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-biodisk A dilution test based on the diffusion principle No international recognized interpretation guidelines Expensive for routine usage Not approved by CRL AR Disk diffusion CLSI interpretation guidelines for only two antimicrobials Limited interpretation only detect resistance Not approved in the monitoring by the Commission

Methodologies
Agar dilution Approved for four antimicrobials according to CLSI and 12 according to eucast Laborious and time consuming Broth dilution (microbroth dilution by Trek, VetMic etc.) The Golden Standard Approved for four antimicrobials according to CLSI and 12 according to eucast Expensive 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% O2 and 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 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% O2 and 85%N2 No recommended antimicrobial susceptibility interpretive criteria. According to AB biodisk; resistant isolates can be identified based on the CLSI interpretive criteria for Enterobacteriaceae

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

Agar dilution, cont.

Sterile saline. Mller Hinton II agar. 1000ml Cattleblood. 50ml Solvent 9ml 10ml 3ml Antimicrobial 1280ug/ml 21ml 3ml 90ml 6ml 6ml 10ml 8ug/ml 90ml 9ml 3ml 10ml 90ml 21ml 3ml 10ml 90ml 6ml 6ml 90ml 1ug/ml 10ml 9ml 3ml 90ml 10ml 0,5ug/ml 2ug/ml 4ug/ml 90ml 10ml 16ug/ml 32ug/ml Labeled:

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

Agar dilution, cont.

Agar preparation according to CLSI M7-A7 Inoculation of 1-2 ul of a 10 fold dilution from MacFarland 0.5 use a multipoint inoculation
5 6 4

21

20

22

E.faecalis ATCC 29212

1 19 23 10

E.coli ATCC 25922

S.aureus ATCC 29213

11

Ps.aeruginosa. ATCC 27853

24 26 12

18

17

25 13

16 15

14

Agar dilution, cont.

Agar preparation according to CLSI M7-A7 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% O2 and 85%N2

=> 0,5 = 0,5 < 0,25

Agar dilution, cont.

Reading the Agar dilution plates
5 6 5 6 5 6

21

21

21

20

20

20

22

22

22

E.faecalis ATCC 29212

1 19 23 10 1 19

E.faecalis ATCC 29212

23 10 1 19

E.faecalis ATCC 29212

23 10

E.coli ATCC 25922

S.aureus ATCC 29213

E.coli ATCC 25922

S.aureus ATCC 29213

E.coli ATCC 25922

S.aureus ATCC 29213

11

11

11

Ps.aeruginosa. ATCC 27853

24 26 12

Ps.aeruginosa. ATCC 27853

24 26 12

Ps.aeruginosa. ATCC 27853

24 26 12

18

18

18

17

25 13

17

25 13

17

25 13

16 15

14

16 15

14

16 15

14

Control

0,25

0,5

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

Micro-broth dilution, cont.

Testing condition according to CLSI M7-A7 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

Micro-broth dilution, cont.

Testing condition according to CLSI M7-A7 Seal the plates with pre-manufactured punctured holes Stack no more than two plates 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% O2 and 85%N2

Micro-broth dilution, cont.

Reading the micro-broth dilution plates Semi or full automatic reading
1 A B C D E F G H 2 3 4 5 6 7

Interpretation of MIC values

Epidemiological 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 values

Epidemiological 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 Erythromycin Ciprofloxacin Tetracycline Streptomycin Gentamicin Chloramphenicol* Nalicixic acid*
*Not part of the EFSA monitoring programme

MIC (g/mL) MIC (g/mL) R is > R is > C. jejuni C. coli 4 16 1 1 2 2 2 4 1 2 16 16 16 32

Interpretation of MIC values

Cut off values by eucast www.eucast.org

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 (rshe@food.dtu.dk) www.antimicrobialresistance.dk