EU-RL- Campylobacter

Proficiency test No 8 2011

Detection and enumeration of Campylobacter spp. in minced meat

Ingrid Hansson, EU-RL Campylobacter

Proficiency test No 8 April 2011

•Detection of Campylobacter spp. by enrichment and culture on selective agar plates

•Enumeration by direct culture on selective agar plates

•Confirmation and speciation by phenotypic and/or molecular methods

•Subculture of suspected Campylobacter colonies on non selective agar plates

The proficiency test involved:

Proficiency test No 8 2011

Incubation time and temperatures; according to ISO

10272,– Part 1: Detection method” and – Part 2: Colony-

count technique”.

Species identification; Catalase, Hippurate hydrolysis,

Indoxyl acetate hydrolysis, Nalidixic acid (optional),

Cephalotin (optional), PCR (optional)

Culture media; mCCDA and other selective media

Confirmation tests; morphology, motility, growth at 25ºC

(microaerobic) and oxidase

Enrichment; Bolton and Preston broth

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

1 27 3 2 6 15 3 1 6 4 2 6 1 7 3 9 1 11 1 15 1 28 1

The samples were distributed from the CRL on the 4th 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.

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

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

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

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

Proficiency test No 8 2011

No of participted NRL

Direct

culture

Enrichment in

Bolton broth

24 hours*

Enrichment in

Bolton broth

48 hours

Enrichment in

Preston broth

24 hours

mCCDA mCCDA

2nd

selective

medium

mCCDA

2nd

selective

medium

mCCDA

2nd

selective

medium

No of

NRL´s 32 23 21 34 33 34 27

= Optional

Correct reported detection in PT 8, 2011

No of

correctsamples

BB 24h mCCDA

NRL´s

BB 24h 2nd

NRL´s

BB 48h mCCDANRL´s

BB 48h 2nd

NRL´s

PB 24h mCCDA

NRL´s

PB 24h 2nd

NRL´s

10 9 12 24 22 24 18

9 7 5 5 7 5 4

8 4 2 4 3 2 1

7 1 1 1

6 1

5

4 1 1

3 1 1

Not done 11 13 - 1 - 7

Excluded 1 1 1 1 1 1

Reported results from detection PT 8 2011

Sample

No

BB 24h mCCDA

+/ng*/other

BB 24h 2nd

+/ng*/other

BB 48h mCCDA

+/ng*/other

BB 48h

2nd

+/ng*/other

1. C. lari 21 / 1 / - 19 / 1 / - 33 / - / - 32 / - / -

2. E.coli - / 1 / 21 - / 6 / 14 - / 3 / 30 - / 7 / 25

3. 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 / - / -

10. C.j + E.c 10 / 2 / 10 14 / 2 / 4 25 / - / 8 24 / 1 / 7

Reported results from detection PT 8 2011

Sample No PB 24h mCCDA

+/no growth/other

PB 24h 2nd

+/no growth/other

1. C. lari 30 / 3 / - 23 / 2 / 1

2. E. coli - / 14 / 19 - / 10 / 16

3. 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 / 2

10. C. jejuni + E. coli 31 / 2 / - 23 / 2 / 1

Reported results from detection PT 8, 2011

Sample

No

BB 48h mCCDA

+/no growth/other

BB 48h 2nd

+/no growth/other

PB 24h mCCDA

+/no growth/other

1. C. lari 33 / - / - 32 / - / - 30 / 3 / -

2. E.coli - / 3 / 30 - / 7 / 25 - / 14 / 19

3. 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 / -

10. C.j +E.c 25 / - / 8 24 / 1 / 7 31 / 2 / -

No of correct detected/ not detected in PT 8 2011

Results from 33 NRLs after enrichment

0

2

4

6

8

10

12

1305

07A

B

EB

0C

EA

3F

085B

344C

288D

F0

C1

063A

3320

31

FA

1D

CF

754

26

E3

2907

2E

41

2657

1946

2C

14

F5

C9

E8

C4

802

3C

9D

3E

2C

19C

7

F1

35

2610

2B

5B

05F

1

2A

8D

37

CF

39E

A

36

D3

Enr BB 48 h mCCDA

Enr BB 48 h 2nd

Enr PB 24 h mCCDA

Species identification, phenotyping after enrichment in

Bolton and isolation from mCCDA/second selective

25 NRLs reported species identification

Reported

species 1. C

. lar

i

2. E

. col

i

3. C

. co

li

4. C

. lar

i

5. C

. col

i +

E. c

oli

6. H

elic

obac

ter

7. N

egat

ive

8. C

. jej

uni

9. C

. col

i

10 C

.jeju

ni +

E. c

oli

C. jejuni 2 1 1 1 1 1 25 21

C. coli 24 1 22 25 1

C. lari 24 24 1 1

C. upsaliensis 1

Other 23 2 1 1 1 3

No Growth 3 24 24 1

25 NRLs reported species identification by phenotyping

25 NRLs reported species identification by phenotyping

Species identification, phenotyping after

enrichment in Preston Broth

Reported

species 1. C

. lar

i

2. E

. col

i

3. C

. co

li

4. C

. lar

i

5. C

. col

i +

E. c

oli

6. H

elic

obac

ter

7. N

egat

ive

8. C

. jej

uni

9. C

. col

i

10 C

.jeju

ni +

E. c

oli

C. jejuni 2 1 1 2 24 23

C. coli 21 1 21 20

C. lari 20 21 1 1

C. upsaliensis 2

Other 2 16 3 2 1 2 1

No Growth 1 9 1 2 22 23 2 1

Species identification, according molecular methods

18 NRLs reported species identification from Bolton broth and isolation from selective agar by PCR

Reported

species 1. C

. lar

i

2. E

. col

i

3. C

. co

li

4. C

. lar

i

5. C

. col

i +

E. c

oli

6. H

elic

obac

ter

7. N

egat

ive

8. C

. jej

uni

9. C

. col

i

10 C

.jeju

ni +

E. c

oli

C. jejuni 18 15

C. coli 17 17 16 1 17

C. lari 16

Thermo. Camp. 1 1 2 1

Negative 17 18

Other 18 2 3

Species identification, according molecular methods

17 NRLs reported species identification from Preston broth and isolation from selective agar by PCR

Reported

species 1. C

. lar

i

2. E

. col

i

3. C

. co

li

4. C

. lar

i

5. C

. col

i +

E. c

oli

6. H

elic

obac

ter

7. N

egat

ive

8. C

. jej

uni

9. C

. col

i

10 C

.jeju

ni +

E. c

oli

C. jejuni 17 17

C. coli 15 16 16 15

C. lari 15

Thermo. Camp. 1 1 1 1 1

No growth 1 9 1 17 17 1

Other 8

Correct species identification of Campylobacter 2011

0

2

4

6

8

10

12

1305

07A

B

EB

0C

EA

3F

085B

344C

288D

F0C

1

063A

3320

31FA

1DC

F

754

26 E

3

2907

2E 4

1

2657

1946

2C14

F5C

9

E8C

4

802

3C9D

3E2C

19C

7

F13

5

2610

2B5B

05F

1

2A8D

37C

F

39E

A

36D

3

spec BB

spec PB

spec PCR BB

spec PCR PB

No of Campylobacter spp. enumerated by 33 NRLs in the 10 samples in Proficiency Test No 8, April 2011

False pos: 1 False neg: 1 1 2 1

0

1

2

3

4

5

6

7

8

1.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.

log cfu/g

Conclusions

Proficiency test No 8, April 2011

No of

Correct samples

Dir PD mCCDA

BB 24h mCCDA

BB 24h 2nd

BB 48h mCCDA

BB 48h 2nd

PB 24h mCCDA

PB 24h 2nd

10 14 9 12 24 22 24 18

9 5 7 5 5 7 5 4

8 3 4 2 4 3 2 1

7 4 1 1 1

6 3 1

5 2

4 1 1

3 1 1

Not done 2 11 13 - 1 - 7

Excluded 1 1 1 1 1 1 1

No of NRLs that correctly reported detection

(45%) (41%) (60%) (73%) (69%) (73%) (69%)

No of NRLs that correctly reported detection

1

. C. l

ari

2. E

.col

i

3. C

.col

i

4. C

.lari

5. C

.col

i +

E.c

oli

6. H

elic

o.

7. N

eg

8.C

.jeju

ni

9. C

.col

i

10 C

.jeju

ni

+ E

. col

i

Not

don

e

Dir PD mCCDA

21 32 31 21 25 32 32 32 21 24 2

BB 24h mCCDA

22 23 23 21 14 23 23 21 21 11 12

BB 24h 2nd 19 21 21 19 15 21 21 19 19 15 13

BB 48h mCCDA

34 34 34 34 29 33 33 33 34 25 -

BB 48h 2nd 33 33 33 33 29 33 33 32 33 24 1

PB 24h mCCDA

31 34 32 31 32 34 34 32 28 33 -

PB 24h 2nd 23 27 25 23 23 27 27 25 22 23 7

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