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Methods of Detecting reduced susceptibility to glycopeptides in S. aureus

Methods of Detecting reduced susceptibility to glycopeptides in S. aureus. BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 13 th May 2010. Presenter: Mandy Wootton. Phenotype. Glycopeptide Resistant Staphylococcus aureus GRSA vanA gene present, MIC VAN >3 2mg/L

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Methods of Detecting reduced susceptibility to glycopeptides in S. aureus

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  1. Methods of Detecting reduced susceptibility to glycopeptides in S. aureus BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 13th May 2010 Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus Presenter: Mandy Wootton

  2. Phenotype • Glycopeptide Resistant Staphylococcus aureusGRSA • vanA gene present, MIC VAN >32mg/L • Glycopeptide Intermediate Staphylococcus aureusGISA • homogeneous resistance, mechanism unknown - MIC VAN ~4/8mg/L (Mu50) • heterogeneous-Glycopeptide Intermediate Staphylococcus aureushGISA • heterogeneous resistance, mechanism unknown - MIC VAN ~1.5/4mg/L (Mu3). Precursor to GISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  3. Prevalence GRSA • < 30 world wide • None in UK GISA/hGISA • UK prevalence: • <1% (EARSS) • 6064 isolates (1 Intermediate) • MIC • 0% (2001-6 BSAC Bacteraemia) • 615 isolates • MIC • Cardiff: • 0.8% • 669 MRSA • PHX, disc, MHA5T, PAP-AUC Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  4. GSSA hGISA GISA BSAC Breakpoints MIC <0.25 0.5 1 2 4 8 16 Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  5. MIC <0.25 0.5 1 2 4 8 16 GSSA hGISA GISA EUCAST Breakpoints Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  6. Disc Diffusion 1 • ISA with 1:10 of 0.5 McFarland inoculum • 5g Vancomycin disc • Incubation at 35-37°C for 18-20h • Interpretive Criteria: • Resistance - 14mm • Susceptible - 15mm • GRSA: Detected • GISA: NOT Reliably Detected • hGISA: NOT Detected Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  7. Disc Diffusion 2 • ISA with 1:10 of 0.5 McFarland inoculum • 5g, 30g Vancomycin disc • 5g, 10g, 20g, 30g Teicoplanin • Incubation at 35-37°C for 18-20h • Separation of phenotypes • Low concn discs of V and T differentiate GISA & GSSA • None accurate for ID of hGISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  8. GSSA hGISA GISA MIC Determination 1 • Standard Etest • 0.5 McFarland on ISA, 18-20h • BPs 2 (S)/ >2 (I/R) GRSA Detected GISA Mostly Detected hGISA mostly missed MIC <0.25 0.5 1 2 4 8 16 Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  9. MIC Determination 2 Automated (PHX) • Repeat 1 MRSA/GSSA x22 • GSSA x10 hGISA x10 GISA x10 Some GISA & hGISA identified Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  10. Summary • Disc • Detects GRSA (vanA) • Some detection of GISA • No reliable detection of hGISA • MIC (Etest & automated) • Detects GRSA • Detects some GISA • Detects few hGISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  11. Macro Etest • 2 McF inoculum • Brain Heart Infusion • 48h incubation at 35-37°C in air • Interpretative Criteria: (NOT MICs) GISA or hGISA if 8mg/L plus 8mg/L Vancomycin + Teicoplanin or 12mg Teicoplanin alone. Detects most hGISA and GISA No distinction between GISA and hGISA 2% false positive Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  12. GRD Etest • 0.5 McFarland inoculum • Mueller Hinton + blood • 48h incubation at 35-37°C in air • Reading at 24h and 48h • Interpretive criteria: GISA or hGISA if VA or TP 8mg/L • GISA if GRD+ and standard VA MIC 4mg/L • hGISA if GRD+ and standard VA MIC <4mg/L Detects all GISA, majority at 24h Detects most hGISA and GISA 2% false positives Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  13. Screening Agar 1 • Multi-centre study • 12 labs (Europe & US) • 48 strains • 3 method • Mueller Hinton Agar + 5mg/L TP (MHA5T) • 10ul of 0.5 McF inoculum • 48h Incubation at 35-37°C in air • Interpretive criteria: hGISA or GISA if ≥1 cols after 48hr • Brain Heart Infusion Agar + 6mg/L VA (BHI6V) • 10μl of 0.5McF inoculum • 24h Incubation at 35-37°C in air • Interpretive criteria: hGISA or GISA if ≥2 cols after 24hr * MHA5T: false positives reduced when 2 cols at 48h used MHA5T Detects Most GISA MHA5T Detects Most hGISA Up to 24% false positives Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  14. Population Analysis Profile – Area Under Curve 1 • “Gold Standard” • 2 plates of BHIA + 0, 0.5, 1, 2, 2.5 & 4mg/L VA • Overnight cultures of test organism and Mu3 in TSB - diluted 1:10-3, 1:10-6 • Incubation at 35-37°C in air for 48hrs 106 / 103 106 / 103 106 / 103 103 / N 103 / N 103 / N Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

  15. PAP-AUC 2 • Log viable count vs. VA concn graph • Ratio = AUC of test isolate/AUC of Mu3 • PAP-AUC ratio criteria: GSSA < 0.9 hGISA 0.9 - 1.29 GISA  1.3 Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

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