Gradient tests on EUCAST media

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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 ong>testsong>

• To evaluate whether there was any systematic

difference between MH-F and the two media

recommended for gradient ong>testsong> by the

manufacturers.


ong>Gradientong> ong>testsong> 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.

ong>Gradientong> ong>testsong> 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

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