Methodological problems for the detection of resistant S. aureus:

MRSA, VISA

Y. Glupczynski

Laboratoire de bactériologieCliniques Universitaires UCL de Mont-Godinne

Université Catholique de Louvain

Symposium on Staphylococcus aureus (Bruxelles 11/01/2007)

Methicillin-resistance in S. aureus

1. Acquisition of exogeneous PBP2a (mecA gene)Cf SCCmec

⇒Transcription of mecA ⇒ synthesis of PBP 2a (transpeptidases with decreased affinity to penicillins, cephalosporins and carbapenems-

2. Non mec A mediated resistance (rare)Hyperproduction of β-lactamases (plasmid)

Production of meticillinases (plasmid)

Hyperproduction of normal PBPs (BORSA)Modification of endogeneous PBPs (1,2&4) (MODSA)

mec complex (A, B, C)ccr complex(A/B) SCCmec I

SCCmec II

SCCmec IV

SCCmec III

SCCmec V

orfXIS431

mecA

Staphylococcal Cassette Chromosome mec (SCCmec)

Tn554

pUB110

pT181mecA on mobile genetic element (21-67 kb)may contain other resistance genes

5 different typesbased on polymorphismiin conserved genes

Heterogeneous expression ofmethicillin resistance

Tomasz A. et al. J Clin Microbiol. 1991; 35:124

Hetero-resistant strains

10-6 resistant subpopulation

Homo-resistant strains

Facteurs influencing transcription of mecA

Auxiliary genes (fem, fmt, sarA, agr…)

mecA regulatory genes (mecI, mecR1)

blaZ penicillinase regulatory genes

(blaI, blaR1)

• [NaCl] ↑

•T°

•Osmolality

•pH

Level of methicillin (Oxa) resistance

Methods for detection of MRSA

• Oxacillin disk test (1µg, 5 µg)

• Oxacillin agar screen

• Cefoxitin disk test

• Automated systems (Vitek2, Phoenix,…)

• MIC (agar, microbroth, E-test)

• MRSA screen (PBP2a latex)

• Detection of mecA gene

Accuracy & TAT of Culture-Based MRSA Detection Tests

15 min>9797-100PBP2a latex

6-12h>9998-100Phoenix BD

6-12h>9988-100Vitek 2

8h>8693-97Microscan

24h>99>98Broth microdilution

24h>9595-98Oxacillin agar screen

18h>9797-98Cefoxitin disk diffusion

TATSpecificitySensitivityMethod

Swenson JCM 2001;39:3785 Felten JCM 2002;40:2766 Nonhoff 14th ECCMID 2004 Flayhart JCM 2005; 43:5536 Brown JAC 2005;56:1000.

Sensitivities of various methods for detection of 83 MRSA clinical isolates

Felten A. et al. JCM 2002 40:2766

Cefoxitin (30 µg) zone diameters • More potent inducer of the mecA

regulatory genes

• Easier to read (18 h instead of 24h)

• Disk diffusion breakpoints– Susceptible > 20 mm for S. aureus– Susceptible > 25 mm for CNS

0

20

40

60

80

100

120

140

160

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 >28

mecA +mecA -

Swenson JM et al. JCM 2005 43:3818

0

20

40

60

80

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 >28

mecA +mecA -

S. aureus

CoNS

96-9790-9979-8994-99CoNS

10098-10074-9986-98S. aureus

SpecSensSpecSens

CefoxitinOxacillinOrganism

CLSI 2007

Zone diameter (mm)

S I R

Oxacillin ≤ 10 11-12 ≥ 13

Cefoxitin ≤ 21 - ≥ 22

Disk diffusion test for prediction of mecA-mediated resistance in Staphylococci

(CLSI M100-S17)S. aureus and S. lugdunensis

Sensitivities of disk diffusion, oxascreen and automated systems for detection of MRSA

0

25

50

75

100

Oxascreen DD Cefox DD oxa Vitek 2 BD Phoenix oxa

BD Phoenixcefox

All MRSA Hetero-MRSA (n = 26) Homo-MRSA (n = 72)

Per

cent

Nonhoff C. et al. 14th ECCMID 2004

Belgian national external quality control193 participating labs

One isolate hetero-resistant to oxacillin– mecA positive with MIC to oxacillin of 4 µg/ml– Susceptible to quinolones, MLS and aminoglycosides– Resistant to fusidic acid

Only 82% reported as MRSA

0

10

20

30

40

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60

Disk oxacillin Disk cefoxitin Vitek 2 BD Phoenix ATB

ResistantSusceptible

N o

f lab

s re

spon

ding

Most prevalence CA-MRSA clonein Belgium (ST80 SCCmec IV)

P: Penicilline GOX: oxacillineFox: cefoxitineVa: vancomycineL: lincomycineE: érythromycinePt: pristinamycineTet: tétracyclineFA: ac. fusidiqueC: chloramphénicolOFX: ofloxacineSxt: cotrimoxazoleFt: furanesRA: rifampicineTM: tobramycineGM: gentamicine

P OX FOX Va

TET

Sxt

GMTM

RAFt

FA

L E Pt

OFXC

Reduced susceptibility to glycopeptides: VISA - VRSA

Glycopeptide cut-off values

MIC for vancomycin(µg/ml)

MIC for teicoplanin(µg/ml)

S I R S I R

CLSI ≤ 2 8-16 ≥ 32 ≤ 8 16 ≥ 32

SFM ≤ 4 8-16 ≥ 32 ≤ 4 8-16 ≥ 32

BSAC ≤ 4 > 4

hVISA - VISA

• With the current CLSI breakpoints, nodifferentiation between VISA and hVISA

• What about strain with MIC between 2 and 4µg/ml (E-test) ?

NB: Etest not recommended by CLSI !

Current methods for determination of susceptibility to glycopeptides

• Disk diffusion• Agar screening

– BHI agar + (4) or 6 µg/ml Vanco (CLSI)– MH agar + 5 µg/ml Teico (SFM, EARSS)

• Automated systems• MIC determination

– Agar– Broth microdilution (CLSI)– E-test

Pittfalls and problems in detection of glycopeptide resistance in

Staphylococci

• Poor diffusion in agar (disk diffusion)

• Expression of resistance slow /low (rapid automate systems !)

• High inoculum effect (teicoplanin)

• Type of medium (Brand/batch) (teicoplanin)

• No distinction in MIC distribution between GSSA and certain GISA

isolates (hGISA)

• No molecular reference tests for categorization of GISA

(population analysis profiles)

Comparative MIC values of 294 putative hGISA/GISA strains

11% hGISA

Screening by growth on BHIA +4 µg teico, Macromethod E test and PAP

Garnier et al. JAC 2006

Screening tests for GISA (I)

• Vancomycin agar screen test (CLSI)– 10 µl of 0.5 MF suspension on BHI agar + 6 µg vanco– Incubation: 35°C / ambiant air / 24 h– Test positive: if ≥ 2 colonies

• Teicoplanin agar screen test (SFM)– 10 µl of 2 MF suspension on MH agar + 5 µg teico– Incubation: 35°C / ambiant air / 24-48 h– Test positive: if ≥ 4 colonies

Positive test = presumed reduced susceptibility !

Screening tests for GISA (II)

• Modified E-test (Macromethod) – 2 MF suspension in MH broth; 200 µl plated on BHI agar– Incubation: 35°C / ambiant air / 48 h– Test positive: MIC vancomycin AND teicoplanin ≥ 8 µg/ml or

MIC teicoplanin ≥ 12 µg/ml

Do not round up MIC value (ie: 6 µg/ml -> 8 µg/ml !)

Positive test = presumed reduced susceptibility !

Walsh et al. JCM 2001

Glycopeptide E-test MICsMacromethod (2 McF / BHI / 48 h)

ATCC29213 (Peni-S MSSA)Vanco MIC 2 µg/mlTeico MIC 3 µg/ml

HIP5827 (GISA)Vanco MIC 16 µg/mlTeico MIC 64 µg/ml

Slide kindly provided by O. Denis

Performance of three screening methods for detecting GISA isolates

Screeningmethod

Sensitivity(%)

Specificity(%)

PPV(%)

NPV(%)

BHIA6V 35.2 97.4 98.4 45.2

MHA5T 85.9 75.5 82.2 79.1

Macro ET 82.0 89.1 94.0 74.4

Wootton et al. JCM 2006

Multicentric study (12 labs, Europe - USA- Australia)48 strains (15 GISA, 15 hGISA, 15 GSSA, 3 control strains)

Algorithm for the detection of GISA/hGISA S. aureus strains

Treatment failure withglycopeptide

Positive Agar Screening test (Vanco or Teico)or MIC of Vanco or teico ≥ 4 µg/ml

Macromethod E-test(BHIA, 2 MF, 48 h)Vanco and teico ≥ 8 µg/mlor Teico ≥ 12 µg/ml

Confirmation by Population

analysis (PAP)+ MIC (MH, 0.5 McF, 24 h)

Hetero-GISA GISA

Susceptible

Negative Positive

Howden et al. EJCMID 2005Denis et al. NosoInfo 2006

Confirmatory tests for GISA

• MIC determination – Microbroth dilution (CLSI), Agar dilution, E-test– Medium: Mueller-Hinton– Inoculum: 0.5 McF– Incubation: 35°C / ambiant air / 24 h

• Population analysis studies– Reference laboratory

Glycopeptide E-test MICsCLSI method (0,5 McF / MH / 24 h)

ATCC29213 (Peni-S MSSA)Vanco MIC 0.75 µg/mlTeico MIC 1 µg/ml

HIP5827 (GISA)Vanco MIC 8 µg/mlTeico MIC 16 µg/ml

Slide kindly provided by O. Denis

Population analysis of VSSA, hVISA, VISA and VRSA isolates

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1

2

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5

6

Log

CFU

/ml

7

8

1 2 3 4 5 6 7 8 9 10 11 12Vancomycin concentration (µg/ml)

VSSA

hVISA

VISA

VRSA

Adapted from Liu c. et al. 2003. Antimicrob Agents Chemother. 47:3040

VRSA

Van MIC>256 µg/ml

Teico MIC24 µg/ml

Vancomycin-resistant MRSA isolate

Slide kindly provided byO. Denis

Automated methods for determination of vancomycin MIC (µg/ml) with VRSA

Microscan Vitek Vitek2

Michigan >16 >32 8*

Pennsylvania 2-4 2 2**

* true MIC of 1024 µg/ml** true MIC of 32 µg/ml

Cheng et al. NEJM 2003; 348: 1342-7Whitener SHEA 2003

Automated systems fails to detect VRSA !

Unacceptable methods

• Automated methods, e.g. VITEK, Phoenix, Microscan: did not identify accurately Hershey & NYC VRSAs with MICs of 32-64 µg/ml

• Disk diffusion alone (for VRSA ?)

• LABORATORIES USING THE ABOVE MUST ADD A VANCO (6 µg/ml) or a TEICO (5 µg/ml) AGAR SCREEN PLATE

Acceptable methodology

• NCCLS broth microdilution

• Agar dilution

• E test (0.5 Mac Farland, 24h incubation)

Non automated methodology

Conclusion

• No single method is perfect for AST of S. aureus

• cefoxitin disk test best currrent single test for detection of MRSA (combination of several methods still needed…in some instances)

• Detection of GISA difficult (limited to isolates from deepseated infection ,chronic/recurrent infections with foreignmaterial)– Screening agar (teico, vanco)– Macromethod E test

• Reduced susceptibility to glycopeptides should beconfirmed by population analysis / MIC in reference lab