidaalbicans [6–13].
2High-Persister(hip)Mutants Althoughpersisterswerediscoveredinthe1940s,theywerelargely ignoreduntiltheearly1980swhenHarrisMoyedandco-workers isolatedhigh-persistence(hip)mutants[14, 15].Theyidentified thegene hipA,whichwasthefirstgeneknowntocontributeto persisterformation;thisgenewaslateridentifiedasthetoxinofan antitoxin/toxin(TA)module.The hipA7 alleleofthegeneproduced1000timesmorepersisterstotwoclassesofantibiotics:betalactamsandfluoroquinolones[16].Thiswasthefirstindication thatsomepersistersaremultidrugtolerant.Importantlythe hipA7 mutanthasthesameminimalinhibitoryconcentration(MIC)as theparentstrain[16],indicatingthattheincreasedlevelofpersistersisnotduetoacquisitionofresistance.
PatientNumberandage(yearsold)whenstrainwasisolated
Fig.2 Ascreenof P.aeruginosa clinicalisolatesforhipmutants:Stationary-phaseculturesofclonalearly/late isolatepairsfrom14patientswereexposedtoofloxacin(50 MIC)for8h,andthesurvivingcellswere determinedbycolonycountandexpressedaspercentsurvival(n ¼ 4).Earlyisolatesareindicatedwith white bars,whilelateisolatesareindicatedwith blackbars.Thepatientnumberandageatwhichthetestedisolates wereobtainedaredisplayedonthe x-axis.Ahipmutantemergedin10ofthe14patients.Ahipmutantdidnot emergeintheisolatesfromthelastfourpatientsdisplayedontherightsideofthegraph.TakenfromMulcahy etal.2010
Hipmutantscanbeidentifiedinvitrobygeneratingamutant libraryandexposingittoseveralroundsofalethaldoseofan antibiotic[17].Thebacterialmutantlibrarycanbeobtainedeither bychemicalmutagenesis(ethylmethanesulfonate)[18]orbytransposonmutagenesis[19].Followingseveralroundsofantibiotic challenge,thegeneticbasisforhipmutantphenotypesisidentified bysubjectingsurvivingcellstowhole-genomesequencingor micro-array-basedgeneticfootprinting[17].
Persistersrepresenttransientphenotypicvariantsthatare geneticallyidenticaltotheirdrug-susceptiblekin.Suchtraitshave provedtaxingforinvivoinvestigationsasthesepersisterstendto resuscitate.Mulcahyetal.tookadifferentapproach[20].They hypothesizedthatifhipmutantshadanadvantagethentheywillbe selectedforinvivoduringinfectionorinresponsetoantibiotic treatment.Longitudinalisolatesof Pseudomonasaeruginosa were obtainedfrompatientsthroughoutthecourseofacysticfibrosis infection.Thestudydemonstratedthatstrainsdevelopedahip phenotypeoverthecourseoftheinfection(Fig. 2).Asimilar findingwasobservedin Candidaalbicans withpatientssuffering fromchronicoralthrush[21].Thesestudiesindicatethatpersisters areclinicallyrelevantandmaycontributetotreatmentfailures.
20SarahE.Roweetal.
3Pre-existingandInducedPersisters Thefrequencyofpersistersinhipstrainsismuchhigherthaninthe wild-type E.coli whichfacilitatesresearchintothemechanismof persisterformation.The hipA7 mutantwasusedforsingle-cell time-lapsemicroscopy,whichshowedthatpersistersareslowor non-growingcells,stochasticallyformedandpre-existinginthe population[22].
Inordertodistinguishbetweengrowingandnon-growing cells,Shahetal.usedanunstableGFPvariantdrivenfromaribosomalpromoter[23].Cellsexpressingthisplasmidweregrownto mid-exponentialphaseandanalyzedbyfluorescent-activatedcell sorting(FACS)(Fig. 3).Twodistinctpopulationswerevisualized usingforwardlightscatter,onethatfluorescedbrightlyandonethat didnot.Cellsfrombothpopulationsweresortedandvisualizedby epifluorescentmicroscopyandchallengedwithofloxacin.Thecells thatdidnotexpressGFPweremuchmorelikelytosurviveantibiotic challenge.Thisstudydescribedanewmechanismforidentifying persistersanddemonstratedthatnon-growingcellsexistinan untreated E.coli population[23].Theseresultssuggestthatthere isapopulationofpersistersthatarepre-existinginthepopulation andarenotformedinaresponsetoantibiotictreatment.
Insupportofthis,ithasbeendemonstratedthatan E.coli culture,ifdilutedseveraltimesandchallengedwitheitherampicillinorofloxacin,displaysagradualdeclineandeventualelimination
Fig.3 Isolationofpersistercellsfromanexponentiallygrowingculture. E.coli cellscontainingadegradable GFPreportercassetteweregrowninLBmediumtomid-exponentialphase(~1 108 cells/ml)at37 Cwith aerationandsortedusingahigh-speedcellsorterequippedwithastandardGFPfilterset.(a)Twopopulations weredetectedusingforwardlightscatter,onethatfluorescedbrightly(R3),andanotherthatdidnot(R4).(b) Thesortedpopulationswerevisualizedbyepifluorescentmicroscopy(bar,5 μm).(c)Cellsweresortedas describedin(a–b).Oncesortedbothpopulationsweretreatedwithofloxacin(5 μg/ml)for3h,diluted,and spottedontoLBagarplatesforcolonycounts.TakenfromShahetal.2006
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Title: The play that won
Author: Ralph Henry Barbour
Illustrator: Walt Louderback
Release date: May 13, 2022 [eBook #68062]
Language: English
Original publication: United States: D. Appleton and Company, 1916
Credits: Donald Cummings and the Online Distributed Proofreading Team at https://www.pgdp.net (This file was produced from images generously made available by The Internet Archive/American Libraries.)
Transcriber’s Note: The cover image was created from the title page by the transcriber and is placed in the public domain.
THE PLAY THAT WON By Ralph Henry Barbour
YARDLEY HALL SERIES Guarding His Goal
Forward Pass
Double Play
Winning His Y For Yardley
Around the End Change Signals
PURPLE PENNANT SERIES The Lucky Seventh
The Secret Play
The Purple Pennant
HILTON SERIES The Half-Back
For the Honor of the School Captain of the Crew
ERSKINE SERIES
Behind the Line
Weatherby’s Inning
On Your Mark
THE “BIG FOUR” SERIES
Four in Camp
Four Afoot
Four Afloat
THE GRAFTON SERIES Rivals for the Team
Winning His Game
Hitting the Line
BOOKS NOT IN SERIES For the Freedom of the Seas
Under the Yankee Ensign
Keeping His Course
The Brother of a Hero
Finkler’s Field
Danforth Plays the Game
The Arrival of Jimpson
Benton’s Venture
The Junior Trophy
The New Boy at Hilltop
The Spirit of the School
The Play that Won
D. APPLETON & CO., Publishers, New York
IT WAS LARRY LOGAN WHO FUMED AND IMPLORED.... THE PLAY THAT WON BY
RALPH HENRY BARBOUR
AUTHOR OF
“FOR THE FREEDOM OF THE SEAS,” “UNDER THE YANKEE ENSIGN,” “THE HALF BACK,” ETC.
COPYRIGHT, 1919, BY
D. APPLETON AND COMPANY Copyright, 1918, 1919, by THE CENTURY COMPANY
Copyright, 1916, 1917, by PERRY MASON COMPANY
Copyright, 1918, by SPRAGUE PUBLISHING COMPANY
Copyright, 1919, by FISK BICYCLE CLUBS OF AMERICA
PRINTED IN THE UNITED STATES OF AMERICA
ILLUSTRATIONS It was Larry Logan who fumed and implored....
Frontispiece FACING PAGE
Then the pistol popped and they were off 94
Somewhere in that mêlée was the runner with the precious ball 152
The bridge tender had half closed the second gate 168
THE PLAY THAT WON When the knock came Ted was slumped on his spine in the Morris chair, the green-shaded lamp beside him and a magazine propped on his chest. It was Saturday night and study was not imperative, for which he was grateful. The baseball game with Prospect Hill in the afternoon had been a hard one, and the victory— for Warwick had won in the tenth—had left him rather tired, and he had passed up a lecture in the school auditorium in favor of rest and solitude at home. Which is why the knock on the door brought a sigh and a frown. Of course, he might remain silent, but the light shining through the transom would be a give-away, and the caller might be Trevor Corwin with his everlasting stamp album: Trev was a sensitive kid and easily hurt. So Ted laid down his magazine and said “Come in!” in no very enthusiastic tone. To his relief, the visitor was Hal Saunders.
“Hello, Bowman,” said Hal, glancing about the study. “George around?” His eyes sought the darkened bedroom as he closed the door behind him.
“Gone home over Sunday,” replied Ted.
“Gone home!” Hal’s tone held so much of dismay that Ted wondered.
“Yes, his father’s been sick for about a week or so, and he got leave from faculty. Went right after the game.”
“Gee!” exclaimed Hal worriedly. “He didn’t say anything to me about it. I wish I’d known. I want to see him about—something important.” To Ted’s discomfiture he seated himself on the windowseat and moodily stared at the lamp. “When’s he coming back?”
“Monday. He got permission to cut morning hours. I guess he will be on the twelve-forty-six.”
“That’ll be too late,” said Hal aggrievedly “By Jove, that’s rotten! I don’t see why he couldn’t let folks know he was going.”
Evidently overwhelmed by the news, he made no move to depart. He was a good-looking fellow of sixteen, well-made, tall and lithe, with light hair and eyes and a fair complexion which even three months of baseball had failed to darken. In contrast, the boy in the Morris chair was a year younger, shorter, heavier, more compact, with dark eyes and hair and a face which, if not handsome, was rather attractive in spite of the fact that sun and weather had tanned it to the hue of leather and that the tip of the nose was peeling. Both boys were members of the School Nine, Ted being right fielder and Hal first-choice pitcher. They were not, however, very good friends. Ted thought Hal traded too much on his ability as a twirler. It was undeniable that he was an exceptionally good one, perhaps the best that the school had ever had, but in Ted’s opinion Hal would do well to forget the fact now and then. He didn’t understand what his roommate, George Tempest, saw in Hal to admire; that is, beyond his playing. Naturally George, being captain of the team, would feel kindly toward a chap who so often pitched to victory, but he needn’t overdo it! Ted was fond of his room-mate and so it is possible that jealousy had something to do with his mild dislike of Hal Saunders.
Presently Hal raised his eyes from a frowning contemplation of his shoes and Ted was surprised at the trouble shown in his face. It was a most unusual thing for the self-satisfied, rather superior Hal Saunders to exhibit anything approaching discomposure. In spite of himself, Ted’s sympathies were touched. “Was it something about the Team?” he asked.
Hal shook his head. “No, it was—something——” He hesitated. Then: “I wanted to borrow some money from him.”
“Oh!” murmured Ted. It was, he reflected, a lot like Hal to make a fuss about an unimportant matter like that. Perhaps the other read the thought, for he suddenly said defensively:
“I’m in a dickens of a hole, Bowman, and I was pretty sure that George could help me out. Now I’m blessed if I know what to do!”
“Won’t Monday do?”
“Monday morning might, but Monday afternoon will be too late— unless——” Hal fell into silence again. Ted wondered if Hal was trying to find courage to ask him for a loan. He almost hoped so. It would be rather a pleasure to refuse it. “It’s Plaister, in the village,” Hal went on after a moment. “He’s got a bill of twelve dollars and eighty cents against me. I’ve been owing the old skinflint some of it since last year. And now he says that if it isn’t paid by to-night he will go and get the money from ‘Jerry.’ And you know what that will mean!”
Ted did know. “Jerry” was the popular name for Doctor Morris, the Principal, and when “Jerry” learned that Hal had transgressed the very strict rule against having bills at the village stores, punishment would be swift and stern. Why, Hal might be dismissed from school! The very least that would happen to him would be probation!
“Maybe he’s just bluffing,” offered Ted, but with little conviction in his voice.
“No such luck,” answered Hal. “He’s threatened twice before and I’ve begged him off. This time he means it. I found a letter from him in the mail this noon. I was going to speak to George before the game, but there wasn’t any chance, and I—I sort of funked it anyway. Besides, I thought there was time enough. Plaister won’t do anything until Monday I was pretty sure George had the money and I guess he’d have let me have it. I meant to beat it over to the village right after chapel Monday morning. I hadn’t any idea he was going away!”
“Too bad,” said Ted, more than half meaning it. “How the dickens did you ever manage to run up a bill like that, Saunders?”
Hal shrugged. “Oh, I don’t know. I’m always buying fool things. Plaister was keen enough to charge ’em until he had a nice big bill against me. Afterwards, too. It got so I was afraid not to buy anything he showed me for fear he’d ask me to pay up.”
“But you get an allowance——”
“A dollar a week,” said Hal slightingly “How far does that go? Mother sends me a little now and then. If she didn’t I wouldn’t have a cent in my pocket, ever. I’m a fool about money, and dad knows it. And he will know it a heap better about next Tuesday!”
“But look here, Saunders. Won’t Plaister stand to lose if he goes to ‘Jerry?’ Faculty always says that shop-keepers giving credit to the fellows will be deprived of the school trade. Seems to me Plaister will think twice before he risks that.”
“Oh, he will tell some hard-luck yarn and ‘Jerry’ will believe him. You know how ‘Jerry’ is. Barks a lot, but doesn’t bite much. Yes, he might be scared to do what he threatens, but his letter sounded mighty earnest. He’s got me going, anyway. I say, Bowman, I don’t suppose you—er—happen to have ten dollars you’d let me have? I’d have to pay it back fifty cents a week, but——”
“Sorry,” said Ted, shaking his head. To his surprise he found that he really was sorry—a little. Hal’s gloom enwrapped him again.
“No, I suppose not. And I don’t guess you’d care much about lending to me if you had it. You don’t particularly love me. Well, I guess I’ll toddle.” He arose and stood uncertainly a moment before he moved toward the door.
“What will you do?” asked Ted anxiously. “If—if you get put on ‘pro’ we’ll be in a nasty fix! Hang it, Saunders, you’ve got to do something, you know. Crouch would last about two innings in the Temple game! Why don’t you see Plaister to-morrow and get him to wait another week? After next Saturday it wouldn’t matter.”
“I’ve talked to him until I’m tired,” replied Hal wearily. “It’s no good. Maybe he won’t do it, or maybe I can scrape up the money by Monday. I’m tired worrying about it. I’d just as lief get fired as have this thing hanging over me all the time.”
“Maybe he would take part of it and wait for the rest.”
“He won’t. I tried that. He says he’s waited long enough and—oh, a lot of drivel. You know the way they talk. Well, good-night. And say, Bowman, just keep this to yourself, like a good chap, will you? I don’t
know why I bothered you with it, but I’d rather you didn’t say anything about it.”
“That’s all right. I won’t talk. Good-night. I hope you—come out all right.”
Hal nodded dejectedly and went. Ted took up his magazine, but after finding his place in it he let it drop once more. If Plaister did what he threatened, and Ted knew the hard-featured little shopkeeper well enough to feel pretty certain that he would, it would be all up with Warwick’s chances for the baseball championship that year With Hal Saunders in the points they might defeat Temple Academy next Saturday. Without him they couldn’t. Neither Crouch nor Bradford was good enough to last three innings against the Blue’s hard-hitting team. The knowledge brought real dismay to Ted. Personally he wanted a victory for the school team, but it was the thought of George’s disappointment that moved him most. George, like every captain, had hoped and worked for a triumph harder than any of the others. For Ted’s part, he would go back next year, but this was George’s last chance. Ted was miserably sorry for his friend. He was such a corking fine fellow Ted recalled the day last September when George, learning that fate in the shape of faculty had wished a strange and two years younger boy on him as roommate, had acted so mighty decent about it. Lots of fellows in George’s place, thought Ted, would have been mad and grouchy, but George had never let Ted guess for a moment that he wasn’t entirely welcome. And all through the year George had been a perfect brick. He had helped Ted in many ways: had got him into Plato Society, helped him at mid-year exams, introduced him to nice fellows, coached him in batting until he had become proficient enough to beat out Whipple for right field position. Ted’s feeling for George Tempest was a mingling of gratitude and hero-worship that amounted to a very real affection, and the thought of George’s unhappiness in case the final game of the school year went against Warwick troubled him greatly. Temple Academy had routed Warwick overwhelmingly last year and the sting of that defeat still remained. Warwick wanted revenge, and her three hundred and odd students had their hearts set on obtaining it next Saturday. But to none did it
mean quite what it meant to Captain Tempest. Ted tossed the magazine aside and stood up. “Something ought to be done,” he muttered.
In the bedroom he produced a small tin box from its hiding place in a dresser drawer and emptied the contents on his bed. Three onedollar bills and many silver coins, when counted, came to exactly fourteen dollars and seventy-five cents. He had been accumulating the hoard ever since Fall with the intention of buying a bicycle when he went home in the Summer. When he had about five dollars more he would have enough. He hadn’t told Hal that he didn’t have the money. He had merely politely refused to make a loan. And he had no idea of changing his mind. Hal’s fix was no affair of his, and Hal could get out of it as best he might. Certainly he couldn’t be expected to give up a whole Summer’s fun for the sake of a fellow he didn’t like much anyway! Resolutely he placed the money back in the box and the box again in concealment. “He will wriggle out of it somehow,” he said to himself.
Sunday was rainy and seemed weeks long, and Ted missed George horribly He saw Hal Saunders at dinner and again in the evening, and it was apparent from Hal’s countenance that he had not yet found a way out of his difficulty. Ted went over to the library after supper feeling very angry with Hal, angry because that youth had endangered the success of the nine, because his foolishness was in a fair way to bring grief to George, and because he had somehow managed to make one Ted Bowman distinctly uncomfortable! Ted surrounded himself with reference books, but all the work he did scarcely paid for the effort.
Ted did not say anything to George, when the latter returned on Monday, about Hal’s affairs. After dinner that day he received a summons to the Office, and although conscious of a clear conscience he couldn’t help feeling a trifle uneasy as he obeyed it. One didn’t get an invitation to confer with “Jerry” unless the matter was one of some importance. Events subsequently justified the uneasiness, for when Ted closed the Office door behind him the second time he was on probation!
