Methodological problems for the detection of resistant S. aureus: - - PowerPoint PPT Presentation

Methodological problems for the detection of resistant S. aureus: MRSA, VISA

• Y. Glupczynski

Laboratoire de bactériologie Cliniques 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 I I SCCmec I V SCCmec I I I SCCmec V

• rfX

IS431 mecA

Staphylococcal Cassette Chromosome mec (SCCmec)

Tn554 pUB110 pT181

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

5 different types based on polymorphismi in conserved genes

Heterogeneous expression of methicillin 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 >97 97-100 PBP2a latex 6-12h >99 98-100 Phoenix BD 6-12h >99 88-100 Vitek 2 8h >86 93-97 Microscan 24h >99 >98 Broth microdilution 24h >95 95-98 Oxacillin agar screen 18h >97 97-98 Cefoxitin disk diffusion TAT Specificity Sensitivity Method

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

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

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

96-97 90-99 79-89 94-99 CoNS 100 98-100 74-99 86-98

• S. aureus

Spec Sens Spec Sens Cefoxitin Oxacillin Organism

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

25 50 75 100 Oxascreen DD Cefox DD oxa Vitek 2 BD Phoenix

• xa

BD Phoenix cefox All MRSA Hetero-MRSA (n = 26) Homo-MRSA (n = 72)

Percent

Nonhoff C. et al. 14th ECCMID 2004

Belgian national external quality control

193 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

10 20 30 40 50 60 Disk oxacillin Disk cefoxitin Vitek 2 BD Phoenix ATB

Resistant Susceptible

N of labs responding

Most prevalence CA-MRSA clone in Belgium (ST80 SCCmec IV)

P: Penicilline G OX: oxacilline Fox: cefoxitine Va: vancomycine L: lincomycine E: érythromycine Pt: pristinamycine Tet: tétracycline FA: ac. fusidique C: chloramphénicol OFX: ofloxacine Sxt: cotrimoxazole Ft: furanes RA: rifampicine TM: tobramycine GM: gentamicine

P OX

FOX

Va

TET

Sxt GM TM RA Ft FA L E Pt

OFX

C

Reduced susceptibility to glycopeptides: VISA - VRSA

Glycopeptide cut-off values

MI C for vancomycin (µg/ ml) MI C 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, no

differentiation between VISA and hVISA

• What about strain with MIC between 2 and 4

µg/ml (E-test) ? NB: Etest not recommended by CLSI !

Current methods for determination

• f 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 MICs

Macromethod (2 McF / BHI / 48 h)

ATCC29213 (Peni-S MSSA) Vanco MIC 2 µg/ml Teico MIC 3 µg/ml HIP5827 (GISA) Vanco MIC 16 µg/ml Teico MIC 64 µg/ml

Slide kindly provided by O. Denis

Performance of three screening methods for detecting GISA isolates

Screening method 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 with glycopeptide Positive Agar Screening test (Vanco or Teico)

• r MIC of Vanco or teico ≥ 4 µg/ml

Macromethod E-test

(BHIA, 2 MF, 48 h) Vanco and teico ≥ 8 µg/ml

• r 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 2005 Denis et al. NosoInfo 2006

Confirmatory tests for GISA

• MI C 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 MICs

CLSI method (0,5 McF / MH / 24 h)

ATCC29213 (Peni-S MSSA) Vanco MIC 0.75 µg/ml Teico MIC 1 µg/ml HIP5827 (GISA) Vanco MIC 8 µg/ml Teico MIC 16 µg/ml

Slide kindly provided by O. Denis

Population analysis of VSSA, hVISA, VISA and VRSA isolates

1 2 3 4 5 6

Log CFU/ml

7 8 1 2 3 4 5 6 7 8 9 10 11 12

Vancomycin concentration (µg/ml) VSSA hVI SA VI SA VRSA

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

VRSA

Van MIC >256 µg/ml Teico MIC 24 µg/ml

Vancomycin-resistant MRSA isolate

Slide kindly provided by

• O. 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: 1342-7 Whitener 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 deep

seated infection ,chronic/recurrent infections with foreign material)

– Screening agar (teico, vanco) – Macromethod E test

• Reduced susceptibility to glycopeptides should be

confirmed by population analysis / MIC in reference lab