Antimicrobial susceptibility testing of Campylobacter jejuni and C. coli

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1 Antimicrobial susceptibility testing of Campylobacter jejuni and C. coli CRL Campylobacter Workshop The 7th -8th of Oct National Veterinary Institute Uppsala, Sweden

2 Legislation The Commission has a general obligation to establish coordinated monitoring programmes if special needs are identified Directive 2003/99/EC; Article 5

3 Legislation The Commission launched a survey on the prevalence and antimicrobial resistance of Campylobacter spp. in caeca / faeces from broiler flocks in the MS Decision 2007/516/EC Mandatory after 2008 in all MS!

4 Legislation The content of Decision 2007/516/EC: 170 C. jejuni and C.coli isolates to be included per MS only one isolate per slaughter batch All isolates should be included in MS which do not meet the target sample size A random selection equal or larger than the sample target should be included by MS exceeding the isolation of 170 strains

5 Legislation The content of Decision 2007/516/EC, cont.: Utilize the methodologies described in CLSI M7-A7 and M45-A The MS are obliged to test at least the following antimicrobials: Erythromycin, Ciprofloxacin, Tetracycline, Streptomycin and Gentamicin Epidemiological cut off values should be applied according to guidelines by EFSA 1 Determine the susceptibility by utilizing appropriate conc. ranges according to EFSA 1 )The Commision reference incorrectly to the CLSI guideline M31-A3 and M100- S16

6 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

7 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.

8 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: 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

9 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

10 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: 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

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

12 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

13 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

14 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 E.faecalis ATCC E.coli ATCC S.aureus ATCC Ps.aeruginosa. ATCC

15 Agar dilution, cont. Agar preparation according to CLSI M7-A7 Incubation temp / time: 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

16 => 0,5 = 0,5 < 0,25 Agar dilution, cont. Reading the Agar dilution plates E.faecalis ATCC E.faecalis ATCC E.faecalis ATCC E.coli ATCC S.aureus ATCC E.coli ATCC S.aureus ATCC E.coli ATCC S.aureus ATCC Ps.aeruginosa. ATCC Ps.aeruginosa. ATCC Ps.aeruginosa. ATCC Control 0,25 0,5

17 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

18 Micro-broth dilution, cont. CRL Plate design cover antimicrobials according to Decision 2007/516/EC

19 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

20 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: 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

21 Micro-broth dilution, cont. Reading the micro-broth dilution plates Semi or full automatic reading A B C D E F G H

22 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!

23 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 ( g/ml) MIC ( g/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* Nalicixic acid* *Not part of the EFSA monitoring programme

24 Interpretation of MIC values Cut off values by eucast

25 Interpretation of MIC values Cut off values by eucast

26 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

27 E-test Cation-adjusted Müller Hinton agar supp. with 5% sheep

28 Thank you! Rene Hendriksen

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