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7/30/2019 Staphylococci Fred Tenover
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Antimicrobial Susceptibility TestingAntimicrobial Susceptibility TestingofofStaphylococcus aureusStaphylococcus aureus
Fred C. Tenover, Ph.D., (D)ABMMFred C. Tenover, Ph.D., (D)ABMM
Senior Director, Scientific AffairsSenior Director, Scientific Affairs
CepheidCepheid
Consulting Professor of PathologyConsulting Professor of Pathology
Stanford UniversityStanford University
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Which of the following is true aboutWhich of the following is true about
Staphylococcal Susceptibility Testing?Staphylococcal Susceptibility Testing? Cefoxitin zone diameters are much easier toCefoxitin zone diameters are much easier to
read and interpret than oxacillin zone diametersread and interpret than oxacillin zone diametersfor coagulasefor coagulase--negative staphylococcinegative staphylococci
The clindamycin induction test is onlyThe clindamycin induction test is onlynecessary if the erythromycin MIC or disk resultnecessary if the erythromycin MIC or disk result
is intermediate and clindamycin result isis intermediate and clindamycin result issusceptiblesusceptible
Susceptibility tests for mupirocin forSusceptibility tests for mupirocin forS. aureusS. aureus
must be tested in the presence of 50must be tested in the presence of 50 mMmM Ca++Ca++to be accurateto be accurate
The wild type distribution was named in honorThe wild type distribution was named in honor
of Derek Brown, Gunnar Kahlmeter, and Rafaelof Derek Brown, Gunnar Kahlmeter, and RafaelCanton (the original wild and crazy guys)Canton (the original wild and crazy guys)
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EUCAST Clinical BreakpointsEUCAST Clinical Breakpoints
Oxacillin MICOxacillin MIC Cefoxitin MICCefoxitin MIC ##
S. aureusS. aureus andand
S.S. lugdunensislugdunensis > 2> 2 g/mlg/ml > 4> 4 g/mlg/ml
CoagulaseCoagulase --
NegativeNegative
StaphylococciStaphylococci > 0.25> 0.25
g/mlg/ml
Do not useDo not use
Same breakpoints as CLSISame breakpoints as CLSI## Report asReport as oxacillinoxacillin--resistantresistant
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Cefoxitin Disk Screening TestCefoxitin Disk Screening Test
Cefoxitin and oxacillin haveCefoxitin and oxacillin have equivalentequivalent
sensitivity for detectingsensitivity for detecting mecAmecA--mediatedmediatedresistance inresistance in S. aureusS. aureus
Cefoxitin hasCefoxitin has much better sensitivitymuch better sensitivity forfor
detectingdetecting mecAmecA--mediated resistance inmediated resistance incoagulasecoagulase--negative staphylococci and the zonenegative staphylococci and the zoneedges are muchedges are much easier to readeasier to read
Most resistant strains can be reported at 16Most resistant strains can be reported at 16--1818hours, but the test needs to be held for 24 hourshours, but the test needs to be held for 24 hoursto findto find heteroresistantheteroresistant strains.strains.
Swenson et al J Clin Microbiol 37:4051-4058, 1999
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CLSI Cefoxitin Disk DiffusionCLSI Cefoxitin Disk Diffusion
Screen TestScreen TestOrganismOrganism Cefoxitin zoneCefoxitin zone 11 Cefoxitin zoneCefoxitin zone 22
S. aureusS. aureus 20 mm20 mm
CoNS*CoNS* 25 mm25 mm
1-Report as oxacillin-resistant
2-Report as oxacillin-susceptible
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EUCAST and CLSI BreakpointsEUCAST and CLSI Breakpoints
forforLinezolidLinezolidEUCASTEUCAST
(S / R)(S / R)
CLSICLSI
(Susceptible(Susceptibleonly)only)
MICMIC 44 / >4 g/mlg/ml 21 mm21 mm
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Where Do You Read a
Linezolid Disk Diffusion Result ?
Outer edge
Inner edge
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Where should you measure aWhere should you measure a
linezolid disk diffusion zone?linezolid disk diffusion zone? From the outer edge of the zone of inhibitionFrom the outer edge of the zone of inhibition
At approximately 80% inhibition of growthAt approximately 80% inhibition of growth From the inner edge of growth observed withFrom the inner edge of growth observed with
reflectedreflected lightlight
From the inner edge of growth observed withFrom the inner edge of growth observed with
transmittedtransmitted lightlight
The linezolid disk diffusion test doesnt workThe linezolid disk diffusion test doesnt workand should not be usedand should not be used
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Testing Linezolid by AgarTesting Linezolid by Agar--BasedBased
Methods Can Be DifficultMethods Can Be Difficult
Issue:Large zones
with indistinct
edgesaround disks
and Etest strips
MIC 21 mm, Susceptible
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Accuracy of Linezolid TestingAccuracy of Linezolid Testing
by Six Methodsby Six Methods
Tested 100 isolates (25Tested 100 isolates (25 S. aureusS. aureus, 25, 25
CoNS, 25CoNS, 25 E. faecalisE. faecalis, 25, 25 E. faeciumE. faecium))
32 of 100 organisms were non32 of 100 organisms were non--
susceptible or resistantsusceptible or resistant
Majority of problem were in detectingMajority of problem were in detecting
linezolidlinezolid nonnon--susceptiblesusceptible S. aureusS. aureus
Reading disk diffusion and Etest usingReading disk diffusion and Etest usingtransmitted lighttransmitted light instead of reflectedinstead of reflected
light improved sensitivitylight improved sensitivityTenover FC. J Clin Microbiol 45:2917-22, 2007
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Error Rates Testing Linezolid withError Rates Testing Linezolid with
Staphylococci and EnterococciStaphylococci and Enterococci
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VM=22/58= 37.9%
MA=7/40= 17.5%
Must confirm non-susceptible disk results by MIC
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EUCASTEUCAST VancomycinVancomycin BreakpointsBreakpoints
forforS. aureusS. aureusSusceptibleSusceptible ResistantResistant
MICMIC
88
g/mlg/ml
Disk diffusionDisk diffusion NANA NANA
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The New Challenge: HeteroThe New Challenge: Hetero--VancomycinVancomycin
Intermediate SIntermediate S. aureus. aureus (hVISA)(hVISA)
Vancomycin Susceptible Intermediate Resistant
MIC (g/ml)
Disk (30 g) 2
15 mm4 8
ND
16ND
VISA VRSA
SusceptibleSusceptible HeteroresistantHeteroresistant
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Which statement is true aboutWhich statement is true about
vancomycin testing ofvancomycin testing ofS. aureusS. aureus??A.A. IsoIso--SensitestSensitest agar is the most sensitive medium foragar is the most sensitive medium for
detecting heterodetecting hetero--VISA strainsVISA strainsB.B. Disk diffusion will identifyDisk diffusion will identify vanAvanA--containing VRSAcontaining VRSA
strains but not VISA strainsstrains but not VISA strains
C.C. MIC results should be interpreted after 16MIC results should be interpreted after 16--18 hours of18 hours of
incubation at 35Cincubation at 35CD.D. The macroThe macro-- Etest can identify heteroEtest can identify hetero--VISA strains asVISA strains as
long as both vancomycin and daptomycin are testedlong as both vancomycin and daptomycin are tested
E.E. Most automated susceptibility testing methodsMost automated susceptibility testing methodsaccurately detect vancomycin MICs of 4 g/ml inaccurately detect vancomycin MICs of 4 g/ml in S.S.aureusaureus
F.F. None of the above are trueNone of the above are true
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VISA Strains Cannot be DetectedVISA Strains Cannot be DetectedBy Disk DiffusionBy Disk Diffusion
Zone Diameter (mm)
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
2
4
6
8
10
GISA/GISS N=12
GSSA/GSSS N=24
Susceptible
Resistant
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Disk diffusionDisk diffusion
and Etest Zonesand Etest Zones
Even the most
difficult to
detect VRSAstrain was
detected by
disk diffusion
Tenover et al. Antimicrob Agents Chemother. 48:275-280, 2004.
CDCCDC ScattergramScattergram ofof S aureusS aureus andand
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PAGE | 17
CDCCDC ScattergramScattergram ofofS. aureusS. aureus andand
Vancomycin; To date all VRSA detectedVancomycin; To date all VRSA detected
Former CLSI breakpoint:
>15 mm susceptible
vanA-VRSA
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PAGE | 18
VR-CoNS (Not vanA)
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Detection of VISA StrainsDetection of VISA StrainsNo. (%)a categorized as No. of discrepancies in testing method
TestSusceptible
(n = 84)
Intermediate
(n = 45)
Category
agre
e
(n = 129)
VISA
called S
VISA
called R
S called
VISA
BMIC BBL 83 (98.9) 40 (88.9) 123 (95.3) 5 1
Agar 82 (97.6) 31 (68.9) 113 (87.6) 14 2
Etest 73 (86.9) 44 (97.8) 117 (90.7) 1 11
MicroScan 74 (88.1) 45 (100) 119 (92.2) 10
Phoenix 64 (76.2) 45 (100) 109 (84.5) 20
Sensititre 84 (100) 29 (64.4) 113 (87.6) 13 1
Vitek 83 (100) 0 (0) 83 (64.8) 35 10
Vitek 2 82 (97.6) 35 (77.8) 117 (90.7) 10 2
aPercentage is based on 85 susceptible strains and 45 vancomycin-intermediate
Staphylococcus aureus (VISA) strains.
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Population Analysis of hVISA and VISAPopulation Analysis of hVISA and VISA
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
0 1 3 5 7 9
MIC (ug/ml)
Suscept.
hVISA
VISA
VISA
SuscepthVISA
Note scale change
Subpopulation of hVISA isolates, for which
MIC=4-8 g/ml, are below detection level
Inoculum
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Fig 1. Population Analysis for Strain 9AJ57 on BHI vs. MHA
Vancom ycin Concentrations (mcg/mL)
0 0.25 0.5 0.75 1 2 3 4 6 8 10 12 14 16 32
Log10
(CFU/mL)
1
2
3
4
5
6
7
8
9
10
ATCC 29213
9AJ57-MHA
9AJ57-BHI
Subpopulation apparent onlyon BHI
MHA
Mueller-Hinton MIC= 1 g/ml
BHI MIC = 4 g/ml
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Macro Etest MethodMacro Etest Method
InoculumInoculum: 2.0 McFarland in Mueller: 2.0 McFarland in Mueller--HintonHinton
BrothBroth
100100 l inoculated onto 90mm BHI agar platel inoculated onto 90mm BHI agar plate
Use vancomycin and teicoplaninUse vancomycin and teicoplanin EtestsEtests;;
incubate at 35incubate at 35CC Read at 24 and 48 hoursRead at 24 and 48 hours
Positive results (hVISA)Positive results (hVISA)zz Vancomycin and teicoplanin:Vancomycin and teicoplanin: >>88 g/mlg/ml
zz Teicoplanin:Teicoplanin: >>1212 g/mlg/ml
Novel EtestNovel Etest
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Novel EtestNovel Etest
MethodMethod -- GRDGRD
Glycopeptide Resistance Detection Strip
TeicoplaninVancomycin
hVISA Cli i l R lhVISA Cli i l R l
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hVISA: Clinical RelevancehVISA: Clinical Relevance
Population Analysis Shows IncreasingPopulation Analysis Shows IncreasingMICsMICs
Vancomycin Concentrations
0 0.25 0.5 0.75 1 2 3 4 6 8 10
Log10
(CF/m
l)
1
2
3
4
5
6
7
8
9
ATCC29213
491-0.5VBHI
492-0.5VBHI
493-0.5VBHI
494-0.5VBHI
522-0.5VBHI
(g/ml)
Gradual
increase
in vancomycin
MICs during
10 weeks of
therapy with
vancomycinfor
endocarditis
1 2
3 4 VISA control
Tenover FC et al. IJAA 2009
Macro Etest
Whi h i NOT f
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Which statement is NOT true ofWhich statement is NOT true of
daptomycin susceptibility testing?daptomycin susceptibility testing?A.A. Daptomycin resistance cannot beDaptomycin resistance cannot be
detected by disk diffusiondetected by disk diffusion
B.B. Daptomycin MIC testing requiresDaptomycin MIC testing requiresadditional calcium in the mediumadditional calcium in the medium
C.C. Daptomycin tests should be incubatedDaptomycin tests should be incubatedfor a full 24 hours before interpretationfor a full 24 hours before interpretation
D.D. Reduced susceptibility to daptomycinReduced susceptibility to daptomycin
may accompany reduced susceptibility tomay accompany reduced susceptibility tovancomycinvancomycin
E.E. All the above are trueAll the above are true
D l t f D t iD l t f D t i
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Development of DaptomycinDevelopment of Daptomycin
NonNon--Susceptibility in Same StrainSusceptibility in Same Strain
Daptomycin Concentrations ( g/ml)
0 0.125 0.25 0.5 0.75 1 2 3 4 6 8 10
Log10(CFU/ml)
1
2
3
4
5
6
7
8
9
ATCC 29213
RWJ 1-DRWJ 2-D
RWJ 3-D
RWJ 4-D
1 2
3 4
Some hVISA andVISA strains
remainsusceptible to
daptomycineven after
vancomycinadministration.
Susceptibilitytesting is critical
for decisionmaking
Non-susceptible
EUCAST d CLSI D t iEUCAST d CLSI D t i
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EUCAST and CLSI DaptomycinEUCAST and CLSI Daptomycin
BreakpointsBreakpoints
EUCASTEUCAST(S / R)(S / R)
CLSICLSI(Susceptible(Susceptible
only)only)
MICMIC 11 / >1 g/mlg/ml
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Comparison ofComparison ofEtestEtest Results to BrothResults to Broth
Microdilution for DaptomycinMicrodilution for Daptomycin1616
88
44 77 22
22 99 4343 1212
11
11 22 4141 10100.50.5 7474 1414
0.250.25 6868 77
0.120.12 22 77 11
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Etest Results for DaptomycinEtest Results for Daptomycin
Overall, 95.3% of Etest results wereOverall, 95.3% of Etest results were
withinwithin ++11 dilution of broth microdilutiondilution of broth microdilution
method (n=376)method (n=376)
35.3% of Etest results were 1 dilution35.3% of Etest results were 1 dilution
lower; 5% were 2lower; 5% were 2--3 dilutions lower3 dilutions lower
90.2% of isolates that were non90.2% of isolates that were non--
susceptible by broth microdilution weresusceptible by broth microdilution werenonnon--susceptible by Etestsusceptible by Etest
Jevitt, L et al. JCM 2006;44:3098-104.
S. aureusS. aureus Mupirocin SusceptibilityMupirocin Susceptibility
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p p yp p y
PhenotypesPhenotypes
PhenotypePhenotypeMICMIC
RangeRange
((g/ml)g/ml)
MolecularMolecular
MechanismMechanism
SusceptibleSusceptible 44 Wild typeWild type
LowLow--levellevel
resistanceresistance88--256256 Mutations in nativeMutations in native
isoleucineisoleucine tRNAtRNA
synthetasesynthetase
HighHigh--levellevel
resistanceresistance
512512 NovelNovel isoleucineisoleucine
tRNAtRNA synthetasesynthetase
M i i B k i tM i i B k i t
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Mupirocin BreakpointsMupirocin Breakpoints
CLSI has approved both MIC and diskCLSI has approved both MIC and disk
diffusion breakpoints fordiffusion breakpoints forhighhigh--levellevelmupirocin resistance inmupirocin resistance in StaphylococcusStaphylococcus
aureusaureus
HighHigh--level mupirocin resistance islevel mupirocin resistance is
indicated by:indicated by:
zz MIC 512 g /mlMIC 512 g /ml
zz No zone of inhibition (6 mm) around aNo zone of inhibition (6 mm) around a
200 g disk200 g diskPAGE | 31
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CONCLUSIONSCONCLUSIONS
Linezolid resistance in staphylococci can beLinezolid resistance in staphylococci can be
hard to detect by agarhard to detect by agar--based methodsbased methods
Hetero vancomycin resistance is still hard toHetero vancomycin resistance is still hard to
detect although the macro Etest and GRD candetect although the macro Etest and GRD can
be usefulbe useful Most automated systems are FDAMost automated systems are FDA--cleared tocleared to
detect VRSA; detecting VISA may still be adetect VRSA; detecting VISA may still be a
problemproblem
Etest a reasonable approach for detecting nonEtest a reasonable approach for detecting non--
susceptibility to daptomycin in staphylococcisusceptibility to daptomycin in staphylococci