Limits of control - Campylobacter quantification - SVA

FEDERAL INSTITUTE
FOR RISK ASSESSMENT
„Limits of control“ –
Campylobacter quantification
Kerstin Stingl
NRL for Campylobacter
Federal Institute for Risk Assessment
Diedersdorfer Weg 1 D-12277 Berlin
phone: +49 30 - 184 12 - 2135
kerstin.stingl@bfr.bund.de

Federal Institute for Risk Assessment (BfR), Berlin, Marienfelde
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 2

Prevalence of Campylobacter in German livestock and food
2004-2010
Stingl et al., Eur. J. Microbiol. Immunol., 2012, Vol.2:88-96
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 3

Quantification of Campylobacter on poultry carcasses
slaughterhouse
retail
ISO 10272
Stingl et al., Eur. J. Microbiol. Immunol., 2012, Vol.2:88-96
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 4

Cultural detection of Campylobacter along the food chain
Primary production slaughterhouse
stress
ISO 10272
vital Campylobacter
high detection rate
cecum = primary production
carcass = slaughterhouse
retail
ISO 10272
stress
Microbiological criterion (EFSA 2011)
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 5
15.5 % of German
broiler carcasses
>1000 CFU/g (2008)

Detection of live thermophilic Campylobacter using
real-time PCR
Lübeck et al., Appl. Environ. Microbiol. 2003
Josefsen et al., Appl. Environ. Microbiol. 2004
• Detection after enrichment according to ISO 10272-1 (qualitative)
⇒ cost- and time-effective
• 16S rRNA target (3 copies per
chromosome)
rather long amplicon (~287 bp)
• Validated by NordVal
• Validation/Publication as §64 method
in Germany expected end of the year
y = -3,68x + 41,624
R 2 = 0,9997
Eff = 1,9
20
0 1 2 3 4 5 6
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 6
Ct value
40
35
30
25
log (copy No. chromosome)

Detection of intact/viable Campylobacter using EMA/PMA
as DNA intercalator dyes
only DNA from viable cells is
detected by real-time PCR
viable = intact membrane
dead = compromised membrane
Rudi et al., Appl. Environ. Microbiol. 2005 (EMA in Campylobacter)
Josefsen et al., Appl. Environ. Microbiol. 2010 (PMA in Campylobacter)
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 7

Membrane permeabilities of intercalator dyes in C. jejuni
C. jejuni DSM4688, exponential phase
530/600 nm fluorescence (AU)
1400
1200
1000
800
600
400
200
0
-200
100 µM EtBr
0 500 1000 1500 2000 2500
control
Efflux inhibitor (PAβN)
protonophor (CCCP)
H 2 O 2
100 µM EtBr
time (s)
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 8
Microscopically
also checked
for the analog
EMA

Membrane permeabilities of intercalator dyes in C. jejuni
C. jejuni DSM4688, exponentielle Phase
5 3 0 /6 0 0 n m f lu o r e s c e n c e ( A U )
700
600
500
400
300
200
100
0
100 100 µM PI PI
0 500 1000 1500 2000 2500
-100
control
Efflux inhibitor (PAβN)
protonophor (CCCP)
H 2 O 2
time (s)
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 9
Microscopically
also checked
for the analog
PMA

„Physiological death“ of Campylobacter
live±PMA/H 2 O 2 cells
Cells 70 °C
Cells 95 °C
Cells H 2 O 2 +PMA
Cells 95°C+PMA
~5 x 10 5 CFU Cj 11168, exponential phase, 1/10 of DNA isolation in real-time PCR
Cells 70°C
+PMA
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 10

Ongoing activities…
• Standard protocol
- comparison of sample ± PMA in order to interpret the
residual signal of PMA for dead cells
- suitable range (microbiological criterion), evtl. concentration
of the target
- suitable controls (dead cells)
• Stability of the assay
- concentration of PMA in relation to tissue/background
- quantitative DNA isolation (IPC)
- light source for crosslink (halogen lamp, Phast blue system)
⇒ We expect (!) and have to accept deviations from a 100 %
correlation with ISO 10272-2 („gold standard“) for samples
from retail
Kerstin Stingl, 2012-10-03, EURL Campylobacter workshop 11

FEDERAL INSTITUTE
FOR RISK ASSESSMENT
Thanks to
…the competent authorities of the Federal States
Lüppo Ellerbroek
Bernd Appel
NRL Campylobacter Team
Christiane Buhler
Marie-Theres Knüver
Nora-Johanna Krüger
Petra Vogt
Thank you for your attention