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 Müller 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: 36-37 C for 48h OR 42 C for 24h (not less than 36 C and not above 42 C) Incubation atm: microaerobic equal to 10% CO 2, 5% O 2 and 85%N 2
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 Müller Hinton agar supp. with 5% sheep blood works also with other types of blood Direct colony suspension, equivalent to MacFarland 1 Incubation temp / time: 36-37 C for 48h OR 42 C for 24h (not less than 36 C and not above 42 C) Incubation atm: microaerobic equal to 10% CO 2, 5% O 2 and 85%N 2 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. Müller Hinton II agar. 1000ml Solvent Cattleblood. 50ml Antimicrobial 1280ug/ml 9ml 3ml 21ml 3ml 6ml 6ml 10ml 90ml 10ml 90ml 10ml 90ml Labeled: 32ug/ml 16ug/ml 8ug/ml 9ml 3ml 10ml 90ml 4ug/ml 21ml 3ml 10ml 90ml 2ug/ml 6ml 6ml 90ml 10ml 1ug/ml 9ml 3ml 90ml 10ml 0,5ug/ml
Agar dilution, cont. Agar preparation according to CLSI M7-A7 Cation-adjusted Müller 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 7 3 21 8 20 2 22 9 E.faecalis ATCC 29212 1 19 23 10 E.coli ATCC 25922 S.aureus ATCC 29213 11 Ps.aeruginosa. ATCC 27853 18 26 24 12 17 25 13 16 14 15
Agar dilution, cont. Agar preparation according to CLSI M7-A7 Incubation temp / time: 36-37 C for 48h OR 42 C for 24h (not less than 36 C and not above 42 C) Incubation atm: microaerobic equal to 10% CO 2, 5% O 2 and 85%N 2
=> 0,5 = 0,5 < 0,25 Agar dilution, cont. Reading the Agar dilution plates 5 6 5 6 5 6 4 7 4 7 4 7 3 21 8 3 21 8 3 21 8 20 20 20 2 22 9 2 22 9 2 22 9 E.faecalis ATCC 29212 E.faecalis ATCC 29212 E.faecalis ATCC 29212 1 19 23 10 1 19 23 10 1 19 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 Ps.aeruginosa. ATCC 27853 Ps.aeruginosa. ATCC 27853 18 26 24 12 18 26 24 12 18 26 24 12 17 25 17 25 17 25 13 13 13 16 14 16 14 16 14 15 15 15 Control 0,25 0,5
Micro-broth dilution Testing condition according to CLSI M7-A7 Cation-adjusted Müller 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: 36-37 C for 48h OR 42 C for 24h (not less than 36 C and not above 42 C) Incubation atm: microaerobic equal to 10% CO 2, 5% O 2 and 85%N 2
Micro-broth dilution, cont. Reading the micro-broth dilution plates Semi or full automatic reading A 1 2 3 4 5 6 7 B C D E F G H
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 MIC (μg/ml) MIC (μg/ml) Antimicrobials for Campylobacter R is > 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
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 Müller Hinton agar supp. with 5% sheep
Thank you! Rene Hendriksen (rshe@food.dtu.dk) www.antimicrobialresistance.dk