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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
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
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
Context

    FBOs                            WG NRLs

                   EURL Lm




 Guidelines on sampling the food processing area &
          equipment for the detection of Lm




4
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
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
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
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
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
Review of the literature




10
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
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
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
European survey
Scrubbing devices



                    Sponges




       Gauze pads             Unwoven pad




14
European survey
Swabs                Brush & gauze pad
           Dry       (only in the event of an
                     alert, only one respondent)




                               +




     wet


15
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
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
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
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
Steps in the Guidelines production

• April – August 2012

  version 3 which is available at:
  http://www.ansespro.fr/eurl-listeria




                                         20
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
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
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
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
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
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
The shadows on the wall do not
          make up reality at all




27
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
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
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
THANK YOU FOR YOUR
    ATTENTION




                     31

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"The new EURL Guidelines on sampling the food processing area and equipment for the detection of Listeria monocytogenes" - Léna Barre (ANSES, Frankreich)

  • 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