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8/7/2019 Antibacterial Sensitivity
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Antimicrobial SusceptibilityAntimicrobial SusceptibilityTesting for BacteriologyTesting for Bacteriology
LaboratoriesLaboratories
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Objectives:Objectives:At the conclusion of this presentation participants will be able toAt the conclusion of this presentation participants will be able to
jj Explain how to implement currentExplain how to implement current NCCLSNCCLS antimicrobialantimicrobialsusceptibility testing (AST) and reportingsusceptibility testing (AST) and reportingrecommendations.recommendations.
jj DescribeDescribe reliable methodsreliable methods for detecting resistancefor detecting resistanceamong:among:
EnterobacteriaceaeEnterobacteriaceae
Pseudomonas aeruginosaPseudomonas aeruginosa
Staphylococcus aureusStaphylococcus aureus
Enterococcus spp.Enterococcus spp.
Streptococcus pneumoniaeStreptococcus pneumoniae andand Streptococcus spp.Streptococcus spp.
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At the conclusion of this presentationAt the conclusion of this presentation
Participants will be able to(cont)Participants will be able to(cont)
jjDescribeDescribe effective reportingeffective reporting of ASTof AST
results.results.jj List steps that can be taken toList steps that can be taken to verifyverify
AST resultsAST results obtained on bacteriaobtained on bacteria
isolated from patients.isolated from patients.
jjDiscuss NCCLSDiscuss NCCLS--recommendedrecommended QCQCproceduresprocedures and how to troubleand how to trouble--shootshoot
outout--ofof--control QC resultscontrol QC results
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What Does the Laboratory Need to KnowWhat Does the Laboratory Need to Know
about Antimicrobial Susceptibility Testing (AST)about Antimicrobial Susceptibility Testing (AST)??
jjWhichWhich organismsorganisms to test?to test?
jjWhatWhat methodsmethods to use?to use?
jjWhatWhat antibioticsantibiotics to test?to test?
jjHow toHow to reportreport results?results?
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What Does a Laboratory Need to KnowWhat Does a Laboratory Need to Know
about AST? (cont)about AST? (cont)
jjHow to determine theHow to determine the clinical significanceclinical significance
of results?of results?jjHow to ensureHow to ensure accuracyaccuracy of results?of results?
Quality control / quality assuranceQuality control / quality assurance
jjWhen to call the MD, infection control,When to call the MD, infection control,
public healthpublic health??
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What Does a Laboratory Need to KnowWhat Does a Laboratory Need to Know
about AST? (cont)about AST? (cont)
jjWhen toWhen to ask forask for helphelp??
jjWhere to go forWhere to go forhelphelp??
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Brief Review of Routine ASTBrief Review of Routine AST
MethodsMethods
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Routine Susceptibility TestsRoutine Susceptibility Tests
jjDisk diffusion (Kirby Bauer)Disk diffusion (Kirby Bauer)
jjBroth microBroth micro--dilution MICdilution MIC
NCCLS reference methodNCCLS reference method
jjEtestEtest
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Disk DiffusionDisk Diffusion
TestTest
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Select colonies
Prepare inoculum
suspension
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Mix well
Standardize inoculum
suspension
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Swab plate
Remove sample
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Add disks
Incubate overnight
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Measure ZonesMeasure ZonesTransmitted
LightReflected
Light
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Measure ZonesMeasure ZonesTransmitted Light*Transmitted Light*
jj
StaphylococciStaphylococci
oxacillin, vancomycinoxacillin, vancomycinjjEnterococciEnterococci vancomycinvancomycin
*Measure all other drug/bug*Measure all other drug/bug
combinations with reflected lightcombinations with reflected light
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Zone Interpretive CriteriaZone Interpretive Criteria
(mm)(mm)
DrugDrug
DiskDisk
contentcontent
(ug)(ug)ResRes IntInt SuscSusc
cefazolincefazolin 3030 e 1414 1515--1717 uu 1818
gentamicingentamicin 1010 ee 1212 1313--1414 uu 1515
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Disk Diffusion TestDisk Diffusion Test
jjQualitative resultsQualitative results
SusceptibleSusceptible
IntermediateIntermediate may respond if infectionmay respond if infectionis at body site where drug concentratesis at body site where drug concentrates(e.g. urine) or if higher than normal dose(e.g. urine) or if higher than normal dosecan be safely givencan be safely given
ResistantResistant
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Modify methods forModify methods for
fastidious bacteriafastidious bacteria
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Clinical Conditions when MICs are UsefulClinical Conditions when MICs are Useful
jj EndocarditisEndocarditis
jjMeningitisMeningitis
jj SepticemiaSepticemia
jjOsteomyelitisOsteomyelitis
jj Immunosuppressed patients (HIV, cancer, etc.)Immunosuppressed patients (HIV, cancer, etc.)
jj Prosthetic devicesProsthetic devicesjj Patients not responding despite SPatients not responding despite S
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MICMICjjMinimal inhibitory concentrationMinimal inhibitory concentration
jjThe lowest concentration ofThe lowest concentration of
antimicrobial agent thatantimicrobial agent that inhibitsinhibits thethegrowth of a bacteriumgrowth of a bacterium
jj Interpret:Interpret:
SusceptibleSusceptible IntermediateIntermediate
ResistantResistant
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Inoculum PreparationInoculum Preparation
MIC TestingMIC Testing(NCCLS Reference Method)(NCCLS Reference Method)
jjStandardize inoculum suspensionStandardize inoculum suspension
jjFinal inoculum concentrationFinal inoculum concentration
33 5 x 105 x 1055 CFU/mlCFU/ml
(3(3 5 x 105 x 1044 CFU/well)CFU/well)
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Microdilution MIC tray
Prepare inoculum
suspension
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Dilute & mix inoculum
suspension
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Pour inoculum
into reservoir and
inoculate MIC tray
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Inoculatepurity plate
Incubate overnight
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ExaminingExamining
purity platepurity plate
Reflected lightReflected light
Transmitted lightTransmitted light
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Optimal Use of Purity PlatesOptimal Use of Purity Plates
jj SubSub final test suspensionfinal test suspension to nonto non--selective mediumselective medium
(after inoculating MIC test)(after inoculating MIC test)jj Streak forStreak forisolationisolation (avoid several specimens per plate(avoid several specimens per plate
-- may not reveal contaminants if no isolated colonies)may not reveal contaminants if no isolated colonies)
jj ExamineExamine before reading MICbefore reading MIC (usually at 16(usually at 16--20 h)20 h)
jjReRe--incubateincubate if antibiogram questionableif antibiogram questionable
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Read MICs
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Reporting Time: Disk Diffusion or MICReporting Time: Disk Diffusion or MIC
jjStaphylococciStaphylococci oxacillin, vancomycin, cefoxitinoxacillin, vancomycin, cefoxitin
jjEnterococciEnterococci vancomycinvancomycin
jj ResistanceResistance may be reported any time growth ismay be reported any time growth isobserved after a minimum of 16 h incubationobserved after a minimum of 16 h incubation
jjAfter minimum of 16 h, read test:After minimum of 16 h, read test:
ififRR, report, report
IfIfSS, re, re--incubate and read again at 24 hincubate and read again at 24 h
NCCLS M2NCCLS M2--A8, M7A8, M7--A6A6
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MIC on a stripMIC on a stripabbiodisk.comabbiodisk.com
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S. pneumoniaeS. pneumoniae
Penicillin MIC =Penicillin MIC =
33 QQg/mlg/ml
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MIC Interpretive Criteria (MIC Interpretive Criteria (QQg/ml)g/ml)
DrugDrug SuscSusc IntInt ResRes
cefazolincefazolin ee 88 1616 uu 3232
gentamicingentamicin ee 44 88 uu 1616
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MICs RecommendedMICs Recommended (NCCLS M100(NCCLS M100--S14)S14)
jj ViridansViridans StreptococcusStreptococcus penicillinpenicillin
jj
S. pneumoniaeS. pneumoniae
penicillin, cefotaxime/ceftriaxonepenicillin, cefotaxime/ceftriaxone(sterile sites)(sterile sites)
jj EnterococcusEnterococcus vancomycin Int resultsvancomycin Int results
jj StaphylococcusStaphylococcus vancomycin zonevancomycin zone ee14 mm14 mm
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CreditsCredits
Janet Fick Hindler, MCLS MT(ASCP)Janet Fick Hindler, MCLS MT(ASCP)UCLA Medical CenterUCLA Medical Center
Los Angeles, CALos Angeles, CA