"Listerien-Referenzlabor: Akademischer Luxus oder Public Health Erfordernis?", 19. Oktober 2012 (AGES, Graz) Das Nationale Listerien-Referenzlabor ist innerhalb der AGES an das "Zentrum für lebensmittelbedingte Infektionskrankheiten" / Institut für medizinische Mikrobiologie und Hygiene in Graz übersiedelt. Zum Auftakt am neuen Standort veranstaltete die AGES dieses Fachgespräch, bei dem verschiedene Aspekte in Zusammenhang mit dem potentiellen Auftreten von Listerien aktuell beleuchtet wurden. Erstmals wurden zudem die neuen Richtlinien des Europäischen Referenzlabors (EU-RL) für Listeria monocytogenes zur Harmonisierung der Probennahme vorgestellt. Dokumentation der Fachtagung mit Präsentationen: http://www.ages.at/ages/ages-akademie/stakeholderveranstaltungen/graz-listerien-referenzlabor/

1. European Union
Reference Laboratory
for Listeria
monocytogenes
EURL Guidelines for sampling food preparation
areas and equipment for L. monocytogenes
Léna BARRE & Brigitte CARPENTIER
French Agency for food, environmental & occupational health & safety

2. Context
• RTE foods can be contaminated during processing by
subtypes of Lm which persist in the processing plants
• Sampling processing is thus necessary & mandatory* to:
 detect possible Lm persistence
 eliminate it
 implement corrective actions
*2073/2005, Article 5, paragraph 2:
“Samples shall be taken from processing areas and equipment used in food
production, when such sampling is necessary for ensuring that the criteria are met.
In that sampling the ISO standard 18593 shall be used as a reference method.
2

3. Context
• ISO 18593* on surface sampling methods does not give
sufficient guidance specific to Lm detection
• Great variety of sampling practices and some wrong
practices
 Stress the urgent need for European guidelines
*“Microbiology of food and animal feeding stuffs -- Horizontal methods for
sampling techniques from surfaces using contact plates and swabs”
3

4. Context
FBOs WG NRLs
EURL Lm
 Guidelines on sampling the food processing area &
equipment for the detection of Lm
4

5. Steps in the Guidelines production
1- Review the ISO 18593 Standard
2- Review the literature: need to add to the ISO Standard
specific guidance on sampling techniques for Lm control
3- Conduct a survey: EURL conducted enquiry to survey
the different practices in terms of environmental sampling
techniques
4- Publish the guidelines
5

6. Steps in the Guidelines production
1- Review the ISO 18593 Standard
2- Review the literature: need to add to the ISO Standard
specific guidance on sampling techniques for Lm control
3- Conduct a survey: EURL conducted enquiry to survey
the different practices in terms of environmental sampling
techniques
4- Publish the guidelines
6

7. ISO 18593
The ISO 18593* standard does not describe:
- when sampling should be performed
- what areas should be sampled
The standard ISO 18593 does not provide all the
necessary information
* ISO 18593 : “Microbiology of food and animal feeding stuffs -- Horizontal methods for
sampling techniques from surfaces using contact plates and swabs”
7

8. Steps in the Guidelines production
1- Review the ISO 18593 Standard
2- Review the literature : need to add to the ISO Standard
specific guidance on sampling techniques for Lm control
3- Conduct a survey: EURL conducted enquiry to survey
the different practices in terms of environmental sampling
techniques
4- Publish the guidelines
8

9. Review of the literature
ISO 18 593 FDA Guidance Other references
document (2008)
At least 100 Constant area as large 2000-5000 cm² (Tompkin,
Sampled cm2 preferably as possible 1999)
flat about 1000cm2 - 30-100 cm2 3716 cm2 (Mc Namara, 2007)
surface for sponging Up to 900 cm² (Canadian rec,
area 2010)
- 10-13 cm2 Up to 50 cm² (NSW, 2008)
for swabbing
After the Critical Food-Contact After minimum 3H
Time at disinfection Surfaces: production (Mc Namara,
which contact time both prior to cleanup 2007)
sampling to assess the & no sooner than the
should be performance of middle of production During or at the end of
performed the C &D production (Tompkin, 2004)
programme Critical Non-Food-
Contact Surfaces: During full production or
at any time before equipment clean-up
(NSW, 2008)
9

10. Review of the literature
10

11. Review of the literature
Comparison of the requirements or recommendations on how to perform sampling of
surfaces including those specifically written for Lm
Main ideas:
 Recommendations found in the literature are not all the same
Few published scientific papers reported on results obtained in field
conditions (Kovacevic et al; 2009)
FBOs have great experience in sampling surfaces for Lm detection and
that they do not publish their knowhow
Launching of a European survey to know how they proceed
11

12. Steps in the Guidelines production
1- Review the ISO 18593 Standard
2- Review the literature: need to add to the ISO Standard
specific guidance on sampling techniques for Lm control
3- Conduct a survey: EURL conducted enquiry to survey
the different practices in terms of environmental sampling
techniques
4- Publish the guidelines
12

13. European survey
The EURL conducted a survey in 2010 on the different
practices in terms of environmental sampling techniques
 Enquiry into NRLs, competent authorities and FBOs
 137 respondents, from 15 EU Member States
13

14. European survey
Scrubbing devices
Sponges
Gauze pads Unwoven pad
14

15. European survey
Swabs Brush & gauze pad
Dry (only in the event of an
alert, only one respondent)
+
wet
15

16. What scrubbing device(s) do you use?
3% 3% 1% gauze pad
5%  wet cotton swab only
7% 29% multiple response

sponge
dry cotton swab only
16% ?
dry and wet swabs only

other
20%
16% brush
16

17. European survey
•• Several respondents declared wrong practices
Several respondents declared wrong practices
– sampling only after C&D,
– sampling only after C&D,
– sampling too small areas ...
– sampling too small areas ...
Emphases the need for European guidelines
Emphases the need for European guidelines
17

18. Steps in the Guidelines production
1- Review the ISO 18593 Standard
2- Review the literature: need to add to the ISO Standard
specific guidance on sampling techniques for Lm control
3- Conduct a survey: EURL conducted enquiry to survey
the different practices in terms of environmental sampling
techniques
4- Publish the guidelines
18

19. Steps in the Guidelines production
• March 2011
– Draft 1 sent to NRLs, each NRL circulated it to operators practicing
surface sampling
• September 2011
– 80 comments from 20 reviewers, Draft 2 sent to an European
working group of 20 people (Italy, Denmark, Portugal, Spain,
France, Ireland, Estonia)
• February 2012
– 90 comments from 13 reviewers, Draft 3 sent to the working
group
• March 2012
– 37 comments from 5 reviewers
– Draft 4 sent to the working group for a last discussion at a
web/phone meeting: version 0
– 28 March: NRLs approval version 1 19

20. Steps in the Guidelines production
• April – August 2012
version 3 which is available at:
http://www.ansespro.fr/eurl-listeria
20

21. The Guidelines
http://www.ansespro.fr/eurl-listeria
1. SCOPE
2. NORMATIVE REFERENCES
3. CHOICE OF SAMPLING LOCATIONS
4. TIME AT WHICH SAMPLING SHALL BE PERFORMED
5. DILUENTS TO MOISTEN THE WIPE SAMPLING DEVICES
6. CULTURE MEDIA
7. APPARATUS AND GLASSWARE
8. AREA TO BE SAMPLED
9. PREPARATION OF SAMPLING DEVICES
10.SAMPLING
11.TRANSPORT
12.SAMPLE ANALYSIS
13.EXPRESSION OF RESULTS
14.REFERENCES
21

22. Main differences: guidelines vs ISO 18593
1. SCOPE
– NOTE 2: No advice on
• sampling frequency
• number of sampling points
• validity of compositing (pooling) the samples
• necessity to rotate sampling points
they have to be chosen on a case-by-case basis, with a risk-based approach.
– NOTE 3: These guidelines are not designed to assess C&D
efficiency
2. CHOICE OF SAMPLING LOCATIONS
– A non-exhaustive list of places to choose sampling locations is
given quite similar to those of the FDA (2008) & NSW (2008)
doc.
22

23. Main differences: guidelines vs ISO 18593
3. TIME AT WHICH SAMPLING SHOULD BE
PERFORMED
– Sampling should be performed
• during processing
• after at least 2h of production
• or at the end of production runs
– When raw food is processed, sampling after
C&D or at the beginning of production can be
performed
• Yet, this can lead to a false sense of security
• The detection of L. monocytogenes on food-contact surfaces
after C&D indicates a serious failure in C&D procedures.
23

24. Main differences: guidelines vs ISO 18593
4. DILUENTS TO MOISTEN THE WIPE SAMPLING DEVICES
– No neutralizer when sampling during or at the end of
production except when residues of disinfectants are
expected
– Phosphate buffered diluents are not recommended
5. AREA TO BE SAMPLED
– hard-to-reach/small areas  stick swabs
– large areas  sponge, woven or unwoven cloth or
gauze pad
• The total sampled area should be as large as possible
• It is advised to sample between 1000 cm² and 3000 cm² when the areas
are open and flat (conveyors, shelves, etc.)
24

25. Main differences : guidelines vs ISO 18593
6. PREPARATION OF SAMPLING DEVICES
– No equipment which has been inside a microbiology
laboratory should be taken into a food production area
due to the risks of introducing contamination
– Operators should ensure that no item used for sampling
is left in the production area. For this purpose, counting
these items before and after sampling is recommended.
25

26. Main differences : guidelines vs ISO 18593
7. TRANSPORT, STORAGE, STARTING ANALYSIS
– Several reviewers asked for length of time longer than
24h before sample analysis (48h, 72h)
– Decision: Examine the samples ASAP, preferably not
later than 24h after receipt at the lab & in any case not
later than 36h after sampling (ISO 7218).
8. SAMPLE ANALYSIS
– ½ Fraser broth in the sampling device,
EN ISO 11290-1 or any alternative validated method
26

27. The shadows on the wall do not
make up reality at all
27

28. Next steps
• Analysis of the 2nd part of the survey on the
sample analysis, review of the literature and
decision whether there is a need for specific
recommendations, not included in the
EN ISO 11290-1&2 Standards
• Proposal : an experimental study to assess
– The impact of using a neutralizer on the probability to detect Lm
– The best diluent/neutralizer to avoid loss of culturability of
stressed Lm cells
– The best wiping device : sponge, woven on unwoven cloth,
gauze pad
– Etc.
28

29. IN COLLABORATION WITH
DENMARK
L. Gram
SPAIN
ESTONIA J. Canet Noguera
J. Toomas Kramarenko A. Español Pueyo
J. Lago
FRANCE J. Pardos Bosch
C. Dufour J. Sanchez Hernandez
T. Grégori
P. Leglise
N. Sanchez
V. Stahl PORTUGAL
H. da Silva Mateus Fernandes Guedes
A. L. Ferreira Tavares
IRELAND S. Orfão Ferraz
B. Hickey C. M. Pires Gomes
M. Sol
M. M. Tenreiro dos Santos Monteiro Saraiva
ITALY
S. Barbuti
UE DG-SANCO
29

30. References
ISO 18593. Microbiology of food and animal feeding stuffs -- Horizontal methods for sampling
techniques from surfaces using contact plates and swabs.
ISO 7218 Microbiology of food and animal feeding stuffs — General requirements and
guidance for microbiological examinations
FDA. 2008. Guidance for Industry: Control of Listeria monocytogenes in Refrigerated or
Frozen Ready-To-Eat Foods; Draft Guidance. U.S. Food and Drug Administration,
Department of Health and Human Services
http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/F
oodProcessingHACCP/ucm073110.htm, accessed 1 April 2010.
NSW. 2008. Food Authority for meat processors and retail meat licensees. Listeria
Management Program NSW/FA/FI034/0809.
http://www.foodauthority.nsw.gov.au/_Documents/industry_pdf/listeria-management-
program.pdf accessed 27 January 2011:40 pp.
http://www.ansespro.fr/eurl-listeria
30

31. THANK YOU FOR YOUR
ATTENTION
31