Susceptibility Testing
Anaerobes
Jenny Andrews
The BSAC Standardized Method
Development Centre
Birmingham UK
BSAC Anaerobe Study
Gunnar Kahlmeter & Lena Bieber
SRGA
Jenny Andrews & Becky Walker
SMDC
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
Emergence of resistance in
B. fragilis
Carbapenem
(metallo -lactamase)
Metronidazole
(nim gene)
NB. Resistance is rare to these agents
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 2002
(A survey of susceptibility testing of anaerobes
in the United Kingdom. Andrews & Wise. JAC
2002; 50:757)
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
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)
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%
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
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
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
4-80C for up to 14 days (SRGA)
No significant difference
Disc testing control strains SRGA & SMDC
Within ± 2mm
Materials for disc testing
75 B. fragilis
25 B. thetaiotaomicron
50 C. perfringens
Controls:
B. thetaiotaomicron
NCTC 9343/ATCC 25285 B. fragilis
NCTC 8359/ATCC 12915 C. perfringens
ATCC 29741
ISA + 5% blood + 20 mg/L NAD
Prepared in-house
bioMérieux pre-poured
Oxoid pre-poured
Discs
Co-amoxiclav
30 g
Clindamycin
2 g
Meropenem
10 g
Metronidazole
5 g
Pip/tazobactam 85 g
Penicillin
1 unit
Incubation for 18-20 hours
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
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 18-20 h incubation
General comments about disc
testing
Unable to supply plates to both centres
from the same batches
Organisms grew less well on the prepoured plates from Oxoid & bioMérieux
Zones for metronidazole and
clindamycin difficult to measure
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)
Combined SRGA & SMDC data for C. perfringens:
penicillin 1 unit disc
NB B. fragilis and thetaiotaomicron resistant to penicillin
Co-amoxiclav 30 g disc
•Susceptible MIC BP 8 mg/L
16
•ZD BP 28 mm for B. fragilis &
C. perfringens only.
12
14
B. thetaiotaomicron
Number
10
8
6
•For B. thetaiotaomicron a poor
relationship between MIC and
zone diameter
4
2
0
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Zone diam eter (m m )
Piperacillin/tazobactam
75/10 g disc
70
60
B. fragilis
50
Number
40
30
20
10
0
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Zone diameter (mm)
18
16
B. thetaiotaomicron
14
12
Number
•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
10
8
6
4
2
0
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Zone diam eter (m m )
23
24
25
26
27
28
29
30
31
32
33
34
Meropenem 10 g disc
100
90
80
B. fragilis
70
I
Number
60
50
I
40
30
20
10
0
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Zone diameter (mm)
18
16
14
B. thetaiotaomicron
12
Number
•EUCAST MIC BP 2/8 mg/L
•26 mm = S; 19-25 = I; 18 = R
•Carbapenem resistant B. fragilis
(due to the presence of
metallo-β-lactamase) have no zone of
inhibition
10
8
6
4
2
0
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Zone diam eter (m m )
Clindamycin 2 g disc
40
35
B. fragilis
30
Number
25
20
15
10
5
0
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Zone diam eter (m m )
40
•Susceptible MIC BP 4 mg/L
•ZD BP 10 mm
35
30
B. thetaiotaomicron
Number
25
20
15
10
5
0
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Zone diam eter (m m )
Metronidazole 5 g disc
Recommendations have been on the website since January 2006
70
60
B. fragilis
Number
50
40
nim+ ve
30
20
10
0
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Zone diam eter (m m )
25
20
B. thetaiotaomicron
15
Number
MIC BP 8 mg/L
ZD BP 18 mm
10
5
0
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Zone diam eter (m m )
43
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
40-45
Meropenem
10
42-50
36-43
39-45
Penicillin
1 unit
6
6
26-30
Piperacillin/tazo
bactam
75/10
41-48
29-34
37-43
NB. Metronidazole ranges available on the website since January 2006
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
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