Antimicrobial Susceptibility Testing
Objectives
Attheendofthesessionstudentswillbe ableto:
Explainthesignificanceofantibioticsensitivity testing Describethedifferentmethodsusedin antibioticsensitivity ytesting g Describetheuse,principleandmethodsof antibioticsusceptibilitytestingbybothdisc diffusionanddilutiontechniques.
Prepared by: Fathimath Shiranee
Antimicrobial Susceptibility Testing
Aims Ai
Significance
Antimicrobialsusceptibilitytestingisperformed onisolatesfromclinicalspecimensif:
Thebacterialisolateistheprobablecauseofthe patientsinfection
Toprovidepredictionoflikelyoutcomeoftreatment withparticularantimicrobial Toidentifyalternativedrugstowhichorganismis Detectspecificresistancesanddetectemergenceof resistanceandguidewithanappropriatetreatment
Significance
Factorstoconsidertodeterminewhether susceptibilitytestingisappropriateforaparticular isolate:
Clinicalsignificance g of Presenceofotherbacteriaandq quality yofspecimen p fromwhichbacteriawasisolated.
Significance:
Clinical Cli i lsignificance: i ifi
Istheisolatenormalfloraorpathogenatbodysite fromwhichtheorganismwasisolated? Abilityofthebacteriatocause Detection/abundanceoforganismindirectGram smear,________________and dgrowth thof fan organismwithsamemorphologyinculture.
Significance
Presenceofotherbacteriaandqualityofspecimen.
Organismsisolatedinpureculturearelesslikelyto representcontaminationthanamixedculture.
Methods
Methodsinclude: Dilutionmethods:determinetheMIC
Hoststatus
Organismsusuallyknowntobenormalfloramaybe responsibleforinfectionin
Diskdiffusion:directlymeasureantibioticactivity Alltestingmethodsareinvitro andwillnot always l correspond dto t th theeffect ff t
Components of susceptibility testing that needs standardization include:
Bacterialinoculum size Growthmedium
pH Cation concentration Supplements Thymidine content
Bacterial inoculum
B Bacterial t i linoculum i l areprepared dto t a0.5 0 5 McFarlandstandard. 0.5McFarlandstandard:
Standardpreparedbyadding99.5ml 99 5ml1%sulphuric acid and______________bariumchloridetoobtainbarium sulphate solutionofspecificopticaldensity.
Incubation
Atmosphere Temperature
Coloniesfromapurecultureplateareinoculated inbrothorsalineandthencomparedwiththe McFarland0.5standardtostandardizethe inoculum,whichis
Growth medium
M Mueller ll Hinton Hi t b broth thand dagararethe th standard t d d mediausedfordilutionanddiskdiffusionmethods respectively p y
Growth medium contd contd
Supplements: pp
Supplementsarerequiredinthemediawhentesting theantimicrobialsusceptibilityoffastidiousorganisms. Example:5%sheepbloodinMuellerHintonbase fortestingstreptococciandsomeotherfastidious bacteria. bacteria
pH: Cation concentration:
Cations affecttheactivityofsomeantibiotics.
Thymidinecontent:
hasaninhibitoryeffectonactivityofsome g antimicrobialagents certainingredientsneedtobeaddedtothemediato neutralizethiseffect
Incubation
Forth F themost tcommonly l t tested t dorganisms i i including l di Enterobacteriaceae,Pseudomonas andstaphylococci,theincubation conditionsare: Atmosphere: h Temperature: Time: Specialconditionsareprovidedformorefastidiousorganisms. Example: E l [Link] requires___________forgrowth. FortestingMRSAamongstaphylococci,incubation temperatureof300Cshouldbeused. Someorganismsmayrequireprolongedincubation.
Antimicrobial concentration
Theappropriateconcentrationsofantimicrobial [Link]? Fordiscdiffusionmethodantimicrobialdiscsare availablecommerciallywithpredetermined concentrations Whenperformingdilutiontechniquescareful preparationanddilutionofantimicrobialsis essential
Selection of antimicrobial agents
OrganismIDorgroup
Antimicrobialstowhichorganismsareintrinsically areexcluded
Dilution methods: Broth dilution
Method:
Twomethods: Twofoldserialdilutionofantimicrobialagentsintesting media(MuellerHintonbrothismostcommonlyused)is prepared. d Standardizedsuspensionofinoculum isaddedtoeach dilutiontoobtainafinalconcentrationof Controls:agrowthcontrol(broth+inoculum)andanun inoculatedcontrol(brothonly)areusedineachtest. test Incubateovernightat
Acquiredresistancepatternscommontolocalmicrobialflora Antimicrobialsusceptibilitytestingmethodused
[Link] resistancetocefotaxime canbedetectedbydilution methodsonly
Siteofinfection
E.g. g Nitrofurantoin only yachieveeffectivelevelsinurine
Availabilityofantimicrobialagentsinformulary
Dilution methods: Broth dilution
Interpretation: I t t ti
MICisdeterminedvisuallyasthelowest concentrationthatinhibitsgrowthoforganisms Theorganismisinterpretedassusceptible, intermediateorresistanttoeachagenttestedby usingatable
Advantages
Allowsforbothquantitativeandqualitativeresults
Dilution methods: Broth dilution
Minimumbactericidalconcentration(MBC)
Theconcentrationofantimicrobialwhichkills oftheorganisms Dilutionswithnogrowthcanbeinoculatedon agarplatestodeterminetheMBC.
Disadvantages
Complexprocedure,Dilutions
Dilution methods: Agar dilution
Method:
Doublingdilutionofantimicrobialagentisincorporatedinto agarplates aga p ates(single (s g eplate p ateper pe dilution). d ut o ) Aseriesofplatescontainingvariousantimicrobial concentrations,andgrowthcontrolplatesareprepared. Standardnumberoftestorganisms(inoculum)arespot inoculated. Manydifferentisolatescanbeinoculatedononeplate.
Disk diffusion
Method M th d(Kirby (Ki b Bauer): B )
Standardizedinoculumislawninoculatedonthesurfaceof anagarplate. Diskimpregnatedwithdifferentantibioticsareplacedon theplate. Moistureabsorbedfromagarallowsantimicrobialsto diffuseoutintothemedium,producingaconcentration gradientofantibiotic Incubateovernight Measurethe h diameter di zoneof finhibition i hibi i of fgrowth haround d theantibioticdisk.
Interpretation:
MICisinterpreted p asthe ___________________ofantimicrobialagent thatinhibitedthegrowthoforganism.
Disk diffusion
Interpretation:
Themeasurementsarecomparedtochartsandarereportedassensitive, intermediateorresistantaccordingtopresetcriteria. Visiblegrowthoccurwheretheconcentrationofantibiotichasfallen belowtheMIC.
Etest
Advantages
Technically ysimple p Inexpensive Reliable
Disadvantages
Mustbecarefullycontrolled Lackofinterpretativecriteriafororganismsnotincludedininterpretation charts. h
Disk diffusion Factors affecting zone size
Inoculum I l Timing Ti i of fapplication li ti Incubation I b ti Medium M di Control C t lstrains t i Antibiotic Antibioti discs dis s
