1. BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 25th October 2010
Methods of Detecting reduced susceptibility to glycopeptides in S. aureus
Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus
Presenter: Mandy Wootton

2. 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

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

4. BSAC Breakpoints GSSA hGISA GISA
MIC <
GSSA
hGISA
GISA
Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

5. EUCAST Breakpoints GSSA hGISA GISA
MIC <
GSSA
hGISA
GISA
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. MIC Determination 1 GRSA Detected GISA Mostly Detected
Standard Etest
0.5 McFarland on ISA, 18-20h
BPs 2 (S)/ >2 (I/R)
GRSA Detected
GISA Mostly Detected
hGISA mostly missed
MIC <
GSSA
hGISA
GISA
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 MIC (Etest & automated) 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 Interpretative Criteria: (NOT MICs)
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 MHA5T Detects Most GISA MHA5T Detects Most hGISA
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
hGISA or GISA if ≥2 cols after 24hr
Multi-centre study
12 labs (Europe & US)
48 strains
3 method
* 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 / / / / N / N / 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
GISA  1.3
Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus