EU-RL- Campylobacter Proficiency test No 8 2011...4. ri 5. + i 6 7 g 8. ni 9 10 ni + E. i done Dir...
Transcript of EU-RL- Campylobacter Proficiency test No 8 2011...4. ri 5. + i 6 7 g 8. ni 9 10 ni + E. i done Dir...
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