ANTIBIOTIC SUSCEPTABILITY TESTING

(KirbyBauerDiskDiffusiontest) PURPOSE: Itisusedtoassestheantibioticsensitivityofacertain bacterialisolate. SPECIMEN : Purebacterialisolatefromfreshcultureplate. MATERIALS :

1.

Nutrientbrothforfastidiousorganismsorsterilesalinefor nonfastidiousorganisms.

2.

0.5McFarlandstandardforadjustingtheturbidityofthe inoculums.

3. 4.
5.

Vortexmixerforsuspensionoftheinoculum. Viewboxforcomparisonofbrothwithstandard MuellerHintonagarplatesunsupplementedfornon fastidiousorganismsorsupplementedwithRBCsina concentrationof5%forfastidiousorganisms(90mmdiameterfor sevendisks;150mmdiameterforamaximumof12disks)froma

1

lotthatgivesasatisfactoryqualitycontrolresults.ThePHmust be7.2to7.4,andthedepthmustbeapproximately4mm.
6.

NonCO23537Cincubatorfornonfastidiousorganismsor 5%CO2incubationforH.infS.pneumoniaeN.meningititis QUALITY CONTROL Antibioticdiscsforsusceptibilitytestingarecheckedweekly utilizingappropriateATCCreferencestrains.Inaddition,QC testingwillbeperformedanytimewhenantibioticwithanewlot numberisusedrepeatthetesting.Documentanycorrective actionintheQClogbook.ThediscstestedforQCmustbethe samediscsusedwiththepatientspecimens.Tolerancelimitsfor antimicrobialpotencyarebasedonCLSIguidelines.Ifthezone rangelimitsareexceed,theLabDirectormustbeimmediately notifiedandnosensitivityresultswillbereported.

E coli ATCC 25922 Pseudomonas aeroginosae ATCC 27853 S. aureus ATCC 29213

ALSO

ORGANISMS

QC

STRAINS FOR

ESBL & FASTIDIOUS

MAINTENANCE:

MUST BE INCLUDED

QC ORGANISMS

Avoid repeated subculture

Store stock isolates at -60C or below

2

Prepare working culture weekly & stored at -20C

PROCEDURES
1.

Preparationofinoculum : a. Withasterilewireloop,touchthetopoftwotofive similarappearing,wellisolatedcoloniesonanagar platecultureaccordingtothesizeofcoloniesasfollows: largecoloniesascitrobactertouchonlythe quarterofitssize,smallcoloniesasstrepttouch fivecolonies,whilemoderatesizedcolonies touchonlytwocolonies. b. Emulsifythemin5mLofsterilephysiologicalsalineor nutrientbrothwiththehelpofvortex. c. Theturbidityoftheemulsificationisadjustedto

0.5McFarlandstandard.Turbidityismatched
againstaprintedcardorsheetofpaperinagoodlight.
3

d.

Within15minutesofadjustingtheturbidityofthe inoculumssuspension,addthesuspensiontotheplate bypouringthesuspensiononthesurfaceoftheagar plate,andthendiscardtheexcessinwastecontainer whichcontainadisinfectantReplacethelidofthe dish.Allowatleast5minutesbutnolongerthan15 minutesforthesurfacetodrybeforeaddingthe antibioticdisks

2.

Testingofantibiotics:
a.

Placetheappropriateantimicrobialimpregnateddisks withspecificconcentrationaccordingto(CLSI recommendation,age,pregnancy,inpatientvs outpatient,typeofspecimens)onthesurfaceofthe agar,usingforceps.Disksmustbeevenlydistributed ontheagarsothattheyarenocloserthan25mmfrom centertocenterandabout15mmfromtheedgeofthe agarplate.

b.

Gentlytampeachdiskdownontotheagartoprovide uniformcontact.
4

c.

Within15minutesofapplyingthedisks,invertthe plateandincubateitaerobically(ambientair)at37C for1618hours.Examinetheplatesaftertheovernight incubationexceptforstaph&streptupto24hours

INTERPRETATION
Withtheuseofaruleroratemplate,thezonesof completegrowthinhibitionaroundeachofthedisksarecarefully measuredtowithinthenearestmillimeter;Allmeasurementare madebytheunaidedeyewhileviewingthebackofthepetridish withreflectedlightagainstablack,nonreflectingbackground. Theplatesshouldbeviewedfromadirectlyverticallineofsightto avoidanyparallaxthatmayresultinmisreading. Aninterpretivecorrelate(susceptible,moderately susceptible,intermediateorresistant)isprovidedbyreferenceto publishedCLSIguidelines.

LIMITATIONS
1 Donotmoveadiskonceithascontactedtheagar,becausesomeof thedrugdiffusesalmostimmediately. 2 Susceptibilityplatespreparedwithbloodmustbeviewedfromthe agarsurfaceandmeasurementsmadewiththecoverofthePetri dishremoved.
3-

Zonesthatfallintotheintermediaterangeshouldbeconsidered equivocal;iftherapywiththedrugisdesired,adilution susceptibilitytestshouldbeperformedtoclarifytheissue.

Whentestingstaphylococciagainstmethicillinoroxacillinor enterococciagainstvancomycin,incubationshouldbefor24hours.

MotileorganismssuchasProteusmirabilisorP.vulgarismay swarmwhengrowingonagarsurfaces,resultinginathinveilthat maypenetrateintothezonesofinhibitionaroundantimicrobial agentsusceptibilitydisks.Thiszoneofswarmingshouldbeignored; theoutermargin,whichisusuallyclearlyoutlined,shouldbe measured.Similarly,withsulfonamidedisks,growthmaynotbe completelyinhibitedattheoutermargin,resultinginafaintveil, where80%ormoreoftheorganismsareinhibited.Theclearzoneof ~80%inhibitionshouldbereadasthezonediameter.

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Presenceofdistinctcolonieswithinthezoneofinhibition(2ry colonies)representeithermutantofthesamespeciesthataremore resistanttotheantimicrobialagentthanthemajorportionofthe bacterialstrainbeingtestedorthecultureisnotpureandthe separatecoloniesareofadifferentspecies.Ifitisdeterminedthat theseparatecoloniesrepresentavariantofamutantstrain,the bacterialspeciesbeingtestedmustbeconsideredresistant.Ifitis determinedtobeadifferentspecies,returntotheculturePetridish andrealizewhetheritisamissedcolonyoracontamination.If missed,doaseparateantibiogramfortheisolate.

7-

Whenthereisoverlappingbetweenadjacentagentzones,zones extendbeyondthemarginofthePetridishoroval(elliptical) zones;,thetestmustberepeatedwithmorecarefulplacementof theantimicrobialagentdiskssothatoverlappingwillnotoccur .Whentheplateisstreakedpoorly,thiswillleadtoindistinct zonesandthetestmustberepeated

 CHOICE OF ANTIBIOTICS IN ANTIBIOGRAM:

DrugsarelistedbyCLSIin4groups:
123-

GROUPA:Testing&reportingagainstallisolate. GROUPB:TestingwhenisolateisresistanttogroupA. GROUPC:Supplementaloralternativeagentthatcanbe tested&reportedininstitutionsthatharborresistantstrains.

4-

GROUPU:Agentsthatshouldbetested&reportedonlyon isolatesfromurine.

56-

Group O : Group I : Protocol of antibiotics choice in mic. lab

IN THE FIRST DAY GROUP A & B (GROUB 1) are tested in non urine isolates & (GROUP U) in urine isolates.

IN THE SECOND DAY

GROUP C ( GROUP 2) for H.Infleuanza & Enterobacteriacae & antimicrobial combinations will be done . IN THE THRID DAY Another combination will be tested in multi resistant strains

Antimicrobial agents with FDA clinical indication that should be considered for routine testing
Acinetobacter Fortum(CAZ) Tienam(IPM)orMeronam(MEM) Unasyn(SAM) Ciprofloxacin(CIP)orLevofloxacin(LEV)

Orofloxacin(OFX) Gentamycin(CN)ortobramycin(TOB)or amikin(AK) SUTRIM(SXT)

Sulperazone(SCF) Cefotaxime(CTX)orRocephine(CRO) Tazocin(TZP) Polymyxinlocalonlyineye,ears Doxycycline(Do)orTetracycline(TE)

H.Influenza Ampicillin(AMP) Sutrim(SXT) Unasyn(SAM) Cefuroxime(CXM) Cefotaxime(CTX) Rocephine(CRO) Fortum(CAZ) Azithromycin(AZM) Augmentin(AMC) Cefopodoxime(CPD) Ciprofloxacin(CIP) orLevofloxacinLEV Tienamormeronam

NisseriaGonorrhea

Cefopodixime(CPD) Rocephine(CRO) Ciprofloxacin(CIP) Penicilin(P) Tetracyclin(TE) Ofloxacin(OFX) Fortum(CAZ) Cefotaxime(CTX)

NB : Testing of B lactamase is mandatory for both isolates using either penicillin disc or nitrocefin sticks .

Sutrim(SXT)

Burkholderia

Sutrim(SXT) levofloxacin minocycline

Stenotrophomonas

Fortum(CAZ) Meronam(MEM) minocycline

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 .Haemolyticstrept(pyogens)
Ampicillin(AMP) Pencillin(P) Erythromycin(E)/AZM Clindamycin(Cd) Maxipime(FEP)

Strep.Viridance Ampicillin(AMP) Maxipime(FEP) PencillinP(MIC)only Cefotaxime(CTX) Rocephine(CRO) Vancomycin(VA) Erythromycin/AZM Clindamycin(Cd)

S.pneumoniae

Erythromycin(E)/AZM Oxacillin(ox)testing penicillin Sutrim(SXT) Clindamycin(Cd) Levofloxacin(LEV) Ofloxacian(OFX) Tetracycline(TE) Vancomycin(VA) OptochineOP

Cefotaxime(CTX)or Vancomycin(VA) Levofloxacin(LEV)or

Rocephine(CRO)

Ofloxacin(OFX) BacitracinBC

N.B If oxacillin sensitive S.pneumoniae report blindly all penicillins & cephalosporins sensitive but if resistant MIC for 3rd generation cephalosoprins is mandatory

11

Enterococci(nonurine) Penicillin(P) Ampicillin(AMP) Vancomycin(VA) CN120g(highlevel Erythromycinor Azithromycin screen)orStreptomycin

Staph(nonurine)

Cefoxitin(Fox30g) Penicillin(P)ifsensitive reportallpenicillins cephalospoines&carbapenemsare sensitiveapprovedbyFDA

Sutrim(SXT)

Clindamycin(Cd)testofMLS resistanceisrecommended Azithromycin(AZM)or Vancomycin(VA) only

Tetracycline(TE)

Erythromycin(E)

CIPorOFXorLEVinMSSA DOorTE

Gentamycin(CN) Caphalothin(CF)

Enterococci(urine) Ciprofloxacin(CIP) Levofloxacin Norfloxacin Furadantin(F) P

Staph(urine) Norfloxacin(NOR) Levofloxacin FOX P VA Ofloxacin(OFX)

Tetracycline(TE) AMP Vancomycin

Furadantin(F) Sutrim(SXT)

Quinolones are not recommended in ttt of MRSA

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ESBLconfirmationinKlebsiella,Ecoli&Proteus
(bloodisolatesonly):

DISCAPPROXIMATIONTEST
AMCbetterorSAM(atthecenteroftheplate) CPDaloneorCAZ&CTXtogether(2.5cmaroundAMC fromcentertocenter) FOX30g(besidecephalosporin) TZPORSCF FEP(mandatory) IPMORMEM(besidecephalosporin) OFXORLEV/NORinurineonly CNORAK Inurineaddfuradantin

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CAZ CTX, CFP FOX 30g Or FEP CPD

OFX OR LEV /NOR in urine only

AMC MAINLY OR SAM OR

IPM OR MEM

CN or AK/ Furadantin in urine

TZP or SCF

CHART FOR

ESBL SCREENING & CONFIRMATION Furadantin in urine only

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Proteus&Enterobacter, Citrobacter Tobramycin(TOB)or Amikin(AK) Unasyn(SAM)or Tazocin(TZP) Gentamycin(CN)or

Pseudomonas Fortum(CAZ)

Gentamycin(CN)or Amikin(AK)orTobramycin Tazacin(TZP) CIPorLEV SCF Cefobid(CFP) IPMorMEM FEP

Augmentin(AMC) Cefepime(FEP)

cefotrioxne(CRO)or Cefobid(CFP)

Cefotaxime(CTX) Sulperazone(SCF)

Ciprofloxacinofloxacinor Tienam(IPM)or

Levofloxacin(LEV) Meronam(MEM) Sutrim(SXT) Tetracycline(TE)

rd

NB:fortumis3 generationcephalosporinwithstrong antipseudomonalactivitywhilerocephine/claforanhave weakantipseudomonalactivity.

Coryneformbacteria(Diphtheroid) o o o o o o o Sutrim Penicillin Levo/oflox Erythromycin/azythromycin Vancomycin Tetracycline/doxyycline Augmentin/unasyn

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GramnegativebacilliotherthanEcoli&Klebsiellain urine:

Norfloxacin(NOR) OFX/LEV/CIP Furadantin(F) Lomefloxacin Sutrim(SXT) Cefobid(CFP) Augmentin(AMC) Gentamycin(CN) Carbencillin Tazocin FEP SCF

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CLSIrecommendations:
1. Thefollowingantimicrobialagentmaybeappearactivein vitrobutarenoteffectiveclinicallyandshouldnotreportedas susceptible:
Salmonellaand Shigella MRSA 1stand2ndgenerationcephalosporinsand aminoglycoside All.Lactam,carpenams,

Penicillin AllpenicillinsexceptBlactamase inhibitors,cephms,&carbapenems resistantoxacillin sensitivestaph Enterococcus Aminoglycoside(excepthighconc.) 17

cephalosporins,clindamycin,SXT Yersinia Listeria ESBL Ampicillin resistant enteroccoci .Lactam Cephalospornis Penicillin,cephalosporins&azactam Penicillin,Blactamaseinhibitor(AMC, SAM)&carbapenemsthemechanismis alteredPBPs

Penicillin(Oxacillin) Penicillins,cephalosporins,carpenams resistantpneumoniae except3rdgenerationcephalosporinsmust doMIC

2.

Warning(CSF):Thefollowingantimicrobialagentshould notberoutinelyreported: a.
b.

Agentsad.Byoralroutes. 1stand2ndgenerceph.except(CXM). Clindamycin. MacroLides. Tetracyclines. Fluroquinolones.

c.
d.

e. f.
3.

Susceptibilitytestingofpenicillinsandotherlactams approvedbyFDAfortreatmentofstrep.Pyogenesand agalactiaeisnotnecessaryforclinicalpurposes.

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RecommendationP/AMP/cefazolin/clindamycin/ erythromycin. 4. P.mirabilisshouldbeaddedtoE.coliandK.pneumoniaein screeningforESBLinbacterimicisolatesonly(blood)because reportsofESBLinnonbacterimicisolateshavebeen relativelyrareduetolowfrequencyofplasmidconjugate. 5. Levofloxacinshouldbeusedforstenotrophomonaswith SXT. 6. Susceptibilitytestingisnotrecommendedfor S.saprophyticusinurinaryisolatesNOV(R)<16mm. 7. LabshouldidentifyS.lugdunensis(anuncommon)butone causeofendocarditis:(PYRtest+veandornithinede carboxylase+ve).
8.

ScreeningofMRSAbycefoxitin30or10gbydiscdiffusion whilebyoxacillinMICbyEtestorbrothdilution.ForCONS, thecefoxitindisktesthasgreaterspecificitythanoxacillin andequalsensitivity,althoughitmaymisssomestrainsof mecAdetectionandthelatextestforPBP2aarethemost accuratepredictorsofmecAmediatedresistance.

19

9.

Fororganisms(campy,corynebacterium,bacillusspp.) consultationwithaninfectiousdiseasespecialistis recommendedforguidanceindeterminingtheneedfor susceptibilitytestingandintheinterpretationofresults, publishedreportsinmedicalliteratureandcurrentconsensus recommendationsfortherapyofuncommonisolates,may obviatetheneedfortesting.Ifnecessaryadilutionmethod usuallywillbethemostappropriatetestingmethodandthis mayrequiresubmittingtheorganismtoareferencelab. NB:
CephalothinCF/CF/CListhedisc

representativeforthe1st,2ndgenercephaland cefopodoxime
Tetracycline:isrepresentativeforDo,

minocyclin.
Erythromycine:isrepresentativefor

macrohide.
10.

Oxacillinscreeningdiskdiffusionusedtodetecthighrate ofpenicillinresistanceinS.pneumoniae(>20mm
20

susceptible,<19mm(doMICtesting)andcorrelatewiththe bodysiteitiscollectedfrom.
11.

S.pneumoniaeisolatesfromCSF,itisrecommendedtesting penicillin,cefotaximeceftrioxone,meronamandvancomycinby brothdilutionmethodasdiscdiffusionwithcarbapenemsor cephalosporinesforS.pneumoniaedonotexist.Butfornonlife threateninginfections,agentstobeconsiderarepenicillin, erythromycinsutrim,bybrothanddiscdiffusionmethod.

12.

Staphisolatesthatareresistanttoerythromycinbut susceptibletoclindamycinshouldbetestedforinducible resistnacetoclindamycin(MLS)resistancemediatedby"erm" geneusingtheDzoneapproximationtestwithclosely approximatederythromycinandclindamycintest.

13.

Streptviridansanyisolatefromasterilebodysiteor implicatedinaseriousinfectionasendocarditisshouldbe testedforpenicillinsusceptibilityandcephalosporinsespeci especially3rdgenerationassomeviridansmayexhibitrelative resistance.Vancomycinistherecommendedalternativeto lactamAbs.

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14.Forfecalisolatesofsalmonellaandshigella:(ampicillin, fluroquinolones,SXT(only).whileinExtraintestinalisolates: Chloramphenicol,3rdgenerationcephalosporins.

15.AsregardsESBLdetection:

Duetovariableaffinityoftheseenzymesfordiffusion subsandinoculumeffect,someESBLproducingorganism with3rdgenerationcephalosporinsmayresultinclinical failureifinfectionis(outsidetheurinarytract).

TestingofcephamycinsisrecommendedinESBL producingisolates.

Cefpodoximeandceftazidimehavebeenproposedas indictorsofESBLproductionascomparedtocefotaxime andceftrioxone.

TheseenzymescanbeinducedbycertainAbs,AAs,or bodyfluids.

ItispossibleforonespecimentocontainbothESBL producingandnonESBLproducingcellsofthesame species.So,itmusttestseveralcoloniesforaprimary cultureplate.

22

LatestguidelinesrecommendedscreeningofESBLwith aMIC 2mg/dLagainstcefpodoximeceftazidime, aztreonam,ceftaximeorceftrixone.

ThreeindicatorsofESBL: An8foldreductioninMICinthepresenceof clavulonicacidbybrothdilutionmethod. Potentiationoftheinhibitorzonebyclavulonicacid >5mmindiameterofinhibitionbydiscdiffusion.

Discapproximationtestbyusingofcefoxitin(inducer)

placedatadistanceof2.5cmfromcephalosporindisc flatteningofthezoneofinhibitionofcephalosporin disctowardsinducerdisc>1mm.

AsregardstreatmentofESBLcarbapenensarethe mosteffectiveandreliableastheyarehighlyresistantto hydrolyticactivityofallESBLenzymesduetotrans6 hydroxyethylgroup.

MeronamisthemostactivewithMICsgenerallylower thanthoseofIPM(0.030.12mg/mlvs0.060.5mg/ml).

AlsoESBLactivityisinhibitedbyclavulonicacid,the onlyinfectionsthatcanbetreatedsafelywith
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lactamaseinhibitorarethoseinvolvingtheurinarytract inwhichtheconcentrationhighenoughtocounteractthe hydrolyticactivityofESBL. Clavulonicacidappearsmoreefficientthansulbactam ittakesabouteighttimesmoretoobtainaprotective similartothatbyC.acid.

PlasmidsresponsibleforESBLproductiontendtobe largeandcarryresistancetoseveralagentsan importantlimitationinthedesignoftreatment.Themost frequentcoresistanceareaminoglycosides, flouroquinolones,TE,chloramphenicolandsutrim

ANTIMICROBIAL

COMBINATION BY DIFFUSION METHODS:

Diskapproximationtest :
24

Principle: Thismethodhasbeenexploredtoassessprimarilyina qualitativefashiontheinteractionofantimicrobialsasthey diffusethroughagarplatesseededwithatestorganism. Advantages: Simple. Theuseofreadilyavailablematerials(discsand MullerHintonagar). Disadvantages: Qualitativemethodonly. Lowsensitivityandspecificitycomparedtodilution methods. i.e.: Theresultsofthistestmaydifferfromresultsobtainedwhen thesameagentsandorganismaretestedinliquidmedia.

Procedure:
25

Thistechniqueusesthesamestandardinoculumsand MullerHintonagarasaroutineBauerKirbysusceptibility test.

Toassesspossibleinteractionsbetweentwodrugs(A andB)diskscontainingthesedrugsareplacedonaplatethat hasbeeninoculatedwithatestedorganism.

Thedistancebywhichthedisksareseparatedmaybe varied,butitshouldgenerallybeequaltoorslightlygreater thanthesumoftheradiiofthezonesofinhibitionofthedrugs whenexaminedalone(mostly15mmfromcentreto centre).ONLYFIVECOMBINATIONARETESTEDINTHE 100mmPLATE

Afterovernightincubation(1618hrs)at37Ctheplate arereadyforexamination.

26

Example of antibiotic combinations used for multi resistant organisms "by Dilution methods":
1. Pseudomonas: Bactericidal: Ciprofloxacinandtienam(CIPandIPM). CiprofloxacinandAmikin(CIPandAK). CiprofloxacinandAzactam(CIPandATM). CiprofloxacinandFortum(CIPandCAZ).
LevofloxacinandMaxipime(LEVandFEP).

CiprofloxacinandMaxipime(CIPandFEP).
LevofloxacinandMeronam(LEVandMEM).

CiprofloxacinandTazocin(CIPandTZP).
LevofloxacinandTazocin(LEVandTZP). LevofloxacinandGentamycin(LEVandCN). TazocinandGentamycin(TZPandCN).important 27

 TazocinandTienam(TZPandIPM). Bacteriostatic:
AugmentineandAmpicillin(AMCandAMP).notused VanocomycinandCarbencillin(VAandPip).notused

AzactamandMaxipime(ATMandFEP).

2.

Acinetobacter: Bactericidal : DoxycyclinandAmikin(DoandAK). CiprofloxacinandFortum(CIPandCAZ). CiprofloxacinandMeranam(CIPandMEM). CiprofloxacinandAzactam(CIPandATM). TazocinandGentamycin(TZPandCN). CiprofloxacinandTazocin(CIPandTZP).

Bacteriostatic : TienamandAmikin(IPMandAK). UnasynandAmikin(SAMandAK).

3. o

Enterobacteriaceae: TazocinandAmikinorGentamycin(TZPandAKor CN).

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CefotaximandAmikinorGentamycin(CTXandAKor CN). AzactamandTienam(ATMandAPM). AzactamandMaxipime(ATMandFEP). CeftazidimeandOflaxocin(CTZandOFO). CefoxitinandAmikin(FOXandAK). CiprofloxacinandFortum(CIPandCAZ). CiprofloxacinandTazocin(CIPandTZP).

o o o o o o

4.

Proteus: o TazocinandAmikin(TZPandAK). o TienamandAmikin(IPMandAK).

5.

Enteroccoci: .lactam(penicillin)andaminoglycoside.(gentamycin) Glycopeptide(VAorTEC)andaminoglycoside. TeinamandTeicoplanin(IPMandTEC). TazocinandGentamycin(TZPandCN). TazocinandCiprofloxacin(TZPandCIP).

29

Glycopeptideand.lactam(TECandP). CiproflxocinandVancomycinorPenicillin(CIPand VA).

CiproflxocinandAmpicllin(CIPandAMP).

30

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