Staphylococci Fred Tenover

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

Staphylococci Fred Tenover

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