BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 13th May 2010 Methods of Detecting reduced susceptibility to glycopeptides in S. aureus Methods of Detecting Reduced Susceptibility to Presenter: Mandy Wootton Glycopeptides in S. aureus Phenotype • Glycopeptide Resistant Staphylococcus aureus GRSA – vanA gene present, MIC VAN >32mg/L • Glycopeptide Intermediate Staphylococcus aureus GISA – homogeneous resistance, mechanism unknown - MIC VAN ~4/8mg/L (Mu50) • heterogeneous-Glycopeptide Intermediate Staphylococcus aureus hGISA – heterogeneous resistance, mechanism unknown - MIC VAN ~1.5/4mg/L (Mu3). Precursor to GISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 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 BSAC Breakpoints MIC <0.25 0.5 GSSA 1 2 hGISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 4 8 GISA 16 EUCAST Breakpoints MIC <0.25 0.5 GSSA 1 2 4 hGISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 8 GISA 16 Disc Diffusion 1 • ISA with 1:10 of 0.5 McFarland inoculum • 5g Vancomycin disc • Incubation at 35-37°C for 1820h • Interpretive Criteria: Resistance Susceptible - 14mm - 15mm GRSA: Detected GISA: NOT Reliably Detected hGISA: NOT Detected Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus Disc Diffusion 2 Separation of phenotypes 30 Zone Diammeter (mm) • ISA with 1:10 of 0.5 McFarland inoculum • 5g, 30g Vancomycin disc • 5g, 10g, 20g, 30g Teicoplanin • Incubation at 35-37°C for 1820h GSSA hGISA GISA 20 10 0 V5 V30 • 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 T5 T10 T20 Disc Concentration T30 MIC Determination 1 • Standard Etest –0.5 McFarland on ISA, 18-20h –BPs 2 (S)/ >2 (I/R) 3 6 2 0 0 0 0 % Correctly Classified 100% 0 0 1 41 7 1 0 16% 0 0 0 2 8 0 7 88% 0.5 0.75 MRSA/GSSA x10 hGISA x50 GISA x17 MIC 1 2 4 6 8 <0.25 0.5 GSSA GRSA Detected GISA Mostly Detected hGISA mostly missed 1 2 4 hGISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 8 GISA 16 MIC Determination 2 Automated (PHX) MRSA/GSSA x22 repeats • Repeat 1 MRSA/GSSA x22 GISA • GSSA x10 hGISA x10 GISA x10 MRSA/GSSA x10 hGISA x10 GISA x10 1 2 4 8 9 1 0 0 % Correctly Classified 100% 0 6 4 0 40% 0 5 4 1 50% 1 2 4 18 4 0 hGISA PAPAUC PHX V MIC Etest V MIC PAPAUC 3.01 1.33 1.32 1.47 1.4 1.31 1.32 1.32 1.57 1.41 2 2 2 2 8 4 2 4 4 4 4 4 4 4 8 4 4 4 4 8 1 0.94 1.14 1.23 1.04 0.94 0.97 1.29 1.1 1.14 GSSA PHX Etest V V MIC MIC 4 2 4 4 2 2 2 2 4 2 % Correctly Classified 100% 4 2 2 2 2 2 2 2 2 2 PAPAUC 0.58 0.74 0.68 0.73 0.73 0.74 0.45 0.58 0.65 0.62 PHX Etest V V MIC MIC ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 2 ≤1 ≤1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Some GISA & hGISA identified Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 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 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 GRD Etest • • • 0.5 McFarland inoculum Mueller Hinton + blood 48h incubation at 3537°C in air – • Reading at 24h and 48h Interpretive criteria: GISA or hGISA if VA or TP 8mg/L 1) 2) GISA if GRD+ and standard VA MIC 4mg/L hGISA if GRD+ and standard VA MIC <4mg/L MRSA/GSSA x50 repeats hGISA x51 GISA X26 % Correctly Classified No + at 24h 0 No + at 48 1 30 41 80.4% 24 26 100% 98% Detects all GISA, majority at 24h Detects most hGISA and GISA 2% false positives Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus Screening Agar 1 • Mueller Hinton Agar + 5mg/L TP (MHA5T) • Multi-centre study – 12 labs (Europe & US) – 48 strains – 3 method – 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 BHIA6V MHA5T Macro Etest % GSSA Correctly Classified 97.4% 76% 98% % hGISA Correctly Classified 11.5% 78.7% 70.3% % GISA Correctly Classified 58.9% 93.8% 97.4% * 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 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 Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 103 / N • Log viable count vs. VA concn graph • Ratio = AUC of test isolate/AUC of Mu3 10 1 0 Viable count Log cfu/ml PAP-AUC 2 PAP of Mu 3 (hGISA) , Mu50 (GISA), GSSA and Oxford Staphylococcus aureus Mu 3 (hGISA) Mu50 (GISA) NCTC 6571 (GSSA) GSSA 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 0.5 1 2 2.5 4 8 Vancomycin concentration mg/L GSSA < 0.9 hGISA 0.9 - 1.29 GISA 1.3 PAP-AUC • PAP-AUC ratio criteria: 1.5 hVISA VISA VSSA 1.0 0.5 0.0 0 1 2 3 Observation number Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus 4