Antimicrobial Susceptibility Testing of Campylobacter

Antimicrobial susceptibility testing of Campylobacter jejuni and C. coli

CRL Training course in AST

Copenhagen, Denmark

23 - 27th Feb. 2009

Methodologies

E-test by AB-biodiskA dilution test based on the diffusion principleNo international recognized interpretation guidelinesExpensive for routine usageNot approved by CRL AR

Disk diffusion CLSI interpretation guidelines for only two antimicrobials Limited interpretation only detect resistanceNot approved in the monitoring by the Commission

Methodologies

Agar dilutionApproved for four antimicrobials according to CLSI and 12

according to eucastLaborious and time consuming

Broth dilution (microbroth dilution by Trek, VetMic etc.)The Golden StandardApproved for four antimicrobials according to CLSI and 12

according to eucastExpensive but easy to use.

Disk diffusion

Testing condition according to CLSI M45-A

Cation-adjusted Mller Hinton agar supp. with 5% sheep blood (Depth 4mm, works also with other types of blood)

Direct colony suspension, equivalent to MacFarland 0.5

Incubation temp / time: 36-37C for 48h OR 42C for 24h (not less than 36C and not above 42C)

Incubation atm: microaerobic equal to 10% CO2, 5% O2and 85%N2

Disk diffusion, cont.

Testing condition according to CLSI M45-A

Antimicrobials: Erythromycin (disk content=15ug) and Ciprofloxacin (disk content=5ug) -approved by CLSI

Limited interpretation: no zone indicate resistance however appearance of any zone would require MIC determination to categorize the susceptibility

E-test

Cation-adjusted Mller Hinton agar supp. with 5% sheep blood works also with other types of blood

Direct colony suspension, equivalent to MacFarland 1



No recommended antimicrobial susceptibility interpretive criteria. According to AB biodisk; resistant isolates can be identified based on the CLSI interpretive criteria for Enterobacteriaceae

Agar dilutionAgar and plate preparation according to CLSI M7-A7 / M100-S18

Sterile saline. Mller Hinton II agar. 1000ml

Cattleblood. 50ml

Antimicrobial1280ug/ml

Solvent

32ug/ml

16ug/ml

8ug/ml

4ug/ml

2ug/ml

1ug/ml

0,5ug/ml

3ml

3ml

9ml

21ml

10ml

10ml

90ml

90ml

Labeled:

6ml

3ml

3ml

6ml

9ml

21ml

90ml

10ml

10ml

90ml

90ml

10ml

90ml

90ml

6ml

3ml

6ml

9ml

10ml

10ml

Agar dilution, cont.


Agar preparation according to CLSI M7-A7

Cation-adjusted Mller Hinton agar supp. with 5% sheep blood works also with other types of blood



Inoculation of 1-2 ul of a 10 fold dilution from MacFarland0.5 use a multipoint inoculation

Ps.aeruginosa.ATCC 27853

E.coliATCC 25922

E.faecalisATCC 29212

S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

24

25

26

19

17

15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

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25

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15

20


Reading the Agar dilution plates


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

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19

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15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

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25

26

19

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15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

24

25

26

19

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15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

24

25

26

19

17

15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

24

25

26

19

17

15

20


E.coliATCC 25922


S.aureusATCC 29213

7

8

9

10

11

12

13

1416

18

1

2

3

4

5 6

21

22

23

24

25

26

19

17

15

20

Control 0,25 0,5

=> 0,5= 0,5

< 0,25

Micro-broth dilution

Testing condition according to CLSI M7-A7

Cation-adjusted Mller Hinton broth supp. with 5% sheep blood

Remember to adjust the content of cations and lyse the blood (also horse and cattle blood works fine)

Prepare the inoculum by direct colony suspension, equivalent to MacFarland 0.5

Micro-broth dilution, cont.

CRL Plate design cover antimicrobials according to Decision 2007/516/EC



Dilute the suspension 50ul per 10 ml of broth (200 fold).

Inoculate 100 ul of the final inoculum per well depending on the system used



Seal the plates with pre-manufactured punctured holes

Stack no more than two plates


Incubation atm: microaerobic equal to 10% CO2, 5% O2 and 85%N2


Reading the micro-broth dilution plates

Semi or full automatic reading

1 2 3 4 5 6 7A

B

C

D

E

F

G

H

Interpretation of MIC valuesEpidemiological cut off values should be used for interpretation

according to EFSA and Decision 2007/516/EC

What is an epi cut off value!

Interpretation of MIC valuesEpidemiological cut off values should be used for interpretation

according to EFSA and Decision 2007/516/EC

Cut off values by eucast recommended by EFSA

Antimicrobials for Campylobacter MIC (g/mL)R is > MIC (g/mL)

R is > C. jejuni C. coli

Erythromycin 4 16 Ciprofloxacin 1 1 Tetracycline 2 2 Streptomycin 2 4 Gentamicin 1 2 Chloramphenicol* 16 16 Nalicixic acid* 16 32 *Not part of the EFSA monitoring programme

Interpretation of MIC values

Cut off values by eucast www.eucast.org

Quality Control

Monitor quality of saline, broth, lysed blood, Macfarland st., temp. and atm.

Check the purity of the test suspension for each isolate

Use the reference strain C. jejuni ATCC 33560

E-test

Cation-adjusted Mller Hinton agar supp. with 5% sheep

Thank you!

Rene Hendriksen ([email protected])

www.antimicrobialresistance.dk

Antimicrobial susceptibility testing of Campylobacter jejuni and C. coliMethodologiesMethodologiesDisk diffusionDisk diffusion, cont.E-testAgar dilutionAgar dilution, cont.Agar dilution, cont.Agar dilution, cont.Agar dilution, cont.Micro-broth dilutionMicro-broth dilution, cont.Micro-broth dilution, cont.Micro-broth dilution, cont.Micro-broth dilution, cont.Interpretation of MIC valuesInterpretation of MIC valuesInterpretation of MIC valuesInterpretation of MIC valuesQuality ControlE-testAntimicrobial susceptibility testing of Campylobacter jejuni and C. coliMethodologiesMethodologiesDisk diffusionDisk diffusion, cont.E-testAgar dilutionAgar dilution, cont.Agar dilution, cont.Agar dilution, cont.Agar dilution, cont.Micro-broth dilutionMicro-broth dilution, cont.Micro-broth dilution, cont.Micro-broth dilution, cont.Micro-broth dilution, cont.Interpretation of MIC valuesInterpretation of MIC valuesInterpretation of MIC valuesInterpretation of MIC valuesQuality ControlE-test

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Antimicrobial Susceptibility Testing of Campylobacter