Agarandbrothdilutionmethodstodeterminetheminimalinhibitoryconcentration(MIC)ofantimicrobialsubstances
IrithWiegand,KaiHilpert&RobertEWHancock
CentreforMicrobialDiseasesandImmunityResearch,UniversityofBritishColumbia,2259LowerMallResearchStation,Vancouver,BritishColumbia,V6T1Z4,Canada.CorrespondenceshouldbeaddressedtoR.E.W.H.(bob@cmdr.ubc.ca).
©2008 Nature Publishing Group http://www.nature.com/natureprotocolsPublishedonline17January2008;doi:10.1038/nprot.2007.521
Theaimofbrothandagardilutionmethodsistodeterminethelowestconcentrationoftheassayedantimicrobialagent(minimalinhibitoryconcentration,MIC)that,underdefinedtestconditions,inhibitsthevisiblegrowthofthebacteriumbeinginvestigated.MICvaluesareusedtodeterminesusceptibilitiesofbacteriatodrugsandalsotoevaluatetheactivityofnewantimicrobialagents.Agardilutioninvolvestheincorporationofdifferentconcentrationsoftheantimicrobialsubstanceintoanutrientagarmediumfollowedbytheapplicationofastandardizednumberofcellstothesurfaceoftheagarplate.Forbrothdilution,oftendeterminedin96-wellmicrotiterplateformat,bacteriaareinoculatedintoaliquidgrowthmediuminthepresenceofdifferentconcentrationsofanantimicrobialagent.Growthisassessedafterincubationforadefinedperiodoftime(16–20h)andtheMICvalueisread.Thisprotocolappliesonlytoaerobicbacteriaandcanbecompletedin3d.
INTRODUCTION
Agardilutionandbrothdilutionarethemostcommonlyusedtechniquestodeterminetheminimalinhibitoryconcentration(MIC)ofantimicrobialagents,includingantibioticsandothersubstancesthatkill(bactericidalactivity)orinhibitthegrowth(bacteriostaticactivity)ofbacteria.Themethodsdescribedherearetargetedfortestingsusceptibilitytoantibioticagentsasopposedtootherantimicrobialbiocidessuchaspreservativesanddisinfectants.However,therearenomajorreasonswhytheycannotbeusedfortheseotherantimicrobials.Foragardilution,solutionswithdefinednumbersofbacterialcellsarespotteddirectlyontothenutrientagarplatesthathaveincorporateddifferentantibioticconcentrations.Afterincubation,thepresenceofbacterialcoloniesontheplatesindicatesgrowthoftheorganism.Brothdilutionusesliquidgrowthmediumcontaininggeometricallyincreasingconcentrations(typi-callyatwofolddilutionseries)oftheantimicrobialagent,whichisinoculatedwithadefinednumberofbacterialcells.Thefinalvolumeofthetestdefineswhetherthemethodistermedmacro-dilution,whenusingatotalvolumeof2ml,ormicrodilution,ifperformedinmicrotiterplatesusingr500mlperwell.Afterincubation,thepresenceofturbidityorasedimentindicatesgrowthoftheorganism.Inboththeagarandthebrothdilutionapproaches,theMICisdefinedasthelowestconcentration(inmglÀ1)oftheantimicrobialagentthatpreventsvisiblegrowthofamicroorganismunderdefinedconditions.
Inclinicalpractice,thisinvitroparameterisusedtoclassifythetestedmicroorganismaseitherclinicallysusceptible,intermediateorresistanttothetesteddrug.Theinterpretativestandardsfortheseclassificationsarepublishedbydifferentnationalorgani-zationssuchastheClinicalandLaboratoryStandardsInstitute(CLSI)1intheUSAandtheEuropeanCommitteeonAntimicrobialSusceptibilityTesting(EUCAST)2.Breakpoints(theparticularMICthatdifferentiatessusceptible,andassuminglytreatable,fromresistantandassuminglyuntreatableorganisms)arederivedfrommicrobiologicalandclinicalexperience,andcanvaryaccordingtotheparticularspeciesbeingexaminedandtheparticular
antimicrobialagent.FeaturesthatdefinethesebreakpointsareMICdistributionsofrelevantspecies,pharmacodynamicsandpharmacokineticsoftheantimicrobialagent,andclinicaloutcomedata.Resistance(abovethebreakpoint)isassociatedwithahighlikelihoodoftherapeuticfailure,whereassusceptibilityisassociatedwithagreaterprobabilityoftherapeuticsuccess.Forisolatesclassifiedasintermediate,thetherapeuticeffectisuncertain3.MICdeterminationscanbeusedformonitoringthedevelop-mentofantibioticdrugresistance.MICwild-typedistributiondatabasesareavailableforrelevantspecies–drugcombinations(http://www.eucast.org).ThehighestMICofthewild-typepopu-lationisdefinedasthe‘epidemiologicalcut-offvalue’orwild-type(WT)cut-offvalue3(Fig.1).OrganismswithacquiredresistancecanbeeasilyidentifiedbyshowinghigherMICvaluesthantheepidemiologicalcut-offvalue.Asevenslightchangesmaybecomeclinicallyrelevant,thedeterminationofMICisavaluablemeansforresistancesurveillance,aswellasprovidingavaluablecomparator
500450Number of tested isolates4003503002502001501005000.060.130.250.512816MIC (mg/l)432641282565121,024Species BSpecies AWild-typepopulationSensitiveBreakpointsWild-typepopulationnaturallyresistantIntermediateResistantIsolates withacquiredresistanceEpidemiologicalcut-off valuespecies AEpidemiologicalcut-off valuespecies BFigure1|DistributionofMICvaluesfordifferentisolatesforgivenspecies(modifiedfromWiegandandWiedemann27).
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forvariantsofagivenantimicrobialagentand/orspecieswithdifferentialsusceptibility.Indeedfornewdrugcandidates,theMICdeterminationisoneofthefirststepstoevaluatetheantimicrobialpotential.
Specializedprotocolscanalsoallowinferencestobedrawnregardingresistancemechanisms.Forexample,resultsfrombrothdilutionMICdeterminationwithcertainb-lactamantibiotics(cefotaxime,cefpodoximeand/orceftazidime)inthepresenceorabsenceofaninhibitor(clavulanicacid)canindicatetheproduc-tionofextended-spectrumb-lactamaseswhenMICsareatleastthreetwofoldconcentrationstepslowerinthepresenceoftheb-lactamaseinhibitor1.Epidemiologicalresistancedatafurther-slomoreprovidethebasisforappropriatefirst-linetherapyrecom-cotmendationsforempiricaltreatment.
orpDilutionmethodsareconsideredasreferencemethodsforeruinvitrosusceptibilitytestingandarealsousedtoevaluatethetaperformanceofothermethodsofsusceptibilitytesting.
n/mocCrucialparameters
.eruAsthetestresultsvarywidelyunderdifferenttestconditions,thetaprocedureshavetobestandardizedforintra-andinter-laboratorynw.reproducibility.Theprotocolsdescribedhereareadjustedtoastep-wwby-stepformatandfollowtheguidelinesofthetwoestablished//:porganizationsandcommittees,theCLSI1andEUCAST2.Modifica-tthtionsareintroducedfortestingthesusceptibilitytocationic pantimicrobialpeptidesandothercationicagentsthattendtoubindtosurfaces.IfimplementedrigorouslyaccordingtotheroGproceduresdescribedherein,thesemodificationsallowthe gngenerationofreliabledatathatwillbecomparablebetweenihsdifferentlaboratories.
ilbTheuseofallmethodsofthisprotocolislimitedtoaerobicuPbacteriathatgrowwellwithin24hintheCLSIandEUCAST errecommendedtestMueller–Hintongrowthmedium.Mueller–utaHintonbroth(MHB)isageneralpurposemediumthatcanbeNusedforcultivationofawidevarietyofnonfastidiousmicroorgan- 80isms.Forgrowthoffastidiousorganisms,suchasStreptococcusspp.,02Haemophilusinfluenzae,Neisseriagonorrheae,Helicobacterpylori©andCampylobacterspp.,thebrothneedstobesupplemented;furthermore,enrichmentoftheincubationatmospherewithCO2andanextensionoftheincubationtimemaybenecessaryforgrowth.Forthesespecies,specificrecommendationsformediumcompositionandfortestconditionscanbefoundintheCLSIguidelines1.
Medium
Toproduceaccurateandreproducibleresults,anumberofaddi-tionalrequirementsmustbefulfilledbythetestmediumforcertainantibioticsorantibiotic/speciescombinations:
Correctsusceptibilitytestingoftetracyclines4,5,daptomycinforgram-positivebacteria6andaminoglycosidesforPseudomonasaeruginosa5inbrothmediumisdependentonthecontentofCa2+andMg2+ions.Non-cation-adjusted(unsupplemented)MHBcontainsingeneralinadequateamountsofCa2+andMg2+ions(informationgivenbymanufacturer).Thebroth,therefore,needstobesupplementedwithdivalentcationswhentestingtheabovementionedantibioticsand/orantibiotic-speciescombina-tions.Thefinalconcentrationshouldbe20–25mgCa2+and10–12.5mgMg2+perliter1,whichreflectsthedivalentcationconcentrationinblood.Cation-adjustedMHBiscommercially
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availableandonlyneedstobefurthersupplementedwithCa2+incaseofdaptomycinsusceptibilitytesting,astherecommendedcalciumconcentrationfortestingthisantibioticis50mglÀ1(ref.1).Pleasenotehoweverthatcation-adjustedMHBshouldnotbeusedwhentestingtheactivityofcationicantimicrobialpeptides,asthepresenceofCa2+andMg2+ionscausesasubstantialinhibitionofthecationicpeptides’activity7,8.
Mueller–Hintonagar(MHA)needstobesupplementedwith2%(wt/vol)sodiumchloridefortestingsusceptibilityofStaphylococcusaureustomethicillin,oxacillinandnafcillin1.Methicillin-resistantS.aureus(MRSA)areoftenheteroresistantwithresistantandsusceptiblecellsinthesamecultureandsupplementationwithNaClenhancestheexpressionofhetero-geneousresistance9.
Toavoidtheadsorptionofdalbavancintoplasticsurfaces,theadditionofpolysorbate80tobrothatafinalconcentrationof0.002%(vol/vol)isrecommended.RefertotheCLSIguidelines1whentestingthisantibiotic.
Highlevelsofthymidineandthymineinterferewithsuscept-ibilitytestingofsulfonamidesandtrimethoprim10.ContrarytotheDifcoMHB(notcation-adjusted),theBBLMHB(notcation-adjusted)isnotexplicitlyformulatedtohavealowthymineandthymidinecontent.So,accordingtothemanufac-turer,onlytheformermaybeusedforbrothdilutionantimi-crobialsusceptibilitytesting.
Tigecyclineispronetooxidation,anditseemsthatitsactivityisaffectedbytheamountofdissolvedoxygeninthemedium,whichincreaseswiththeageofthebroth.So,forbrothdilutionMICtestswithtigecycline,itisnecessarytousefreshcation-adjustedMHB(o12hafterautoclaving)11.
Bacteria
Thebacteriasubjectedtoantimicrobialsusceptibilitytestingmustbeisolatedinpurecultureandshouldhavebeenidentifiedatthegenusandspecieslevel.Mostorganismsareavailablefromhospitallaboratories,theAmericanTypeCultureCollectionorothernationalcollections(seeTable1).
Inoculum
Thestandardizationofthebacterialcellnumberusedforsuscept-ibilitytestingisofcriticalimportanceforobtainingaccurateandreproducibleresults.Therecommendedfinalinoculumsizeforbrothdilutionis5Â105colony-formingunits(cfu)mlÀ1;the
TABLE1|Controlorganismsforantimicrobialsusceptibilitytesting.IdenticaltoATCCstrainEscherichiacoliATCC25922NCTC12241,CIP76.24,DSM1103
PseudomonasaeruginosaATCC27853NCTC12934,CIP76.110,DSM1117
StaphylococcusaureusATCC29213NCTC12973,CIP103429,DSM2569
EnterococcusfaecalisATCC29212
NCTC12697,CIP103214,DSM2570
ATCC,AmericanTypeCultureCollection,P.O.Box1549,Manassas,VA20108,USA;NTCT,National
CollectionofTypeCultures,HealthProtectionAgency,61ColindaleAvenue,LondonNW95EQ,UK;CIP,Collectiondel’InstitutPasteur,25–28RuedeDocteurRoux,75724ParisCedex15,France;DSMZ,DeutscheSammlungvonMikroorganismenundZellkulturen,Inhoffenstrae7B,38124Braunschweig,Germany.
PROTOCOL
appropriatecellnumberinagardilutionexperimentsissetat104cfuperspot.
HigherinoculacanleadtoanincreaseintheMICparticularlyifthetestedbacteriumproducesanenzymecapableofdestroyingtheantibiotic.Aninoculumeffect(e.g.,aneightfoldorgreaterMICincreaseupontestingwitha100-foldhigherinoculumthanrecommended)isfrequentlyseenwhentestingb-lactamsuscept-ibilityforisolatesthatproduceb-lactamasesthatareabletoinactivateb-lactamantibiotics12.Lighterinoculathanrecom-mendedmaygiveartificiallylowerMICs.Useofinoculawitho5Â105cfumlÀ1inbrothmicrodilutioncanleadtofalse-susceptibleresultsasdescribedforthedetectionofmethicillinresistanceinS.aureus13andforresistancetocertainb-lactamsinb-lactamaseoverproducingKlebsiellaoxytocaisolates14.
Afreshpurecultureshouldbeusedforthepreparationoftheinoculum.Toavoidtheselectionofanatypicalvariantclone,bacteriafromfourtofivenormal-appearingcoloniesaretakentoprepareabacterialsuspensionwithadensityequivalentto108cfumlÀ1,whichislaterusedforinoculation.
Severaloptionsareavailableforthegenerationofthebacterialsuspension(directcolonysuspensionintoliquidandgrowthmethodsusingeitherfreshorovernightcultures).Thedensityofthecellsuspensioncanbeassessedspectrophotometri-callyfortestingasmallnumberofdifferentbacterialisolates(no5).Foralargernumberofdifferentbacterialisolates,tosavetime,aturbiditystandardcanbeusedasavisualyardstick.Comparisonbetweenthestandardsandtheturbidityofthebacterialsuspensionswillinfactpointtheresearchertowardtheappropriatedilutionforthesuspension.Theturbidityofaso-calledMcFarland0.5standardisequalto1–2Â108cfumlÀ1.McFarland0.5turbiditystandardsarecommerciallyavailablefromseveralmanufacturers(e.g.,bioMerieux,cat.no.70900orScientificDeviceLaboratory).Alternatively,aBaSO4turbiditystandardequalingtheMcFarland0.5standardcanbepreparedasdescribedbelow.
Oncethebacterialsuspensionisadjusted,itmustbeusedwithin30mintoavoidchangesinthecellnumber2.
Allprotocolsdescribedherecontainaparagraphonhowtodeterminewhetherthecorrectinoculumdensitywasusedforthesusceptibilitytesting.IftheMICtestsarecarriedoutinalaboratoryonaroutinebasis,thecellcountsoftheinoculumneedtobedeterminedonlyperiodically.Forallotherusers,werecommendvalidatingtheaccuracyofproceduresforeverytest.
Qualitycontrol
Toverifythatthesusceptibilityresultsareaccurate,itisnecessarytoincludeatleastonecontrolorganismwitheverybatchofMICdeterminations.Controlorganismsareavailablefromdifferentstraincollections(Table1).TheMICsforroutinelyusedantibioticsforthequalitycontrolorganismsarepublished1,2andthetestvaluesforthecontrolstrainsshouldbewithinthepublishedrangetobeconsideredacceptable.
Limitations
TheMICvaluedoesnotgiveanindicationofthemodeofaction(cidalorstatic)oftheantimicrobialagent.WithintheMICwellortubeorontheagarplatewithnovisiblegrowth,theremaystillbeviablecellsifthedrughadabacteriostaticeffectonthebacterialspeciestested.Growthmayresumeaftertheremovalofthedrug.Alternatively,theremaybepartialinhibitionresultinginimpairedandreducedgrowthratesandconsequentlynovisiblegrowthwithinthetimegiven.Bothphenomenaaredifferentfromtheactionofabactericidaldrug,whichcausesirreversibledamageleadingtocelldeath.
Furthermore,evenwiththeknowledgeofthemodeofactionofanantimicrobialagent,theMICvaluealoneisapoorpredictoroftheefficacyofthedruginvivo.Factorsthataffecttheresponsetotherapyarefarmorecomplexandincludehostdefensemechan-isms,underlyingdiseasesofthepatient,thesiteofinfection,andthepharmacokineticandpharmacodynamicpropertiesofthedrugs15.AlternativemethodfordeterminingMICvalues
MICsforcommonlyusedantibioticscanbeobtainedusinganagardiffusionmethodwithcommerciallyavailablestripscontaininganexponentialgradientofantibiotic(Etest;ABBiodisk).Theantibioticdiffusesintotheagarmediuminoculatedwithalawncultureofthetestorganism.Afterovernightincubation,theMICisreadatthepointofintersectionofanellipticalgrowthinhibitionzonewiththestripthathasanMICscaleprintedonit.Thistesthasbeenevaluatedforavarietyofbacteria/antibioticcombinations16–21andisrapidandeasytouse;however,itislimitedtotheantibioticrangesuppliedbythemanufacturerandisanexpensivetesttouseforscreening.Severalautomatedsystemsforantimicrobialsusceptibilitytest-ingandidentificationofclinicallyrelevantbacteriaarenowcommerciallyavailable,e.g.,PhoenixAutomatedMicrobiology
´r-System(BDDiagnosticSystems),theVITEK2System(bioMeieux)andtheMicroScanWalkAway-96System(DadeBehring).Thesesystemsarecosteffectiveforclinicallaboratorieswithahighthroughputofclinicalspecimens.Fullyautomatedsystemsreducethetimeforsetupand,dependingonthesystem,alsoreducethetimetoproduceresultscomparedtoconventionaltests.Moreover,theyofferconvenientinterfaceswithlaboratoryandhospitalinformationsystems.However,whentestingcertainorganism-antimicrobialcombinationslimitationsontheaccuracyoftheassessmentofMICvaluesbythesesystemsareknown22,23.Experimentaldesign
Astherearealternativeroutesforgeneratingthebacterialsuspen-sionwithdifferenttimerequirementsandalternativemethodstodetermineMICvalueswithpotentialpausepointoptionsthatrequireadvanceplanningoftheworkflow,theexperimentshouldbecarefullydesignedbytheuserbeforestartingtheprotocol.TheflowchartinFigure2illustrateshowthedifferentstagesarecoordinated.Pleaseexaminethisfigurecarefullytomakeaninformedchoiceastowhichexperimentalapproachtoembarkon.
©2008 Nature Publishing Group http://www.nature.com/natureprotocolsMATERIALS
.MHB(Difco;BDDiagnostics,cat.no.275730)sterilizedbyautoclaving.Mueller–HintonIIbroth(cation-adjusted(CAMHB);BBL,BDDiagnostics,
cat.no.212322)sterilizedbyautoclaving
REAGENTS
.MHA(Difco;BDDiagnostics)
.Agar,Technical(Difco;BDDiagnostics,cat.no.281230)
.SolutionA:0.02%aceticacid(Fisher)containing0.4%BSA(Boehringer
Mannheim)
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Pure cultures ofbacterial isolates(test and control)
or
Prepare overnight cultures
Prior to testing(2 d required)
Determine the cell count in
overnight cultures
Prepare media (store at 4 °C) and antibiotic
stock solutions (freeze)
Day 1
Preparation of bacterial suspension
Incubate on nonselective
agar overnight
or
Prepare overnight cultures in
liquid
Day 2
or4–6 h growth method
Prepare agarplates withantibiotics
Antimicrobial susceptibility testing— part Ior
Prepare
or
broth macrodilutions ofantibiotic
Preparebroth microdilutions ofantibiotic
or
Preparebroth microdilutions ofpeptide
©2008 Nature Publishing Group http://www.nature.com/natureprotocolsColony suspension
Growth method usingovernight cultures
[Day 1 may bepossible
depending on theantibiotic (store at 4 °C)]
[Day 1 may bepossible (freeze)]
Adjust turbidity using aMcFarland Standard 0.5
or
Adjust turbidity using aspectrophotometer
Suspension with 1–2 × 10 cfu/ml
8
Take sample for cell count (if
used for agar dilution)
Antimicrobial susceptibility testing— part ll
Inoculate agar
plates
or
Inoculatebroth macrodilutions ofantibiotic
or
Inoculatebroth microdilutions ofantibiotic
or
Inoculatebroth microdilutions ofpeptide
Take sample for cell count
Day 3
Determine cell count
Read MICs
Figure2|Flowchartforantimicrobialsusceptibilitytesting.
.SolutionB:0.01%aceticacidcontaining0.2%BSA
.PreparationofMcFarlandStandard0.5:BaCl2Á2H2O(Sigma-Aldrich,cat.
.Shaker,suitablefortesttubes13Â100mm
.Parafilm(PechineyPlasticPackaging;FisherScientific,
.Glasstubes,13Â100mm(VWRInternational,cat.no.47729-572)with.Erlenmeyerflasks
.Sterilepetridishes,15Â100mm(Fisherbrand;FisherScientific,.0.2-mmporesizecelluloseacetatefilters(Nalgene;FisherScientific,.Cellspreader(Fisherbrand;FisherScientific,cat.no.08-100-11).Inoculationlooporcottonswabs(sterilizedbyautoclaving).Vortexmixer
REAGENTSETUP
PreparationofMcFarland0.5BaSO4turbiditystandardPreparea1.175%(wt/vol)bariumchloridedihydrate(BaCl2Á2H2O)solution(0.048mollÀ1BaCl2)anda1%(vol/vol)sulfuricacid(H2SO4)solution(0.18mollÀ1,0.36N).Add0.5mlofthe1.175%BaCl2solutionto99.5mlofthe1%H2SO4solutionwithconstantstirringtogetasuspension.Measuretheopticaldensityoftheturbiditystandardusingaspectrophotometerwitha1cmlightpathlength.Thecorrectabsorbanceat625nmshouldbe0.08–0.13.Aliquot4–6mlintoscrew-cappedglasstubes.Thetubesshouldhavethesamesizeasthoseforpreparingthebacterialsuspensionforinoculation.Sealtubestightlywith
Parafilmandstoreinthedarkatroomtemperature(20–23.51C).Standardsarecat.no.190-2520)cat.no.08-75-712)
cap(UtechProducts,cat.no.1017622),sterilizedcat.no.13-374-10)
no.B0750),H2SO4(Fluka,cat.no.84721)!CAUTIONH2SO4isverycorrosive/toxic;handlingmustbeperformedunderthehood;wearacid-resistantglovesandprotectiveclothing(seeREAGENTSETUP).
.Cationadjustment:MgCl2Á6H2O(Fluka,cat.no.63072),CaCl2Á2H2O(Fluka,cat.no.21101)
.Physiologicalsaline[0.9%(wt/vol)NaCl]sterilizedbyautoclavingEQUIPMENT
.Spectrophotometersuitableformeasuringatwavelengthsof600and625nm
.Forantibiotics:sterile96-wellmicrotiterplates.Werecommendpolystyreneplates(BDFalcon;FisherScientific,cat.no.351177)formostantimicrobialsastheseareeasiertoreadattheendoftheexperiment
.Forcationicantimicrobialagentssuchaspeptides:96-wellpolypropylenemicrotiterplates(Costar,cat.no.3790)!CAUTIONAvoidtissueculturetreatedorpolystyreneplatesasthesearestronglynegativelychargedandwillnonspecificallybindpeptides.
.Eppendorfpolypropylenemicrocentrifugetubes,1.5ml(FisherScientific,cat.no.05-402-24B),sterilized
.Screw-cappedglasstubes,13Â100mm(FisherScientific,cat.no.14-930-10A)
.48-Pinreplicator(BoekelScientific,cat.no.140501)forinoculatingagardilutionplates
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PROTOCOL
stableforatleastamonth.!CAUTIONH2SO4iscorrosive/toxic;wearappro-priatesafetyclothingwhenhandlingconcentratedH2SO4.
Preparationofantibiotic-freenutrient-richagarplatesPrepareagarmed-iumaccordingtothemanufacturer’sinstructions.Alternatively,usenutrient-richbrothaccordingtothemanufacturer’sinstructionsandadd1.7%agar(17gagarperliter)beforeautoclaving.Approximately20–25mlisnecessarytopourone15Â100mmpetridish.Afterautoclaving(e.g.,1211C,15min,1bar),coolthemediumto50–601C.Pouragarintothepetridishesandallowtoset.Drythesurfaceoftheagarplateseitherinanincubatororinalaminarairflowhoodfor30minwiththelidajar.Storeagarplatesinplasticbagsininvertedposition(bottomfacingup)at41C.
AdjustmentofcationcontentofMHBmedium(20–25mgCa2+and10–12.5mgMg2+perliter)Preparea10mgmlÀ1Mg2+stocksolutionbydissolving8.36gofMgCl2Á6H2Oin100mldeionizedwater.Preparea10mgmlÀ1Ca2+stocksolutionbydissolving3.68gofCaCl2Á2H2Oin100mldeionizedwater.Filter-sterilizebothstocksolutionsusing0.2-mmporesizecellulose-acetatefilters.PrepareMHBaccordingtothemanufacturer’sinstructions,autoclaveandcoolthemediumto2–81Cbeforetheadditionofthecationsolutions.Add100mlofstocksolutionper1mglÀ1neededfor1lofmedium.Forexample,add2mlofCa2+stocksolutionif20mgneedstobeaddedto1lMHB.StocksolutionoftheantimicrobialagentAntimicrobialagentsshouldbestoredinthedarkat41Cinsealedcontainerscontainingadesiccantunlessrecommendedotherwisebythemanufacturer.Weadvisethestorageof
antimicrobialpeptidesat201C.Beforeweighingtheantimicrobialagent,letthecontainerwarmatroomtemperatureforB2htoavoidcondensationofwateronthepowder.Antibioticsaregenerallysuppliedbythemanufacturerwiththeinformationaboutthepotency(mgpermgpowder)thatneedstobetakenintoconsiderationwhenweighingtheagent.Forantibiotics,prepareastocksolutionat10mgmlÀ1whenplanningtouseitforagardilution(Step4A).Forbrothdilutiontests(Steps4Band4C)setupastocksolutionwithaconcentrationat
leasttentimeshigherthanthehighestconcentrationtobetested.Fortestingantimicrobialpeptides(Steps4Dand4E),preparea20-foldconcentratedstocksolution.Usethefollowingformulaforcalculatingtherightamountofantibiotictobeweighed(thisdoesnotapplytoantimicrobialpeptides):W¼
ðCÂVÞP
©2008 Nature Publishing Group http://www.nature.com/natureprotocolswhere,W¼weightofantimicrobialagentinmilligramtobedissolved;
V¼desiredvolume(ml);C¼finalconcentrationofstocksolution(mgmlÀ1);P¼potencygivenbythemanufacturer(mgmgÀ1).
Usesterilecontainersandspatulaforweighingtheantimicrobialagentanddissolveinsteriledistilledwaterorintherecommendedsolvent.Alistof
solventsforfrequentlyusedantibioticsisfoundinref.24.Antibioticsolutionscanbefilter-sterilizedusinga0.2-mmporesizecellulose-acetatefilter.
However,ithastobeascertainedthattheantibioticdoesnotbindtocelluloseacetate(informationthatissometimesgivenbythemanufacturer).Donotfilter-sterilizeantimicrobialpeptides,whichtendtobindtoanionicsurfaceslikecelluloseacetate.Alwaysusethefreshantibioticstocksolutionforbrothmicrodilutionifitisplannedtofreezetheantibioticcontainingmicrotiterplatesat701Cforlaterusage.Forotherapplications,aliquotthestock
solution.Thevolumeofthealiquotsdependsonthedownstreamapplicationsandingeneralonealiquotshouldcontainthevolumeneededforonetest.Containersneedtobesterile,coldresistantandshouldsealtightly(e.g.,forsmallervolumessterileEppendorftubescanbeused).Storethealiquotsat201Corbelowunlessitisinstructedotherwisebythemanufacturer.MostantimicrobialagentsarestableatÀ601Cforatleast6months.Stabilityandstorageinformationforfrequentlyusedantibioticscanbefoundinref.24.Donotrefreezethawedstocksolutions.Someantimicrobials,particularlyb-lactamsantibiotics,candegradewhenthawedandrefrozenrepeatedly1.
PROCEDURE
PreparationofthebacterialsuspensionTIMINGB5minperisolate/overnightpausepoint
1|Streakthebacterialisolatestobetested(includingacontrolorganism)ontonutrient-rich(e.g.,Mueller–Hinton)agarplateswithoutinhibitortoobtainsinglecolonies.
2|Incubateplatesfor18–24hat371C.
3|Differentmethodsforthepreparationoftheinoculumcanbeused.Directsuspensionofovernightcoloniesintobrothorsterilesalinesolution(optionA)isaveryconvenientmethodthatcanbeusedformostbacterialspecies.Itisparticularly
recommendedforfastidiousorganismssuchasStreptococcusspp.,Haemophilusspp.andNeisseriaspp.Forsomestrainswithinaspecies,unpredictableclumpingcanoccurwithoptionA.Consequently,whencoloniesaredifficulttosuspendandanevensuspensionisdifficulttoachieve,freshlygrownbrothculturescanbediluted(optionB).Asanalternativetoafreshlygrownculture,anovernightbrothculturecanalsobeused(optionC)accordingtotheuser’spreference.ThismethodisnotpartofCLSIorEUCASTrecommendations;however,theoptiontouseovernightculturesisgivenintheguidetoantimicrobialsusceptibilitytestingoftheBritishSocietyofAntimicrobialChemotherapy24.(A)ColonysuspensionmethodTIMINGB5minperisolate(i)Preparetheantibioticorpeptidedilutions.
(ii)Foreachisolate,selectthreetofivemorphologicallysimilarcoloniesfromthefreshagarplatefromStep2andtouch
thetopofeachselectedcolonyusingasterilelooporcottonswab.Transferthegrowthintoasterilecappedglasstubecontainingsterilebrothorsalinesolution.Mixusingavortexmixer.
(iii)TurbiditycanbeassessedvisuallybycomparingthetestandtheMcFarlandStandard.MixtheMcFarland0.5BaSO4stan-dardvigorouslyusingavortexmixer.Pleasenotethatcommerciallyavailablestandardscontaininglatexparticlesshouldnotbevortexed,butgentlyinvertedseveraltimes.Comparisonagainstawhitebackgroundwithcontrastingblacklinesandgoodlightingarehelpful.Alternatively,theturbiditycanbeverifiedmeasuringtheabsorbanceofthesuspensionspectrophotometrically.TheabsorbanceshouldbeinthesamerangeasthatoftheMcFarlandstandard0.5(OD625nmshouldbeat0.08–0.13).
(iv)Adjustthesuspension’sturbiditytothatofaMcFarlandStandard0.5byaddingsteriledistilledwater,salineorbroth,if
theturbidityistoohigh,orbyaddingmorebacterialmaterialifistoolow.
mCRITICALSTEPAfterturbidityadjustment,thebacterialsuspensionshouldbeusedwithin30min,asthecellnumbermightotherwisechange.
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(B)GrowthmethodTIMING2–6h
(i)Foreachisolate,selectthreetofiveisolatedcoloniesofthesamemorphologicalappearancefromthefreshagarplate
(fromStep2),touchthemusingasterilelooporswabandtransferintoatubecontaining3–4mlofasuitablenutrient-richmedium(e.g.,MHB).Mixusingavortexmixer.
(ii)Incubatethebrothat35–371Cinashakerat225r.p.m.untilitreachesavisibleturbiditythatisequaltoorgreater
thantheturbidityofaMcFarlandStandard0.5.Pleasenotethattheculturegrowthwillrequire2–6hdependingongrowthrateofthebacteriatobetested.Thispauseperiodcanbeusedtopreparetheantibioticorpeptidedilutions.(iii)ChecktheturbidityaccordingtoStep3A(iii).
(iv)Adjustthesuspension’sturbiditytothatofaMcFarlandStandard0.5byaddingsteriledistilledwater,salineorbroth,if
theturbidityistoohigh,orbyfurtherincubatingthebacterialcultureiftheturbidityistoolow.
mCRITICALSTEPAfterturbidityadjustment,thebacterialsuspensionshouldbeusedwithin30minasthecellnumbermightotherwisechange.
(C)GrowthmethodusingovernightculturesTIMINGB5minperisolateSteps(i)–(iii)/overnightpause
point/B15minperisolateSteps(iv)–(viii)/overnightpausepoint/B20minperisolateSteps(ix)–(xiv)/overnightpausepoint/B5minperisolateSteps(xv)–(xvi)
(i)DeterminethecorrelationbetweenOD600ofanovernightcultureandthemicrobialnumberforeach
isolateperformingthefollowingsteps(Steps(ii)through(xi))intriplicate.Thisprocedureisnecessary
becauseliquidovernightculturesmayprovelessviablethanfreshlygrownbrothculturesmakingasimpleturbiditytestunreliable.
(ii)Selectonecolonyfromthefreshagarplate,touchitusingasterilelooporswabandinoculateasterileglasstubewith
cap(13Â100mm)containing3mlnutrient-richmedium(e.g.,MHB).(iii)Incubatethetubesovernightinashakerat225r.p.m.at371C.
(iv)MeasuretheOD600oftheovernightcultures.BecauseofthelossoflinearityatOD600values41.0,itisnecessaryto
dilutethesampleuntiltheOD600valueisbelow1.0.Formostbacterialspecies,adilutionof1:30mightbeappropriate(33mlin967mlbroth).
(v)Dilutetheovernightculture1:100usingsteriletubesandnutrition-richbrothorsterilesalinesolution(dilution:10–2)(vi)DilutestepwisethesolutionfromStep(v)fivetimes1:10,usingsteriletubesandnutritionrichbrothorsterilesaline
solutionuntilyoureachadilutionof10–7.
(vii)Plate100mlofthelastfourofthe1:10dilutions(10–4to10–7)evenlyontonutrient-richagarplatesusingasterile
cellspreaderstartingwiththedilution10–7.(viii)Incubateplatesovernightat371C.
(ix)Countcoloniesonplates(takingintoaccountonlyplateswithupto500colonies).
(x)ItisbesttodeterminetherelationshipbetweenOD600andthemicrobialnumberbasingthecalculationonplates
displayinganumberofcoloniesbetween100and400.Highernumbersdonotaccuratelyrepresenttheoriginalsuspensionaserrorscreatedbycoincidenceofcoloniesandnutrientlimitationleadtonumbersthatarelowerthanexpected.Relyingonfewerthan100coloniesmight,ontheotherhand,leadtoincorrectconclusionsduetotheadditionofstatisticalandmethodologicalerrorswhileperformingthedilutions.
(xi)Calculatethecfupermlthatwereintheovernightcultureaccordingtothefollowingformula:
©2008 Nature Publishing Group http://www.nature.com/natureprotocols
N¼
CÂ1010ÀD(xii)
(xiii)
(xiv)(xv)(xvi)
where,N¼cfumlÀ1;C¼numberofcoloniesperplate;D¼numberofthe1:10dilution.
AveragetheresultsfromthreetestsperformedwiththemicrobialisolateandcorrelatethisnumberwiththeOD600valueobtainedfromtheovernightculture.Thisrelationshipholdstrueforsubsequentculturesofthesamebacteriumgrowninthesameway.
ForthegenerationoftheovernightculturestobeusedforpreparationofthebacterialsuspensionforMICtests,selectthreetofivemorphologicallysimilarcoloniesforeachisolatefromafreshagarplate.Touchthetopofeachselectedcolonyusingasterilelooporcottonswab.Inoculateasterileglasstubewithcap(13Â100mm)containing3mlnutrient-richmedium(e.g.,MHB).
Incubateovernightat371C.BeforestartingwithStep3C(xv),preparetheantibioticorpeptidedilutions.MeasuretheOD600oftheovernightculture(diluteyoursampleuntilOD600valueiso1.0).
CalculateadilutionfactoroftheovernightcultureusingthecorrelationfromStep3C(xii)toobtainasolutionthatwillcontain1Â108cfumlÀ1andadjustaccordingly.
mCRITICALSTEPAfterturbidityadjustment,thebacterialsuspensionshouldbeusedwithin30minasthecellnumbermightotherwisechange.
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Antimicrobialtesting
4|Brothandagardilutionareroutinelyusedmethodsforantimicrobialsusceptibilitytesting.Aslargervolumesof
antimicrobialagentsareneededforagardilution,thismethodisapplicabletoantibioticsavailableinampleamounts.Themainadvantageoftheagardilution(optionAbelow)istheoptiontotestalargenumberofbacterialisolatessimultaneouslyunderexactlyidenticalconditions(e.g.,48spotscanbeplacedontooneagarplateusingpetridishesof100Â15mm).Agardilutionisnotthemethodofchoiceifthesusceptibilitytoawiderangeofdifferentantibioticsistobetestedonasmallernumberofbacterialisolates.Brothdilution(optionsBandCbelow)isequallyapplicableforeithertestingdifferentantibioticsoralargenumberofbacterialisolates.Usingamicrotiterformathastheadvantagethatplatescanbepreparedinadvance,storedat201Candcanbeusedatthetimepointneeded.Brothmacrodilution(optionB)requireslargevolumesofantibiotic.Werecommendusingbrothmicrodilution(optionC)formostroutineprocedures.PleasenotethatneitheroptionBnoroptionC,butmodifiedversionsofthemicrodilutionprocedure(optionsDandE)shouldbeusedforantimicrobialpeptidesandhighlycationicantibiotics(e.g.,polymyxinB,gramicidinS,cecropin).Themodificationsinthemicrodilutionprocedureforantimicrobialpeptidesandothercationicantimicrobials(optionsDandE)areintendedtoavoidthepossibilityofthe
antimicrobialsbindingtotheplasticdishesandfurthermoretoreducetheamountofsubstanceneededfortesting,whilestillproducingacceptableresults.Table2illustratesthesubstantialvariationsinMICthatcanoccurwithdifferentpeptidesolventsandwhentheincorrecttypeoftestvesselisutilized.SomepeptidescanprecipitatewhendilutedintoMueller–Hintonmediumwhichcanbeavoidedbyaddingaceticacid/BSA25.Whetherantimicrobialpeptidesrequire(optionD)ornot(optionE)theadditionofaceticacid/BSAhastobedeterminedbytrialanderror.IftheMICofanantimicrobialpeptidedeterminedbyoptionsDandEforthesametestisolateistwoormoredilutionstepshigherforoptionE(noBSA/aceticacid)comparedtooptionD(withtheadditionofaceticacid/BSA)itcanbeassumedthattheantimicrobialpeptiderequiresaceticacid/BSA.Irrespectiveofthemethodused,twofolddilutionserieswitharound10–12dilutionsupand/ordownfrom1mglÀ1
(1mgmlÀ1)areconventionallyusedformostantibacterialagents.However,theconcentrationrangewilldependonthebacterialisolatestobetestedandtheantibacterialagent(seeref.24forsuggestions).(A)AgardilutionTIMING4–6h
(i)PrepareMHAmediumaccordingtothemanufacturer’sinstructions.Alternatively,usetheMHBaccordingtothe
manufacturer’sinstructionsandadd1.7%agar(17gagarperliter)beforeautoclaving.Around25mlisnecessarytopourone15Â100mmpetridishtoproducetherequireddepthof3–4mm.(ii)Afterautoclaving(e.g.,1211C,15min,1bar),coolthemediumto501C.
mCRITICALSTEPHighertemperaturesmightinactivatetheantibiotic,whereasatlowertemperaturestheagarwillbeginformingsolidclumpsmakingitdifficulttopourhomogeneousplates.
(iii)Asthemediumcoolsdown,calculatetheamountofantibioticsolutions(10,1and0.1mglÀ1)needed.Table3gives
anexampleofthevolumesneededforaconcentrationrangebetween0.125and128mglÀ1forone25mlagarplateperconcentration.Adjustifadifferentconcentrationrangeisneeded.Thevolumesofantibioticandagarcanalsobevarieddependingonthenumberofplatestobepoured.Aseachagarplatecanbeusedforupto48tests,morethanoneplatemightbenecessaryifalargeramountofisolatesisgoingtobetested.
(iv)Label12(adjustingthenumberifmoreorlessthan12differentconcentrationsaregoingtobetested)sterilecontainers
appropriately(e.g.,glassErlenmeyerflasksclosedwithmetalcaps,tinfoilcapsorcottonbuds)withthefinalantibioticconcentration.Thesizeoftheflaskdependsonthetotalvolumeneeded;e.g.,usea100-mlflaskfor50mlofagar.(v)Dilutethe10mgmlÀ1antibioticstocksolution1:10insterilebrothorwatertoachievea1mgmlÀ1solution.(vi)Dilutethe1mgmlÀ1solution1:10insterilebrothorwatertoachievea0.1mgmlÀ1solution.(vii)Dispenseappropriateamountsofantibioticsolution(Table3)intotherespectivecontainers.
(viii)Foreachagarplate,add25mlagar(nowatatemperatureofB501C)intothecontainer,mixwell(avoidbubbles)and
pour25mlintoapetridishlabeledwiththerespectiveantibioticconcentration.Usea25-mlpipetteifthecontainercontains425mlagarinsituationswheremorethanoneplatewiththesameantibioticconcentrationisgoingtobepoured(seeStep3A(iii))
(ix)Pouracontrolagarplatewithoutanyantibiotic.Adjustthenumberifnecessary(seeStep3A(iii)).
©2008 Nature Publishing Group http://www.nature.com/natureprotocols
TABLE2|EffectofMICdeterminationconditionsontheMICvaluerecordedforcationicantimicrobialpeptides.PeptidesolventAceticacid/BSAdH2O
AceticacidAceticacid/BSAdH2O
*Describedas‘tissue-culturetreated’.
MicrotiterplatePolypropylenePolypropylenePolypropylenePolystyrene*Polystyrene*
MICofCP-26(lgmlÀ1)(a-helicalpeptide)
0.51.02.01.08.0MICofCP-11CN(lgmlÀ1)(extendedpeptide)
2.04.04.04.016.0
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(x)Allowagartoset.TABLE3|Antibioticdilutionchartforagardilutionmethod.(xi)DrythesurfaceoftheagarplateseitherinanVolumeofantibioticFinalconcentrationAntimicrobial
incubatororinalaminarairflowhoodfor30min.stocksolutionawhenaddingconcentration
À1Leavethelidajar.(ll)25mlagar(mgl)
(xii)Markthebottomoftheagarplatestodefinean10320128
orientation.1016064
108032’PAUSEPOINTPlatesshouldideallybeusedonthe
104016sameday.However,dependingontheantibioticused
2008(seeinformationoccasionallyprovidedbythemanufac-1
11004turerorref.26),theplatescanbestoredat4–81Cfor1502upto5d.
0.12501
(xiii)Mixthebacterialsuspension,adjustedto1Â
0.11250.5
108cfumlÀ1fromStep3A(iv),Step3B(iv)orStep0.162.50.253C(xvi),byvortexinganddiluteit1:10intoacavityof0.131.250.125asterile96-wellmicrotiterplatebypipetting10mlintoawellcontaining90mlofsterilebrothorsaline.
(xiv)Repeatforeachbacterialisolatetobetested.Besuretomakeanoteofthecontentofeachwellandtoinoculatethe
microtiterplateinawaythattheinoculacanbetransferredtotheagarplatesusinga48-pinreplicator.Forupto48tests,inoculateonlywithinrowsA–H,columns1–6ofthemicrotiterplate.Asanalternativetothereplicator,amultichannelmicropipettesetat1mlcanbeusedtodeliverthespots.
mCRITICALSTEPMakesurethattherequirednumberofbacterialcellsisgoingtobetransferred.A48-pinreplicatorwith1.5mmpinsdelivers1ml.Thefinalinoculumforaspotwithasizeof5–8mmshoulddeliverthedesiredcelldensityofaround104CFUperspot.AdjustthedilutionofthebacterialsuspensioninStep4A(xiii)ifareplicatorwithdifferentpindiameterisused.
(xv)Sterilizethe48-pinreplicatorbysoakingthepinsin95%ethanolandpassingthemthroughaBunsenburner
flame.Holdthepinsinuprightpositionuntiltheflameextinguishes.Letthepinscoolinaninvertedpositiontomaintainsterility.
!CAUTIONCareshouldbetakentoholdtheflamingreplicatorawayfromhairandclothing.Incasethealcoholbathaccidentallyignites,haveaglassplateavailablethatcanbeusedtocoverthealcoholbathtoextinguishthefire.(xvi)Placethesterilizedreplicatorintothemicrotiterplatetosoakthepinsandtransferitontotheagarplate.
Startbyinoculatingagrowthcontrolplatewithoutantibiotic.Makesurethateachagarplatehasthesame
orientationwhileinoculatingandtomakeanoteoftheorientationsothatspotsontheagarplatecanbeassignedtotherespectiveisolatetested.
mCRITICALSTEPMakesurethemicrotiterplatecontainingthebacterialsuspensionisnotcross-contaminatedbyliquiddrippingfromthepins.
(xvii)Inoculatetheantibiotic-containingagarplatesstartingwiththelowestconcentration.(xviii)Lettheinoculumspotsdryatroomtemperaturebeforeinvertingtheplates.
(xix)Toconfirmthatthesizeofthebacterialinoculumwasappropriate,determinetheviablecountofthebacterial
suspensionusedforpreparingtheinitialinoculum.Dilutethissuspension1:100bypipetting10mlintosterilecappedEppendorftubescontaining990mlnutrient-richbrothorsterilesalinesolution(dilution:10–2).Dilutethis10–2solutionsequentially1:10threetimesuntilyoureachadilutionof10–5.
(xx)Plate100mlofthelasttwo1:10dilutions(10–4to10–5)evenlyontoantibiotic-freenutrient-richagarplatesusinga
sterilecellspreader.
(xxi)Incubateagarplatesat371Cfor16–20h.
(xxii)CountcoloniesthenextdaywiththesameconsiderationsasdiscussedinStep3C(x).(B)BrothmacrodilutionTIMINGB20–45minperantibiotic
(i)PrepareantibioticdilutionsinsterileMHBinsteriletesttubesaccordingtoTable4.Astheantibioticsolutionislater
inoculatedwithanequalamountofbacteriainbroth,thedilutionsarepreparedataconcentrationtwicethedesiredfinalconcentration.Startbydispensingsterilebrothintotwelvesterile13Â100mmtubesclosedwithmetalcaps.Asingle10mlpipettecanbeusedtopipettethe9mland3mlvolumesofbrothintotherespectivetubes(seeTable4).Useasingle1mlpipetteforpipettingthe1mlofbrothinstages2,5,8and11.
(ii)Itispossibletousethesamepipetteforpipetting1mloftheantibioticstocksolutionintothefirsttesttube.
Mixthoroughlyusingavortexmixer.
(iii)Useseparatepipettes/pipettetipswhenpreparingeachoftheotherantibioticsolutionsaccordingtoTable4.
Mixthoroughlyusingavortexmixer.
©2008 Nature Publishing Group http://www.nature.com/natureprotocols
170|VOL.3NO.2|2008|NATUREPROTOCOLS
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TABLE4|Schemeforpreparingantibioticdilutionstobeusedinbrothdilutionsusceptibilitytests*.
Antimicrobialconcentration(mglÀ1)12801281281281616162220.250.25
Volumeofantibioticstocksolutionw
(ml)(+)
111111111111
Volumesterilebrothw(ml)(¼)
913713713713
Antimicrobialconcentrationobtained12864321684210.50.250.1250.06
Finalconcentration
intest64321684210.50.250.1250.060.03
©2008 Nature Publishing Group http://www.nature.com/natureprotocolsStage123456789101112SourceStockStage1Stage1Stage1Stage4Stage4Stage4Stage7Stage7Stage7Stage10Stage10
*ModifiedfromEricssonandSherris28.wThevolumesusedcanbeanymultipleofthesefiguresdependingonthenumberoftheteststobeperformed.Formacrodilution,1mlofeachantibioticdilutionisneededforonebacterialisolatetobetested.
(iv)Foreverybacterialisolate,labeltwelvesterile13Â100mmtesttubesclosedwithcottonbudsormetalcapswiththe
respectiveantibioticconcentrationtobetested.Alsolabelcontroltubesforbacterialgrowth(oneforeachisolatetested)andasingletubeforsterilitycontrolfortheentiremeasurement.
(v)Add1mlofeachantibioticdilutionintoonetesttubeforeachisolatetobetested.Fillthecontroltubeswith1ml
sterilebrothwithoutantimicrobialagent.Mixthebacterialsuspensionadjustedto1Â108cfumlÀ1fromStep3A(iv),Step3B(iv)orStep3C(xvi)byvortexing,anddiluteitbyafactorof1:100byadding200mlbacterialsuspensionto19.8mlsterileMHBinasterile50mlErlenmeyerflasktopreparea20mlinoculum.Mixwell.Adjustvolumesifnecessary.
(vi)Inoculateeachtesttubecontainingtheantibioticsolutionandonecontroltesttube(growthcontrol)with1mlofthe
bacterialsuspensionfromStep4B(v).Thisresultsinthefinaldesiredinoculumof5Â105cfumlÀ1.mCRITICALSTEPMakesurethatthebacterialsuspensioniswellmixedbeforeinoculatingeachtube.
(vii)Removea10mlsamplefromthegrowthcontroltubeimmediatelyafterinoculatingandpipetteitintoasterile
Eppendorftubeholding990mlofsterilesalineorbroth.Mixwellbyvortexing.Makeafurtherdilutionofthissuspension(1:10)bypipetting100mlinto900mlofsterilesalineorbrothandmixwell.
(viii)Plate100mlofeachofthetwodilutionsfromStep4B(vii)ontotwodifferentnutrient-richagarplates.(ix)Incubateagarplatesandtubesat371Cfor16–20h,shakingthelatterwithat225r.p.m.(x)CountcoloniesthenextdaywiththesameconsiderationsasdiscussedinStep3C(x).(C)BrothmicrodilutionTIMINGB20–45minperantibiotic
(i)FollowSteps4B(i)–(iii)inordertopreparetheantibioticsolutions,andadjustvolumesifnecessary.Notehoweverthat
the96-wellmicrotiterplateformatallowsonlytestingtendifferentconcentrationsiffollowingtheoutlineofFigure3.Forthisprocedure,50mlofeachdilutionisneededforeachbacterialisolatetobetested.Insituationswhereoneplatecontainsonlydilutionsofoneantibioticpermittingthetestingofeightdifferentbacterialisolates,amultichannel
e.g., X = 32 mg l–132
1162Xc /283c X/4c /4c /4c X/4c Y/4c /4c /4c Y/432YYXX44c X/8c /8c /8c X/8c Y/8c /8c /8c Y/816YYXX25160.570.2580.12590.0610GC11SC 12SCSCSCSCSCSCSCSCAntibiotic AAntibiotic BAntibiotic CAntibiotic DAntibiotic EAntibiotic FAntibiotic GAntibiotic HSCFigure3|Outlineofthesetupofamicrotiterplateforantimicrobialsusceptibilitytestingwithdoublingdilutionsofeightdifferentantimicrobialagentsintwodifferentconcentrationrangeswithlabelingsuggestionsinblue.c,concentration;X,Y,thehighestconcentrationstested(mglÀ1);GC,growthcontrol(brothwithbacterialinoculum,noantibiotic);SC,sterilitycontrol(brothonly);blue,actuallabeling.Ifmorethantwo
concentrationranges(e.g.,withfurtherdifferentmaximalconcentrationscZorcW,etc.)aregoingtobetested,writethevalueforcZ,cW,etc.nexttocolumn1thatcontainsthehighestconcentration.
Ac XBc XCc XDc XEFc Yc Yc Xc Xc Xc Xc Yc Yc Yc Yc X/16c X/32c X/64c X/128c X/256c X/512GCc /16c /32c /64c /128c /256c /512GCc /16c /32c /64c /128c /256c /512GCc X/16c X/32c X/64c X/128c X/256c X/512GCc Y/16c Y/32c Y/64c Y/128c Y/256c Y/512GCc /16c /32c /64c /128c /256c /512GCc /16c /32c /64c /128c /256c /512GCc Y/16c Y/32c Y/64c Y/128c Y/256c Y/512GC84210.50.25YYYYYYYYYYYYXXXXXXXXXXXXc /2c /2c X/2c Y/2c /2c /2c Y/264YYXXGc YHc Ye.g., Y = 128 mg l–1128GCNATUREPROTOCOLS|VOL.3NO.2|2008|171
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pipetteoramultisteppipettorcanbeusedtodistributethesolutions.Atleast400mlofeachantibioticdilutionisrequiredperplateinthisscenario.Ifamultichannelpipetteisused,theliquidscanbetransferredintosterilepetridishesfordispensingandtotalvolumesof10mlormorearerecommended.
(ii)Removea96-wellmicrotiterplatefromitssterilepacking.Iftheplateisnotsuppliedwithalid,coveritwithanother
sterilemicrotiterplateorwithamicrotiterplatethathasbeenthoroughlywipedwith70%ethanolatthebottom.mCRITICALSTEPKeepthelidclosedwhennothandlingtheplatetoavoidcontaminationbyairbornepollutants.
(iii)Labelthe96-wellsterilemicrotiterplatewiththerespectiveantibioticconcentrationaccordingtoFigure3.Useone
rowforeachtestwithupto10differentdilutions.Pipette100mlofbrothinthesterilitycontrolwell(column12)and50mlinthegrowthcontrolwell(column11).
(iv)Foreachbacterialisolatetobetested,add50mlofeachantibioticdilutionintotherespectivewell.
(v)Mixthebacterialsuspensionadjustedto1Â108cfumlÀ1fromStep3A(iv),Step3B(iv)orStep3C(xvi)byvortexing,
anddiluteit1:100accordingtoStep4B(v).
(vi)Inoculateeachwellcontainingtheantibioticsolutionandthegrowthcontrolwellwith50mlofthebacterial
suspension.Thisresultsinthefinaldesiredinoculumof5Â105cfumlÀ1.
(vii)Removea10mlsamplefromthegrowthcontrolwellimmediatelyafterinoculatingtheplateandpipettethesampleinto
asterileEppendorftubeholding990mlofsterilesalineorbroth.Mixwellbyvortexing.Makeafurtherdilutionofthissuspension(1:10)bypipetting100mlinto900mlofsterilesalineorbrothandmixwell.
(viii)Plate100mlofeachofthetwodilutionsfromStep4C(vii)ontotwodifferentnutrient-richagarplates.(ix)Incubatemicrotiterplateandagarplatesat371Cfor16–20h.
mCRITICALSTEPMakesurethattheliquidinthemicrotiterplatedoesnotevaporateduringincubation.Iftheplatesareincubatedinanincubatorwithlowhumidity,itisadvisabletoputtheplatesintoacontainerthatincludesamoistpapertowel.Donotstackmorethanfourmicrotiterplatesontopofeachother.(x)CountcoloniesthenextdaywiththesameconsiderationsasdiscussedinStep3C(x).
(D)Brothmicrodilutionforantimicrobialpeptidesthatrequirethepresenceofaceticacid/BSATIMINGB20–45minperantibiotic
(i)Removetwo96-wellsterilepolypropylenemicrotiterplatesfromtheirpacking.Iftheplateisnotsuppliedwitha
lid,coveritwithanothersterilemicrotiterplateorwithamicrotiterplatethathasbeenthoroughlywipedwith70%ethanolatthebottom.
mCRITICALSTEPKeepthelidclosedwhennothandlingtheplatetoavoidcontaminationbyairbornepollutants.
(ii)LabelmicrotiterplateswiththerespectivepeptidedilutionsaccordingtoFigure3.Useonerowforeachtestwithupto
10differentdilutionsandasingletestisolate.
(iii)Add20mlofthe20-foldconcentratedpeptidestocksolutionsintothewellsofcolumn1ofplate1.IftheMICtest
involvesjustonepeptideinsteadofdifferentpeptides,thenthisvolume(V)canbeadjustedupto100mlthatcanbeusedforeightMICtests(10mlisneededforeachsingletest)andonlyonerowofthemicrotiterplateisusedforpreparingthepeptidedilution.
(iv)Addthesamevolume(V)ofsolutionAintothewellsincolumn1.Mixcarefullybypipettingupanddown6–8times.(v)Addhalfthevolume(V/2)ofsolutionBintocolumns2–10.
(vi)WithdrawV/2fromeachwellincolumn1andaddthistothecorrespondingwellsincolumn2.Thismakescolumn2a
twofolddilutionofcolumn1.Mixcarefullybypipettingupanddown6–8times.
(vii)Repeattheproceduredowntocolumn10.Discardthewithdrawnsolutionfromcolumn10.
(viii)Mixthebacterialsuspensionadjustedto1Â108cfumlÀ1fromStep3A(iv),Step3B(iv)orStep3C(xvi)byvortexing
anddiluteit1:200inMHB.Thisresultsinthefinaldesiredinoculumof5Â105cfumlÀ1.Eachtestrequires1,000mlofthisbacterialsolution.Useasterilepetridishoranothersterilereservoirtoholdthesolution.
(ix)Pipette90mlofthemicrobialsolutionintoeachwellofcolumn1–10ofmicrotiterplate2.Add100mlintocolumn11
(growthcontrol).
(x)Pipette100mlofMHBinthesterilitycontrolwell(column12)ofmicrotiterplate2.Useasterilepetridishoranother
sterilereservoirtoholdtheliquid.
(xi)Transfer10mlofeachofthe10wellscontainingthepeptidedilutionsinplate1intotherespectivewellsofthe
microtiterplatecontainingthemicrobialsolution(plate2).Donotaddpeptidesincolumn11or12ofplate2.
(xii)Removea10mlaliquotfromthegrowthcontrolwellimmediatelyafterinoculatingtheplateandpipetteitintoasterile
Eppendorftubeholding990mlofsterilesalineorbroth.Mixwellbyvortexing.Makeafurtherdilutionofthissuspension(1:10)bypipetting100mlinto900mlofsterilesalineorbrothandmixwell.
(xiii)Plate100mlofeachofthetwodilutionsontotwodifferentantibiotic-freenutrient-richagarplates.Useasterilecell
spreadertospreadtheliquid.
(xiv)Incubatemicrotiterplateandagarplatesat371Cfor16–20horuntilsatisfactorygrowthisobtained.
mCRITICALSTEPMakesurethatthevolumeinthemicrotiterplatesdoesnotevaporateduringincubation.Ifthe
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platesareincubatedinanincubatorwithlowhumidity,itisadvisabletoputtheplatesintoacontainerthatincludesamoistpapertowel.Donotstackmorethanfourmicrotiterplatesontopofeachother.(xv)Countcoloniesthenextdaywiththesameconsiderationsasdiscussedin3C(x).
(E)Brothmicrodilutionforantimicrobialpeptidesthatdonotrequirethepresenceofaceticacid/BSATIMINGB20–45minperantibiotic
(i)Removea96-wellsterilepolypropylenemicrotiterplatesfromitspacking.Iftheplateisnotsuppliedwithalid,
coveritwithanothersterilemicrotiterplateorwithamicrotiterplatethathasbeenthoroughlywipedwith70%ethanolatthebottom.
mCRITICALSTEPKeepthelidclosedwhennothandlingtheplatetoavoidcontaminationbyairbornepollutants.
(ii)LabelmicrotiterplateswiththerespectivepeptidedilutionsaccordingtoFigure3.Useonerowforeachtestwithupto
10differentdilutionsandasingletestisolate.
(iii)Pipette50mlofMHBintocolumn2–11ofmicrotiterplate2.Add100mlintocolumn12(sterilitycontrol).
(iv)Dilutethe20-foldconcentratedpeptidestocksolution1:10intoMHBinapolypropyleneEppendorftube.Foreachtest,
100mlofthissolutionisneeded.
(v)Add100mlofthethustwofoldconcentratedpeptidesolutionintothewellsincolumn1.
(vi)Withdraw50mlfromeachwellincolumn1andaddthistothecorrespondingwellsincolumn2.Thismakescolumn2a
twofolddilutionofcolumn1.Mixbycarefullypipettingupanddown6–8times.
(vii)Repeattheproceduredowntocolumn10.Discardthewithdrawnsolutionfromcolumn10.
(viii)Mixthebacterialsuspensionadjustedto1Â108cfumlÀ1fromStep3A(iv),Step3B(iv)orStep3C(xvi)byvortexing
anddiluteit1:100accordingtoStep4B(v).
(ix)Inoculateeachwellcontainingthepeptidesolutionandthegrowthcontrolwellwith50mlofthebacterialsuspension
(columns1–10).Thisresultsinthefinaldesiredinoculumof5Â105cfumlÀ1.
(x)Removea10mlaliquotfromthegrowthcontrolwellimmediatelyafterinoculatingtheplateandpipetteitintoasterile
Eppendorftubeholding990mlofsterilesalineorbroth.Mixwellbyvortexing.Makeafurtherdilutionofthissuspension(1:10)bypipetting100mlinto900mlofsterilesalineorbrothandmixwell.
(xi)Plate100mlofeachofthetwodilutionsontotwodifferentnutrient-richagarplates.Useasterilecellspreaderto
spreadtheliquid.
(xii)Incubatemicrotiterplateandagarplatesat371Cfor16–20horuntilsatisfactorygrowthisobtained.
mCRITICALSTEPMakesurethatthevolumeinthemicrotiterplatesdoesnotevaporateduringincubation.Ifthe
platesareincubatedinanincubatorwithlowhumidity,itisadvisabletoputtheplatesintoacontainerthatincludesamoistpapertowel.Donotstackmorethanfourmicrotiterplatesontopofeachother.(xiii)CountcoloniesthenextdaywiththesameconsiderationsasdiscussedinStep3C(x).
?TROUBLESHOOTING
©2008 Nature Publishing Group http://www.nature.com/natureprotocolsANTICIPATEDRESULTSAgardilution(Step4A)
Inorderforthetesttobevalid,theagarplatesforthecellcount(Step4A(xxii))havetobecheckedtoverifythattherightnumberofcfuwereused.Thepresenceofaround100–200coloniesonthelowerofthetwodilutions(10À5)oftheinitial
bacterialsuspensionisexpectedwhenusingthecorrectbacterialsuspensiondensityof1–2Â108cfumlÀ1.Ifthecellnumbersarewithinthedesiredrange,thetestcanbeanalyzedtodeterminetheMIC.Alsochecktheantibiotic-freegrowthcontrolplate.Visiblegrowthneedstooccurforthetesttobevalid.
TheMICisdefinedasthelowestconcentrationoftheantimicrobialsubstancethatinhibitsvisiblegrowthofthetestediso-late.Thegrowthofasinglecolonyorfaintfilmcausedbytheinoculumshouldbedisregarded.Whengrowthofthetestedorganismoccursonallagarplateswithantimicrobialagent,theMICisrecordedasgreaterthanthehighestconcentrationtested.TheMICisrecordedaslessthanorequaltothelowestconcentrationwhennogrowthoccursonanyoftheagarplatesbutthegrowthcontrol.
Brothdilution(Steps4B–4E)
Inorderforthetesttobevalid,theagarplatesforthecellcount(Steps4B(x),4C(x),4D(xv)or4E(xiii))havetobecheckedtoverifythattherightnumberofcfuwereused.Thepresenceofaround50coloniesonthelowerofthetwodilutions(1:1000oftheinitialinoculum)isexpectedwhenusingthecorrectinoculumdensityof5Â105cfumlÀ1.
Wipeoffthebottomofthemicrotiterplateusingalint-freetissuetoremoveanycondensationthatmaybepresent.Deter-mineifthereissufficientgrowthinthegrowthcontroltubeorinthegrowthcontrolwell.Inorderforthetesttobevalid,adefiniteturbidityorsediments(buttonsizeinmicrotiterplatesZ2mm),needtooccur.DonotreadtheMICvalueifthesterilitycontrol(nobacterialinoculum)isturbid.
NATUREPROTOCOLS|VOL.3NO.2|2008|173
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Figure4|InterpretationofpossiblegrowthpatternsinMICmicrotiterplates.Whengrowthoccursinalldilutionscontainingtheantimicrobialagent,theMICisrecordedasgreaterthanthehighestconcentration.TheMICisrecordedaslessthanorequaltothelowestconcentration,whennogrowthoccursinanyoftheconcentrationstested.Aclearwellinaseriesofwellswithvisiblegrowth,e.g.,growthat1,2and8mgmlÀ1,butnotat4mgmlÀ1,iscalledaskippedwellandshouldbeignored.Spotgrowthinisolatedwellsindicatescontamination.Thetestshouldberepeated.
1ABCDEFGHGrowth4210.50.25control32168Sterility–1Antimicrobial agent in mg lcontrol23456789101112A: MIC = 64 mg l–1B: MIC > 128 mg l–1
C: MIC ≤ 0.25 mg l–1
D: MIC = 1 mg l–1, single spot in column 2 indicatescontamination of well, repeat test–1
E: MIC = 32 mg l ignore skipped well, repeat testE: repeat test, culture might have been contaminatedG: repeat test, medium might have been contaminatedH: repeat test, organism tested did not grow under theapplied conditions12864©2008 Nature Publishing Group http://www.nature.com/natureprotocolsTheMICisdefinedasthelowestconcentrationoftheantimicrobialagentthatinhibitsvisiblegrowthofthetestedisolateasobservedwiththeunaidedeye(Fig.4).TheMICforthequalitycontrolorganismshouldbewithinonetwofolddilutionofpublishedvaluesforroutinelyusedantibiotics1,2.?TROUBLESHOOTING
TroubleshootingadvicecanbefoundinTable5.
TABLE5|Troubleshootingtable.Problem
Microbialgrowthinsterilitycontrol
Possiblereason
Brothwascontaminated
Pipettes/pipettetipsusedwerenotsterile
Containerstoholdsolutionswerenotsterile
Formicrodilution:Contaminationwithmicrobesincolumn11
Nogrowthingrowthcontrol
Inoculumwastoolow
Solution
MakesuretouseanunopenedbatchofsterilizedbrothMakesuretousesterilepipettes/pipettetips
Makesuretousesterilecontainers
Avoidspillsandaerosolformationwhenpipettingthebac-terialsolution
Determinethecellcountofthebacterialsuspensionforeverytestisolateused
Checkifusedbrothandincubationtemperatureissufficientformicrobialgrowth
Growthconditionswerenotappropriateformicrobialgrowth
Steps1and2:B5minperisolate/overnightpausepointStep3A,colonysuspensionmethod:B5minperisolateStep3B,growthmethod:2–6h
Step3C,growthmethodusingovernightcultures:B5minperisolate(Steps3C(i)–(iii))/overnightpausepoint/B15minperisolate(Steps3C(iv)–(viii))/overnightpausepoint/B20minperisolate(Steps3C(ix)–(xiv))/overnightpausepoint/B5minperisolate(Steps3C(xv)–(xvi))Step4A,agardilution:4–6h
Step4B,macrodilution:B20–45minperantibiotic
Step4C,microdilutionforantibiotics:B20–45minperantibiotic
Steps4Dand4E,microdilutionforantimicrobialpeptides:B20–45minperantibiotic
TIMING
174|VOL.3NO.2|2008|NATUREPROTOCOLS
ACKNOWLEDGMENTSWeacknowledgethefinancialassistanceoftheAppliedFoodandMaterialsNetworkandtheCanadianInstitutesofHealthResearch.R.E.W.H.wassupportedbyaCanadaResearchChairaward.K.H.wassupportedbya
fellowshipfromtheCanadianInstitutesofHealthResearch.I.W.wassupportedbytheJuergen-Manchot-Foundation.
Publishedonlineathttp://www.natureprotocols.com
Reprintsandpermissionsinformationisavailableonlineathttp://npg.nature.com/reprintsandpermissions
1.ClinicalandLaboratoryStandardsInstitute.Performancestandardsfor
antimicrobialsusceptibilitytesting;sixteenthinformationalsupplement.CLSIdocumentM100-S16CLSI,Wayne,PA(2006).
2.EuropeanCommitteeforAntimicrobialSusceptibilityTesting(EUCAST)ofthe
EuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases(ESCMID).Determinationofminimuminhibitoryconcentrations(MICs)ofantibacterialslagentsbybrothdilution.Clin.Microbiol.Infect.9,ix–xv(2003).
oc3.Kahlmeter,G.etal.EuropeanharmonizationofMICbreakpointsforantimicrobial
otosusceptibilitytestingofbacteria.J.Antimicrob.Chemother.52,145–148(2003).rp4.Nanavaty,J.,Mortensen,J.E.&Shryock,T.R.Theeffectsofenvironmental
erconditionsontheinvitroactivityofselectedantimicrobialagentsagainstutEscherichiacoli.Curr.Microbiol.36,212–215(1998).
an5.D’amato,R.F.,Thornsberry,C.,Baker,C.N.&Kirven,L.A.Effectofcalciumand
/mmagnesiumionsonthesusceptibilityofPseudomonasspeciestotetracycline,ocgentamicinpolymyxinB,andcarbenicillin.Antimicrob.AgentsChemother.7,.er596–600(1975).
ut6.Rhomberg,P.R.,Sader,H.S.&Jones,R.ReproducibilityofdaptomycinMICresults
anusingdry-formcommercialtrayswithappropriatesupplementalcalciumcontent.w.Int.J.Antimicrob.Agents25,274–276(2005).
ww7.Bowdish,D.M.,Davidson,D.J.&Hancock,R.E.Are-evalutionoftherole
//:ofhostdefencepeptidesinmammalianimmunity.Curr.ProteinPep.Sci.6,35–51pt(2005).
th 8.Hancock,R.E.&Sahl,H.G.Antimicrobialandhost-defensepeptidesasnewanti- pinfectivetherapeuticstrategies.Nat.Biotechnol.24,1551–1557(2006).
u9.Chambers,H.F.&Hackbarth,C.J.EffectofNaClandnafcillinonpenicillin-binding
roGprotein2aandheterogeneousexpressionofmethicillinresistancein
gStaphylococcusaureus.Antimicrob.AgentsChemother.31,1982–1988(1987).n10.Ferguson,R.W.&Weissfeld,A.S.Comparisonofthesuitabilityofthreecommon
ihsbacterialmediaforsusceptibilitytestingoftrimethoprim-sulfamethoxazole.Clin.ilMicrobiol.19,85–86(1984).
bu11.Bradford,P.A.etal.TigecyclineMICtestingbybrothdilutionrequiresuseoffresh
P mediumoradditionofthebiocatalyticoxygen-reducingreagentoxyrasetoerstandardizethetestmethod.Antimicrob.AgentsChemother.49,3903–3909ut(2005).
aN12.Thomson,K.S.&Moland,E.S.Cefepime,piperacillin-tazobactam,andthe
8inoculumeffectintestswithextended-spectrumbeta-lactamase-producing00Enterobacteriaceae.Antimicrob.AgentsChemother.45,3548–3554(2001).213.Chambers,H.F.Methicillin-resistantstaphylococci.Clin.Microbiol.Rev.1,
©173–186(1988).
PROTOCOL
14.Granier,S.A.etal.FalsesusceptibilityofKlebsiellaoxytocatosomeextended-spectrumcephalosporins.J.Antimicrob.Chemother.50,303–304(2002).
15.Pankey,G.A.&Sabath,L.D.Clinicalrelevanceofbacteriostaticversusbactericidal
mechanismsofactioninthetreatmentofGram-positivebacterialinfections.Clin.Infect.Dis.38,864–870(2004).
16.Biedenbach,D.J.,Schermer,I.H.&Jones,R.N.ValidationofEtestforseven
antimicrobialagentsusingregulatorycriteriafortheassessmentofantimicrobialsusceptibilitydevices.Diagn.Microbiol.Infect.Dis.27,1–5(1997).
17.Andrews,J.M.&Wise,R.ComparisonoftheEtestwithaconventionalagar
dilutionmethodinevaluatingtheinvitroactivityofmoxifloxacin.J.Antimicrob.Chemother.45,257–258(2000).
18.Steward,C.D.etal.Antimicrobialsusceptibilitytestingofcarbapenems:
multicentervaliditytestingandaccuracylevelsoffiveantimicrobialtestmethodsfordetectingresistanceinEnterobacteriaceaeandPseudomonasaeruginosaisolates.J.Clin.Microbiol.41,351–358(2003).
19.Biedenbach,D.J.&Jones,R.N.ComparativeassessmentofEtestfortesting
susceptibilitiesofNeisseriagonorrhoeaetopenicillin,tetracycline,ceftriaxone,cefotaxime,andciprofloxacin:investigationusing510(k)reviewcriteria,recommendedbytheFoodandDrugAdministration.J.Clin.Microbiol.34,3214–3217(1996).
20.DiBonaventura,G.etal.ComparisonofEtest,agardilution,brothmicrodilution
anddiskdiffusionmethodsfortestinginvitroactivityoflevofloxacinagainstStaphylococcusspp.isolatedfromneutropeniccancerpatients.Int.J.Antimicrob.Agents.19,147–154(2002).
21.Fritsche,T.R.,Rennie,R.P.,Goldstein,B.P.&Jones,R.N.Comparisonof
dalbavancinMICvaluesdeterminedbyEtest(ABBIODISK)andreferencedilutionmethodsusinggram-positiveorganisms.J.Clin.Microbiol.44,2988–2990(2006).
22.Sader,H.S.,Fritsche,T.R.&Jones,R.N.Accuracyofthreeautomatedsystems
(MicroScanWalkAway,VITEK,andVITEK2)forsusceptibilitytestingof
Pseudomonasaeruginosaagainstfivebroad-spectrumbeta-lactamagents.J.Clin.Microbiol.44,1101–1104(2006).
23.Wiegand,I.etal.Detectionofextended-spectrumbeta-lactamasesamong
Enterobacteriaceaebyuseofsemiautomatedmicrobiologysystemsandmanualdetectionprocedures.J.Clin.Microbiol.45,1167–1174(2007).24.Andrews,JM.Determinationofminimuminhibitoryconcentrations.
J.Antimicrob.Chemother.48(suppl.1):5–16(2001).
25.Steinberg,D.A.,Hurst,M.A.,Fujii,C.A.,Kung,A.H.,Ho,J.F.,Cheng,F.C.,Loury,
D.J.&Fiddes,J.C.Protegrin-1:abroad-spectrum,rapidlymicrobialpeptidewithinvivoactivity.Antimicrob.AgentsChemother.41,1738–1742(1997).26.Wick,WE.Influenceofantibioticstabilityontheresultsofinvitrotesting
procedures.J.Bacteriol.87,1162–1170(1964).
27.Wiegand,I.&Wiedemann,B.Microbialresistancetodrugs.InEncyclopedic
RefererenceofMolecularPharmacology(eds.Offermanns,S.&Rosenthal,W.)594–600(Springer-Verlag,BerlinHeidelberg,2003).
28.Ericsson,HM&Sherris,JC.Antibioticsensitivitytesting.Reportofan
internationalcollaborativestudy.Acta.Pathol.Microbiol.Scand.B.217(suppl.),1–90(1971).
NATUREPROTOCOLS|VOL.3NO.2|2008|175
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