1 / 15

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

abbott
Télécharger la présentation

Methods of Detecting reduced susceptibility to glycopeptides in S. aureus

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related