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 N.meningitidis requires___________forgrowth. FortestingMRSAamongstaphylococci,incubation temperatureof300Cshouldbeused. Someorganismsmayrequireprolongedincubation.

Antimicrobial concentration
Theappropriateconcentrationsofantimicrobial agentsshouldbeused.Why? 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
E.g.S.pneumoniae 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