Methods for colistin testing
What works and what does not?
Erika Matuschek, Ph D
EUCAST Development Laboratory, EDL
3rd joint meeting on AMR in Salmonella and Campylobacter, Copenhagen 7 April 2017
Antimicrobial susceptibility testing
of colistin
• Broth microdilution (BMD)
– International reference method (ISO 20776-1)
• Agar dilution?
• Gradient tests?
– Etest, bioMérieux
– MIC Test Strip (MTS), Liofilchem
• Disk diffusion??
AST of colistin – dilution methods
• BMD (reference)
See EUCAST Guidance Documents
www.eucast.org/guidance_documents/
– Sulphate salts
– Standard polystyrene trays
– No additives or pre-treatment of plates
– In-house prepared or commercial plates
• Agar dilution
– To be evaluated
AST of colistin – diffusion methods
• Gradient tests?
– Poor correlation with reference BMD
– Warning on www.eucast.org
• Disk diffusion?
– Poor separation between resistant and
susceptible isolates
• Problems probably related to poor diffusion
of colistin in agar
Results colistin disk diffusion
Enterobacteriaceae, 50 isolates
Colistin 25 µg
16
8
4
2
1
0.5
≤0.25
Inhibition zone diameter (mm)
MIC
(mg/L)
32
16
15
8
4
10
2
5
1
0.5
0
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No of isolates
20
Inhibition zone diameter (mm)
32
16
8
4
2
1
0.5
Inhibition zone diameter (mm)
Colistin 50 µg
25
No of isolates
32
MIC
(mg/L)
20
18
16
14
12
10
8
6
4
2
0
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
MIC
(mg/L)
18
16
14
12
10
8
6
4
2
0
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No of isolates
Colistin 10 µg
≤0.25
≤0.25
Colistin MIC distributions
http://www.eucast.org/mic_distributions_and_ecoffs/
• All distributions are recently reviewed
– Old distributions with P-80 (tween) removed
– Only non-truncated distributions included
• ECOFFs established for E. coli, K. pneumoniae and P.
aeruginosa
– More data are needed for Acinetobacter and Salmonella
• Clinical breakpoints: S≤2, R>2 mg/L
– Enterobacteriaceae, Pseudomonas spp. and
Acinetobacter spp.
EUCAST evaluation of colistin MIC
methods
• 75 Gram-negative bacteria with varying colistin
MICs (0.25-128 mg/L)
– 14 E. coli
• of which 10 mcr-1 positive
– 18 K. pneumoniae
S≤2 mg/L
R>2 mg/L
15
10
5
MIC (mg/L)
≥512
256
128
64
32
16
8
4
2
1
0.5
0.25
0.125
0.06
0.03
0.008
0.004
0
≤0.002
No of isolates
– 21 P. aeruginosa
– 22 Acinetobacter spp.
20
0.016
• of which 1 mcr-1 positive
EUCAST evaluation of colistin MIC
methods
• BMD
– According to ISO 20776-1 and EUCAST/CLSI
recommendations
– Frozen panels as reference
– Commercial freeze-dried panels
• Sensititre, MICRONAUT-S, MICRONAUT MIC Strip
• Gradient tests
– Etest
• Mueller-Hinton (MH) agar: Oxoid, BBL and MHE
– MIC Test Strip (MTS)
• Mueller-Hinton (MH) agar: Oxoid and BBL
Manual reading
of MICs
96 well microtiter
plate
MICRONAUT
MIC Strip
Typical gradient test result
Etest
Often very narrow
ellipse with a dip
MICs were read at
the bottom of the
ellipse according to
the manufacturer's
instructions
MTS
Often 0.5-1
dilution higher on
the right side of
the strip
MICs were read at
the higher value
according to the
manufacturer's
instructions
Results
Essential agreement for colistin MIC tests (no of MICs being
within ± 1 dilution of reference MIC).
Organism
Essential agreement
(EA)
Colistin MIC range
Sensititre custom plate
MICRONAUT-S
MICRONAUT MIC-Strip
E. coli and
P. aeruginosa
K. pneumoniae
(n=21)
(n=32)
0.25-32
0.25-128
27
31
31
Etest, Oxoid MH
27
Etest, BBL MH
20
Etest, MHE
24
MTS, Oxoid MH
19
MTS, BBL MH
24
*
The total number of tests for calculation of EA was 28 for
aeruginosa due to truncation at ≤0.25 and >32 mg/L.
19
21
21
Acinetobacter
spp.
(n=22)
0.5-32
All isolates
(n=75)
20
20
22
66 (96%)
72 (96%)
74 (99%)
0.25-128
13
13
53 (71%)
11
1
32 (43%)
9
2
35 (47%)
12
9
40 (53%)
12
13
49 (65%)
E. coli/K. pneumoniae and 19 for P.
Essential agreement
BMD: 96-99%
Gradient tests: 43-71% (depending on MH agar used)
Results
Categorical agreement for colistin MIC tests
Organism
Very Major Errors
(VME)
Major Errors
(ME)
Colistin MIC range
(mg/L)
E. coli and
P. aeruginosa
K. pneumoniae
(n=21)
(n=32)
Acinetobacter
spp.
(n=22)
All isolates
(n=75)
0.25-32
0.25-128
0.5-32
0.25-128
Sensititre custom plate
MICRONAUT-S
MICRONAUT MIC-Strip
1
2
2
1
1
0
2
3
3
4
6
5
Etest, Oxoid MH
Etest, BBL MH
Etest, MHE
MTS, Oxoid MH
MTS, BBL MH
2
1
2
0
0
0
0
0
0
0
0
0
0
0
0
2
1
2
0
0
Sensititre custom plate
MICRONAUT-S
MICRONAUT MIC-Strip
0
0
0
0
2
2
0
0
0
0
2
2
Etest, Oxoid MH
Etest, BBL MH
Etest, MHE
MTS, Oxoid MH
MTS, BBL MH
0
1
0
6
5
6
7
5
6
6
6
7
4
4
7
12
15
9
16
18
Results
Quality Control (QC) strains
Colistin MIC method
Reference frozen panel
Sensititre custom plate
MICRONAUT-S
MICRONAUT MIC-Strip
Etest, Oxoid MH
Etest, BBL MH
Etest, MHE
MTS, Oxoid MH
MTS, BBL MH
Escherichia coli
ATCC 25922
0.125 0.25 0.5
1
7
1
*
4
4
5
3
1
6
1
2
5
12
7
6
1
6
* ≤0.25 mg/L
2
1
Colistin MIC (mg/L)
Pseudomonas aeruginosa
ATCC 27853
0.25 0.5
1
2
4
8
1
7
4
4
8
7
4
8
3
4
2
5
4
3
Escherichia coli
NCTC 13846**
2
4
8
1
7
8
7
1
2
6
8
8
5
3
8
8
** mcr-1 positive
QC target value
Upper/lower limit
Red text = MIC out of range
EUCAST QC table v 7.0: ”For E. coli NCTC 13846, the colistin MIC target
value is 4 mg/L and should only on occasion be 2 or 8 mg/L.”
Results
Commercial BMD panels
Sensititre
≤0.25
0.5
1
2
4
8
16
32
≥64
0.25
2
0.5
3
9
2
1
Colistin reference MIC (mg/L)
2
4
8
16
32
1
8
5
2
5
1
1
7
3
2
5
3
5
5
64
128
1
4
1
MICRONAUT MIC Strip
MICRONAUT-S
0.25
0.125
0.25
0.5
1
2
4
8
16
32
64
128
1
1
0.5
1
10
2
1
1
1
7
8
Colistin reference MIC (mg/L)
2
4
8
16
32
1
4
2
2
5
3
5
2
4
6
3
1
2
2
64
128
1
0.125
0.25
0.5
1
2
4
8
16
32
64
128
0.25
1
1
0.5
6
6
2
1
2
10
4
Colistin reference MIC (mg/L)
2
4
8
16
32
2
4
1
2
7
1
7
3
9
1
64
128
3
2
1
Results
Gradient tests
Etest
Oxoid MH
BBL MH
Colistin reference MIC (mg/L)
0.25 0.5 1 2 4 8 16 32 64 128
0.06
0.125
0.25
0.5
1
2
4
8
16
32
64
128
1
1
6
9
1
2
3
2
2
6
2
1
1
3
2
3
1
2
6
1
1
2 2
12 14
5
5
2
1
1
1
1
1
1
2
2
2
7
1
1
2
3
1
1
1
2
4
2
1
2
1
Colistin reference MIC (mg/L)
0.25 0.5 1 2 4 8 16 32 64 128
1
2
7
5
2
8
1
BBL MH
Colistin reference MIC (mg/L)
0.25 0.5 1 2 4 8 16 32 64 128
2
11
2
1
1
1
1
2
MIC Test Strip
Oxoid MH
0.25
0.5
1
2
4
8
16
32
64
128
Colistin reference MIC (mg/L)
0.25 0.5 1 2 4 8 16 32 64 128
2
13
1
MHE
3
4
7
5
1
1
3
2
12 4
2 12
2
5
2
8
4
3
1
7
3
1
2
1
2
1
Colistin reference MIC (mg/L)
0.25 0.5 1 2 4 8 16 32 64 128
1
1 12 4
1
2 2 2 1
1
7 3
1
3
1 2 1
1
2 8 3 5
1 3 3
1 1 1
1
Salmonella spp.
28 isolates (DTU and SSI), gradient tests on Oxoid and BBL MH
Etest
Oxoid MH
BBL MH
≤0.25 0,5
0,125
0,25
0,5
1
2
4
8
16
32
64
1
1
COL BMD
2
4
1
5
6
8
16
32
0,125
0,25
0,5
1
2
4
8
16
32
64
2
2
1
4
2
4
MIC Test Strip
Oxoid MH
≤0.25 0,5
0,125
0,25
0,5
1
2
4
8
16
32
64
1
5
1
1
6
≤0.25 0,5
1
1
5
1
1
3
2
COL BMD
2
4
1
1
8
16
32
2
2
1
4
1
3
BBL MH
COL BMD
2
4
2
4
1
4
8
16
3
1
≤0.25 0,5
32
0,125
0,25
0,5
1
2
4
8
16
32
64
1
6
1
1
5
COL BMD
2
4
2
5
3
1
8
4
16
32
Conclusions
• Correlation with reference MICs was good
for all BMD methods.
• Gradient tests generally underestimated
colistin MICs resulting in very major errors
(false susceptibility).
• The poor performance of gradient tests
could not be detected with QC strains.
Warning on www.eucast.org
• Disk diffusion cannot be used for AST of
colistin.
• Currently available gradient tests
underestimate colistin MIC values (and
resistance) and should be avoided.
• Use BMD for AST of colistin, but perform
extended quality control.
EUCAST recommendations for
colistin QC
• Use both a susceptible strain (E. coli
ATCC 25922 or P. aeruginosa ATCC
27853) AND the colistin resistant E. coli
NCTC 13846 (mcr-1 positive).
– For E. coli NCTC 13846, the colistin MIC
target value is 4 mg/L and should only on
occasion be 2 or 8 mg/L.
EUCAST QC Tables v 7.0, 2017
Further work
• Additional BMD methods will be evaluated
– Colistin SensiTest (Liofilchem)
– UMIC (Biocentric)
Read more!
• Poster 161, ECCMID 2017 (available soon)
– www.eccmid.org
– http://www.eucast.org/presentations_and_stati
stics/eucast_at_eccmid/
• http://www.eucast.org/warnings/
• Check the EUCAST website regularly for
updates on methodology, QC ranges and
breakpoints.
www.eucast.org
• For questions and comments, please contact
[email protected] or the EUCAST
secretariat (see website).