1. Susceptibility Testing Anaerobes
Jenny Andrews
The BSAC Standardized Method Development Centre
Birmingham UK

2. BSAC Anaerobe Study Gunnar Kahlmeter & Lena Bieber SRGA
Jenny Andrews & Becky Walker
SMDC

3. Susceptibility Testing Anaerobes
Very difficult, particularly for slow growing organisms
CLSI have tried to give recommendations for disc testing, but pilot studies have not been encouraging
Now suggest MIC batch testing to see if there are changes in levels of susceptibility

4. Emergence of resistance in B. fragilis
Carbapenem
(metallo -lactamase)
Metronidazole (nim gene)
NB. Resistance is rare to these agents

5. Is there a need for the BSAC to provide recommendations?
Questionnaire sent to approximately 318 taking part in the NEQAS scheme
108 replies (34%)
63 DGH; 16 private laboratories; 11 Teaching Hospitals; 4 Associate Teaching Hospitals; 6 PHLS; 1 Reference Laboratory; 7 other institutions
Questionnaire published in the JAC (A survey of susceptibility testing of anaerobes in the United Kingdom. Andrews & Wise. JAC 2002; 50:757)

6. Information from the Questionnaire
Identification
28% ID anaerobes
45% ID if isolates from blood culture, CSF or sterile site
27% never ID anaerobes
Susceptibility testing
6 never
26% metronidazole on the primary isolation plate. No further testing unless R to metronidazole, a pure culture , or from a blood culture, CSF or sterile site

7. Media used for testing
48% laboratories used ISA supplemented with 5 % defibrinated horse blood and 20 mg/L NAD
21% Fastidious Anaerobe agar
5 % Wilkins & Chalgren agar (medium recommended by the BSAC for MIC determinations)

8. Method of susceptibility testing
90 % disc testing
15 % used a combination of disc testing and Etest
Antibiotics most often tested other than metronidazole:
penicillin 91%; clindamycin 48%; erythromycin 44%; co-amoxiclav 44 %; a carbapenem 3%; piperacillin/tazobactam 3%

9. Reasons for undertaking a BSAC Study
Working parties concern over emerging resistance (carbapenems & metronidazole)
Laboratories were routinely undertaking disc testing, so the BSAC should try to provide recommendations
The Working Party chose 6 antibiotics to be tested based on those generally used for treatment of anaerobic infections
Pragmatic decision to use ISA + 5% defibrinated horse blood and 20 mg/L NAD for testing because this was the medium used most often by diagnostic laboratories (data from the questionnaire)
Only fast growing anaerobes B. fragilis, B. thetaiotaomicron and C. perfringens to be studied

10. Technical considerations
Media used for testing by diagnostic laboratories:
Should media be stored under anaerobic conditions before use?
Were results comparable between the two centres undertaking the study:
This is important when combining data for analysis

11. Initial studies by the SRGA & the SMDC
Disc testing control strains on media prepared on the day and used immediately; prepared and stored anaerobically until use; media stored at C for up to 14 days (SRGA)
No significant difference
Disc testing control strains SRGA & SMDC
Within ± 2mm

12. Materials for disc testing
Discs
Co-amoxiclav 30 g
Clindamycin g
Meropenem g
Metronidazole 5 g
Pip/tazobactam 85 g
Penicillin 1 unit
Incubation for hours
75 B. fragilis
25 B. thetaiotaomicron
50 C. perfringens
Controls:
ATCC B. thetaiotaomicron
NCTC 9343/ATCC B. fragilis
NCTC 8359/ATCC C. perfringens
ISA + 5% blood + 20 mg/L NAD
Prepared in-house
bioMérieux pre-poured
Oxoid pre-poured

13. Disc testing Clinical isolates Sweden & SMDC
Acceptable ranges for control strains
SMDC
Media depth of 3.5, 4 and 4.5 mm
Pre-poured Oxoid & bioMérieux
Tested 5 times on 6 separate occasions

14. MIC testing SMDC ISA + 5% blood + 20 mg/L NAD
Same antibiotics as those chosen for disc testing (antibiotic powders obtained from the pharmaceutical company or Sigma)
Tests read after h incubation

15. General comments about disc testing
Unable to supply plates to both centres from the same batches
Organisms grew less well on the pre-poured plates from Oxoid & bioMérieux
Zones for metronidazole and clindamycin difficult to measure

16. Data analysis
Identification of the `wild sensitive’ population used to suggest zone diameter BPs
EUCAST clinical MIC BPs (where available) or BSAC MIC BPs
If available clinical response data (to be discussed by Robin Howe)

17. Combined SRGA & SMDC data for C. perfringens: penicillin 1 unit disc
NB B. fragilis and thetaiotaomicron resistant to penicillin

18. Co-amoxiclav 30 g disc Susceptible MIC BP 8 mg/L
ZD BP 28 mm for B. fragilis & C. perfringens only.
For B. thetaiotaomicron a poor relationship between MIC and zone diameter

19. Piperacillin/tazobactam 75/10 g disc
Susceptible MIC BP 16 mg/L
ZD BP of 27 mm for C. perfringens &
B. Fragilis only
For B. thetaiotaomicron , unacceptable
merging of S & R populations

20. Meropenem 10 g disc I I EUCAST MIC BP 2/8 mg/L
26 mm = S; = I; 18 = R
Carbapenem resistant B. fragilis
(due to the presence of
metallo-β-lactamase) have no zone of
inhibition

21. Clindamycin 2 g disc Susceptible MIC BP 4 mg/L ZD BP 10 mm
B. thetaiotaomicron

22. Metronidazole 5 g disc Recommendations have been on the website since January 2006
nim+ ve
MIC BP 8 mg/L
ZD BP 18 mm

23. QC
Acceptable range (mm)
Antibiotic
Disc content
(g unless stated)
B. fragilis
NCTC 9343
B. thetaiotaomicron
ATCC 29741
C. Perfringens
NCTC 8359
Clindamycin 2
13-27
11-25
23-28
Co-amoxiclav 30
43-49
40-45
Meropenem 10
42-50
36-43
39-45
Penicillin
1 unit 6
26-30
Piperacillin/tazobactam
75/10
41-48
29-34
37-43
NB. Metronidazole ranges available on the website since January 2006

24. Comments 1
From these data it would appear that supplemented ISA media can be used for testing fast growing anaerobes
Media does not have to be stored under anaerobic conditions before use, but can be stored in the refrigerator at 4-80C
Difficult group of organisms to test
Only fast growing anaerobes B. fragilis, B. thetaiotaomicron & C. perfringens studied
Zones for clindamycin and metronidazole are more difficult to measure than for the other antibiotics tested

25. Comments 2
Unable to give recommendations for B. thetaiotaomicron when testing inhibitor combinations because of the poor relationship between MIC and zone of inhibition
Organisms resistant by disc testing should have resistance confirmed by an MIC
For debate: Should organisms considered resistant be sent to the Anaerobe Reference Laboratory for further investigation?
Look forward to comments from users