Gradient tests on EUCAST media

Can MH-F be used for gradient MIC
determination for streptococci and
Haemophilus influenzae?
Gunnar Kahlmeter
Charlotta Karlsson, Erika Matuschek,
Jenny Åhman
Växjö, Sweden

EUCAST disk diffusion test
recommends two media:
• Unsupplemented Mueller-
Hinton agar for nonfastidious
organisms
• Mueller-Hinton agar with 5%
defibrinated horse blood and
20 mg/L β-NAD for fastidoius
organisms (MH-F)

Aim
It would be practical if routine microbiological laboratories could use
MH and MH-F for both disk diffusion testing and gradient MIC-testing.
• To evaluate whether the MH-F medium used for
disk diffusion testing of streptococci and
Haemophilus influenzae in the EUCAST method
could be used for gradient <strong>tests</strong>
• To evaluate whether there was any systematic
difference between MH-F and the two media
recommended for gradient <strong>tests</strong> by the
manufacturers.

<strong>Gradient</strong> <strong>tests</strong> for MIC determination
• More user friendly than microdilution and agar dilution
assays.
• Instantly available for MIC determination of single
isolates
• Several studies show good agreement with reference
methods; however for some antibiotics systematically
high or low results obtained.
• <strong>Gradient</strong> <strong>tests</strong> investigated:
– Etest (bioMérieux)
– M.I.C.Evaluator (Oxoid)

Manufacturers recommendations
Etest (bioMérieux)
M.I.C.Evaulator, “M.I.C.E.” (Oxoid)
• Mueller-Hinton
– Non-fastidious organisms
• Mueller-Hinton with sheep blood
– Streptococcus pneumoniae
– Streptococcus A, B, C and G
• Haemophilus Test Medium (HTM)
– Haemophilus influenzae

Manufacturer recommendations
compared with EUCAST disk method
Haemophilus influenzae
bioMérieux Oxoid EUCAST
a) HTM
Medium
HTM
b) ISA + 5% horse
blood and
NAD
MH-F
Inoculum
McF 0.5 in broth
(McF 1 if mucoid)
McF 0.5 in
suitable media
McF 0.5 in
saline
Incubation
35ºC, 5% CO 2
20-24 h
35-37ºC, 5% CO 2
18-20 h
35ºC, 5% CO 2 ,
16-20 h

Manufacturer recommendations
compared with EUCAST disk method
Streptococcus pneumoniae
bioMérieux Oxoid EUCAST
Medium
MH + 5% sheep
blood
a) MH + 5%
sheep blood
b) ISA + 5%
horse blood
and NAD
MH-F
Inoculum
McF 0.5 in broth
(McF 1 if mucoid)
McF 0.5-1 in
suitable media
McF 0.5 in saline
Incubation
35ºC, 5% CO 2
20-24 h
35-37ºC, 5% CO 2
a) 20-24 h
b) 18-20 h
35ºC, 5% CO 2 ,
16-20 h

Methodology
• H. influenzae, S. pneumoniae and S. pyogenes
– QC strains and clinical isolates were tested
• MH-F vs. recommended media for Etest and
M.I.C.Evaluator
– HTM for H. influenzae
– MH + 5% sheep blood for streptococci
• Incubation in 5% CO 2 and 35ºC for 20 h.

H. influenzae*
MH-F compared with reference medium
MH-F < HTM MH-F = HTM MH-F > HTM
2 dil 1 dil 0 1 dil 2 dil No of readings
Etest (Number of readings)
Amoxicillin/clavulanic acid 2 33 35
Ampicillin 5 21 9 35
Ciprofloxacin 5 29 1 35
Tetracycline 2 19 13 1 35
SXT * 15 20 35
MICE (Number of readings)
Amoxicillin/clavulanic acid 7 25 3 35
Ampicillin 3 30 1 1 35
Ciprofloxacin 7 26 2 35
Tetracycline 3 15 17 35
* Trimethoprim-sulfamethoxazole (SXT) was not available for MICE
H. Influenzae ATCC 49247, NCTC 8468 and three clinical isolates

H. influenzae
35
0 • MH-F = Reference medium
- 30• MH-F < Reference medium
+ • MH-F > Reference medium
25
Ampicillin
No of readings
20
15
10
E-test
MICE
5
0
-3 -2 -1 0 1 2 3
Dilution steps' difference
Tetracycline
35
30
No of readings
25
20
15
10
E-test
MICE
5
0
-3 -2 -1 0 1 2 3
Dilution steps' difference

S. pyogenes* and S. pneumoniae*
MH-F compared with reference medium
MH-F < MH-Str
MH-F = MH-Str
MH-F > MH-Str
3 dil 2 dil 1 dil 0 1 dil 2 dil No of readings
Etest (Number of readings)
Benzylpenicillin 10 23 2 35
Erythromycin 5 21 9 35
Moxifloxacin* 1 1 20 13 35
Tetracycline 3 27 5 35
SXT * 6 21 5 3 35
MICE (Number of readings)
Benzylpenicillin 4 28 3 35
Erythromycin 3 22 10 35
Tetracycline 1 30 4 35
* Moxifloxacin and Trimethoprim-sulfamethoxazole (SXT) were not available for MICE
*S. pyogenes CCUG 25571, S. pneumoniae ATCC 49619 and three
clinical S. pneumoniae isolates

S. pyogenes and S. pneumoniae
Benzylpenicillin
35
30
No of readings
25
20
15
10
E-test
MICE
5
0
-3 -2 -1 0 1 2 3
Dilution steps' difference
Tetracycline
35
30
No of readings
25
20
15
10
E-test
MICE
5
0
-3 -2 -1 0 1 2 3
Dilution steps' difference
0 • MH-F = Reference medium
- • MH-F < Reference medium
+ • MH-F > Reference medium

Conclusions I
• The correlation between MICs obtained on
MH-F and reference media was good for
Streptococcus pneumoniae, Streptococcus
pyogenes and Haemophilus influenzae but the
number of strains/isolates was very limited
• Results were reproducible and easy to read
• Our data suggest that MH-F may be a suitable
substrate for gradient test MIC determination.

Conclusions II
We call on manufacturers and
others to extend our trials and
validate the use of MH-F for
gradient MIC-testing.

We thank bioMérieux and Oxoid for
supplying the gradient strips