Proficiency test No Detection and enumeration of Campylobacter spp. in minced meat

Transcription

1 EU-RL- Campylobacter Proficiency test No Detection and enumeration of Campylobacter spp. in minced meat Ingrid Hansson, EU-RL Campylobacter

2 Proficiency test No 8 April 2011 The proficiency test involved: Detection of Campylobacter spp. by enrichment and culture on selective agar plates Subculture of suspected Campylobacter colonies on non selective agar plates Confirmation and speciation by phenotypic and/or molecular methods Enumeration by direct culture on selective agar plates

3 Proficiency test No Enrichment; Bolton and Preston broth Culture media; and other selective media Incubation time and temperatures; according to ISO 10272, Part 1: Detection method and Part 2: Colonycount technique. Confirmation tests; morphology, motility, growth at 25ºC (microaerobic) and oxidase Species identification; Catalase, Hippurate hydrolysis, Indoxyl acetate hydrolysis, Nalidixic acid (optional), Cephalotin (optional), PCR (optional)

4 No of days for transport of the PT 8 to the NRLs Days Transport of the ring test: No of NRLs Starting the analysis: No of NRLs The samples were distributed from the CRL on the 4 th of April. The 10 vials and the minced meat were placed in a plastic container (DG container 500, Noax Sys AB), which were placed in a cool insulation box (Noax Sys AB) and delivered by CLX Cargo Logistics Express AB.

5 Proficiency test No 8, April 2011 The proficiency test included detection and enumeration of Campylobacter in 10 samples consisting of minced meat from cattle. The minced meat should be used together with 10 vials, each containing one capsule with or without Campylobacter spp. Three of the vials contained a strain of E. coli resistance to some microbial substances

6 Description of the 10 vials with the mean number of Campylobacter/samples, when testing the samples Sample No Species Mean No of Campylobacter 1 Campylobacter lari 3.6 log cfu/g 2 E. coli - 3 Campylobacter coli 5.6 log cfu/g 4 Campylobacter lari 3.6 log cfu/g 5 Campylobacter coli + E. coli 3.6 log cfu/g 6 Helicobacter spp. - 7 Uninoculated - 8 Campylobacter jejuni 5.7 log cfu/g 9 Campylobacter coli 3.6 log cfu/g 10 Campylobacter jejuni + E. coli 2.6 log cfu/g

7 Proficiency test; Detection and enumeration Second selective media used by NRLs; 9 Karmali 8 Skirrow 5 Preston agar 5 CFA Biomeriouex 3 Butzler 1 Brilliance 1 Campy Cefex 1 CASA 1 Not done

8 Profiency test; Detection and enumeration Microaerobic atmosphere ; 22 Gas-generating kits 10 Microaerobic incubator 5 Anoxomat system 1 Zip-lock bags filled with gas 1 Injections in jars. 1 Don Whitley MACS 1 Air Liquide France

9 Proficiency test No No of participted NRL Direct culture Enrichment in Bolton broth 24 hours* Enrichment in Bolton broth 48 hours Enrichment in Preston broth 24 hours 2 nd selective medium 2 nd selective medium 2 nd selective medium No of NRL s = Optional

10 Correct reported detection in PT 8, 2011 No of correct samples BB 24h NRL s BB 24h 2 nd NRL s BB 48h NRL s BB 48h 2 nd NRL s PB 24h NRL s PB 24h 2 nd NRL s Not done Excluded

11 Reported results from detection PT Sample No BB 24h +/ng*/other BB 24h 2 nd +/ng*/other BB 48h +/ng*/other BB 48h 2 nd +/ng*/other 1. C. lari 21 / 1 / - 19 / 1 / - 33 / - / - 32 / - / - 2. E.coli - / 1 / 21 - / 6 / 14 - / 3 / 30 - / 7 / C.coli 22 / - / - 20 / - / - 33 / - / - 32 / - / - 4. C. lari 20 / 2 / - 19 / 1 / - 33 / - / - 32 / - / - 5. C.c + E.c 14 / - / 8 14 / - / 6 29 / - / 4 28 / 1 / 3 6. Helicob. - / 21 / 1 - / 20 / - 1 / 24 / 8 1 / 23 / 8 7. Negative - / 22 / - - / 20 / - 1 / 31 / 1 - / 30 / 2 8. C. jejuni 21 / 1 / - 19 / 1 / - 33 / - / - 32 / - / - 9. C.coli 20 / 1 / 1 19 / 1 / - 33 / - / - 32 / - / C.j + E.c 10 / 2 / / 2 / 4 25 / - / 8 24 / 1 / 7

12 Reported results from detection PT Sample No PB 24h +/no growth/other PB 24h 2 nd +/no growth/other 1. C. lari 30 / 3 / - 23 / 2 / 1 2. E. coli - / 14 / 19 - / 10 / C. coli 31 / 2 / - 24 / 2 / - 4. C. lari 30 / 3 / - 23 / 3 / - 5. C. coli + E. coli 31 / 1 / 1 23 / 1 / 2 6. Helicobacter - / 32 / 1 - / 21 / 5 7. Negative - / 32 / 1 - / 22 / 4 8. C. jejuni 32 / 1 / - 25 / 1 / - 9. C. coli 27 / 6 / - 21 / 3 / C. jejuni + E. coli 31 / 2 / - 23 / 2 / 1

13 Reported results from detection PT 8, 2011 Sample BB 48h BB 48h 2 nd PB 24h No +/no growth/other +/no growth/other +/no growth/other 1. C. lari 33 / - / - 32 / - / - 30 / 3 / - 2. E.coli - / 3 / 30 - / 7 / 25 - / 14 / C.coli 33 / - / - 32 / - / - 31 / 2 / - 4. C. lari 33 / - / - 32 / - / - 30 / 3 / - 5. C.c + E.c 29 / - / 4 28 / 1 / 3 31 / 1 / 1 6. Helicob. 1 / 24 / 8 1 / 23 / 8 - / 32 / 1 7. Negative 1 / 31 / 1 - / 30 / 2 - / 32 / 1 8. C. jejuni 33 / - / - 32 / - / - 32 / 1 / - 9. C.coli 33 / - / - 32 / - / - 27 / 6 / C.j +E.c 25 / - / 8 24 / 1 / 7 31 / 2 / -

14 AB EB0C EA3F 085B 344C 288D F0C1 063A FA 1DCF E E C14 F5C9 E8C C9D 3E2C 19C7 F B5B 05F1 2A8D 37CF 39EA 36D3 12 No of correct detected/ not detected in PT Results from 33 NRLs after enrichment Enr BB 48 h Enr BB 48 h 2nd Enr PB 24 h

15 1. C. lari 2. E. coli 3. C. coli 4. C. lari 5. C. coli + E. coli 6. Helicobacter 7. Negative 8. C. jejuni 9. C. coli 10 C.jejuni + E. coli Species identification, phenotyping after enrichment in Bolton and isolation from /second selective Reported species C. jejuni C. coli C. lari C. upsaliensis 1 Other No 25 Growth NRLs reported species 3 identification NRLs reported species identification by phenotyping

16 1. C. lari 2. E. coli 3. C. coli 4. C. lari 5. C. coli + E. coli 6. Helicobacter 7. Negative 8. C. jejuni 9. C. coli 10 C.jejuni + E. coli Species identification, phenotyping after enrichment in Preston Broth Reported species C. jejuni C. coli C. lari C. upsaliensis 2 Other No Growth NRLs reported species identification by phenotyping

17 1. C. lari 2. E. coli 3. C. coli 4. C. lari 5. C. coli + E. coli 6. Helicobacter 7. Negative 8. C. jejuni 9. C. coli 10 C.jejuni + E. coli Species identification, according molecular methods Reported species C. jejuni C. coli C. lari 16 Thermo. Camp Negative Other NRLs reported species identification from Bolton broth and isolation from selective agar by PCR

18 1. C. lari 2. E. coli 3. C. coli 4. C. lari 5. C. coli + E. coli 6. Helicobacter 7. Negative 8. C. jejuni 9. C. coli 10 C.jejuni + E. coli Species identification, according molecular methods Reported species C. jejuni C. coli C. lari 15 Thermo. Camp No growth Other 8 17 NRLs reported species identification from Preston broth and isolation from selective agar by PCR

19 AB EB0C EA3F 085B 344C 288D F0C1 063A FA 1DCF E E C14 F5C9 E8C C9D 3E2C 19C7 F B5B 05F1 2A8D 37CF 39EA 36D3 Correct species identification of Campylobacter spec BB spec PB spec PCR BB spec PCR PB

20 No of Campylobacter spp. enumerated by 33 NRLs in the 10 samples in Proficiency Test No 8, April 2011 log cfu/g C. lari 2.E.coli 3.C.coli 4.C. lari 5.C.c.+E.c.6.Helicobacter 7.Neg 8.C. jejuni 9.C. coli 10.C. j+e.c. False pos: 1 False neg:

21 Conclusions Proficiency test No 8, April 2011

22 No of NRLs that correctly reported detection No of Correct samples Dir PD BB 24h BB 24h BB 48h 2 nd BB 48h 2 nd PB 24h PB 24h 2 nd (45%) (41%) (60%) (73%) (69%) (73%) (69%) Not done Excluded

23 1. C. lari 2. E.coli 3. C.coli 4. C.lari 5. C.coli + E.coli 6. Helico. 7. Neg 8.C.jejuni 9. C.coli 10 C.jejuni + E. coli Not done No of NRLs that correctly reported detection Dir PD BB 24h BB 24h 2 nd BB 48h BB 48h 2 nd PB 24h PB 24h 2 nd

24 Proficiency test No 8, April 2011 Acknowledgement To all Member States, Norway, Switzerland and Iceland and FYROM for participating in the proficiency test To the staff of the EURL-Campylobacter To the Commission for funding the work of the EURL