NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA

Transcription

1 NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA Vol. 17 No. 1 April 2011 ISSN Continuation of Newsletter Community Reference Laboratory for Salmonella ISSN

2 Produced by European Union Reference Laboratory for Salmonella National Institute of Public Health and the Environment P.O. Box 1, 3720 BA Bilthoven, The Netherlands phone: (Kirsten Mooijman) fax:

3 CONTENTS Editorial Note 1 Page Contribution of CRL-Salmonella 3 Literature Information 19

4 EDITORIAL NOTE Bilthoven, 8 April 2010 Dear colleagues, One year ago I informed you about the fact that according to the Lisbon Treaty of December 2009, the term Community had to be replaced by European Union. However, it took until March 2011 until it was officially arranged, by the publication of Commission Regulation 208/2011, that the name of the Community Reference Laboratory has been changed into European Union Reference Laboratory. For the official abbreviation to be used no rules are set yet, meaning that we can choose anything like EURL, EU-RL, ERL. For the moment we will use EU-RL, unless DG-Sanco will inform us to do differently. Due to this change of name, also the newsletter has become a new name and because of that also a new International Standard Serial Number (ISSN). However, the content of the newsletter will remain the same, containing information for you as NRLs for Salmonella. Our activities performed in 2010 were recently reported to DG-Sanco. For your information this technical report is also included in this newsletter. In November 2010 the fifteenth interlaboratory comparison study on typing was organised. The results have been analysed and recently you should have received the interim summary report of this study. In February 2011 the fourteenth interlaboratory comparison study on the detection of Salmonella in chicken faeces was organised. In this study for the first time lenticules were used as reference materials. I hope you were satisfied with the use of these new reference materials? The results of the study look good. Only a few NRLs were not able to reach the level of good performance. Also of this study you should recently have received your own results and the interim summary report showing the results of all laboratories. As you may know we are also very busy with the preparation of our annual workshop (19 and 20 May 2011). I hope you will like the new location: Zandvoort aan Zee (translated: Zandvoort at the sea). We hope for nice weather so that you can take a swim in the sea! Our secretariat (Jeanette) has been very busy with booking of the flights and on short term (if not already received) you should receive your e-ticket(s). The programme is almost final. At the moment it undergoes a final check by the speakers. I hope to be able to send you the final programme and information on the location, hotel and how to reach the meeting venue one month before the workshop. I look forward to meet you all in Zandvoort! Best wishes, Kirsten Mooijman 1

5 CONTRIBUTION OF CRL-Salmonella Technical report of 2010 Introduction The work plan of CRL-Salmonella for the year under review, 2010, was submitted to the European Commission in August This report details consequential activities of the CRL according to the agreed work plan. General In 2010 the following activities were carried out: 1. Organisation of three interlaboratory comparison studies 2. Organisation of a workshop with the NRLs-Salmonella 3. Performance of research 4. Giving assistance to the Commission and ad hoc activities 5. Communication 6. Training 7. Missions Since 1 January 2010 the microbiological department of RIKILT Institute for Food Safety has been moved to the RIVM. By this move, 7 additional persons became staff members of the Laboratory for Zoonoses and Environmental Microbiology (LZO), the laboratory at RIVM where the CRL-Salmonella is located. After the introduction of the new staff members it was agreed to reshuffle the input of staff members on different projects, to obtain an optimal exchange of know how in all projects. By this, in total 3 new staff members give input to the CRL-Salmonella: Wilma Jacobs-Reitsema (who was already subcontracted since 1 September 2009), Irene Pol-Hofstad and Wendy van Overbeek. The new staff members took over some planned hours of other staff members, so that the original planned budget for 2010 remained the same. The change in input was communicated to DG-Sanco in January Deliverables RIVM-Reports In 2010 the following RIVM-reports were published: Mooijman, K.A. The fourteenth CRL-Salmonella workshop 25 and 26 May 2009, Bilthoven, the Netherlands. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Kuijpers A.F.A., Veenman C., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food III (2009) - Bacteriological detection of Salmonella in minced chicken meat. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Berk, P.A., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Thirteenth CRL-Salmonella interlaboratory comparison study (2008) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: /

6 Kuijpers A.F.A., Veenman C. and Mooijman, K.A. EU Interlaboratory comparison study Veterinary-XIII (2010) - Bacteriological detection of Salmonella in chicken faeces. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Mooijman, K.A. The fifteenth CRL-Salmonella workshop 27 June, Saint Malo, France. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / The draft report was sent to DG-Sanco on 22 July The presentations were published on the CRL-Salmonella website on 6 July 2010: The final report was published January The following RIVM-reports are under preparation: Jacobs-Reitsema, W.F., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Fourteenth CRL- Salmonella interlaboratory comparison study (2009) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: / The interim summary of this interlaboratory comparison study was published in May The final report is under preparation. Kuijpers A.F.A, van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food IV (2010) - Bacteriological detection of Salmonella in minced meat. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: /. The interim summary of this interlaboratory comparison study was published in November The final report is under preparation. Reports on trainings In January 2010, two staff members of CRL-Salmonella visited an NRL-Salmonella who showed poor performance in several interlaboratory comparison studies on detection of Salmonella. The report of this visit was sent to the NRL and to DG-Sanco in February In April 2010, two staff members of CRL-Salmonella visited an NRL-Salmonella who showed poor performance in several interlaboratory comparison studies on serotyping of Salmonella. The report of this visit was sent to the NRL and to DG-Sanco in May Presentations on symposium A poster was presented and an oral presentation was given by two staff members of CRL- Salmonella at the International Symposium on Salmonella and Salmonellosis (I3S), St. Malo, France on June 2010 ( Furthermore, Kirsten Mooijman was asked to co-chair a session of the symposium. Both presentations were published in the proceedings of the 2010 symposium, by the symposium secretariat ISPAIA, Ploufragan: Mooijman, K.A. Salmonella and its standard methods (oral presentation). Kuijpers, A.F.A and Mooijman, K.A. Detection of Salmonella in food, feed and veterinary sample by different EU laboratories (poster presentation). ISO and CEN A consolidated report of 7 CRLs on the meetings of ISO/TC34/SC9 and CEN/TC275/WG6 held in Buenos Aires, Argentina on 31 May-4 June 2010, was sent to DG-Sanco on 22 July ISO6579-1: Detection of Salmonella A first draft document was prepared by Kirsten Mooijman and sent to ISO-working group (WG)9 in May After receiving comments and additional contributions, a second draft document was prepared in October

7 ISO : Enumeration of Salmonella The document was amended by Kirsten Mooijman and sent to the secretariat of ISO in February The publication for final vote as Technical Specification (TS) has been delayed due to administrative problems at CEN and ISO. ISO : Serotyping of Salmonella The draft guidance document was amended by Kirsten Mooijman and sent to the ISO ad hoc group on serotyping in May Interlaboratory comparison studies In the interlaboratory comparison studies of the CRL-Salmonella, matrices artificially contaminated with Salmonella reference materials are used for many years. The contamination levels has varied during the years, but always low level and high level materials with two Salmonella serovars have been used. The low level is chosen as close to the detection limit as possible. The high level samples are approximately 5-10 times higher than the low level materials. It is expected that when samples with a contamination level close to the detection limit are tested, approximately 50% of the tested samples will be negative. The reference materials are prepared by the CRL and exist of gelatine capsules containing milk powder with Salmonella serovars at different contamination levels. The milk powders are prepared by spray drying milk, artificially contaminated with a Salmonella serovar. The obtained highly contaminated milk powder is mixed with sterile milk powder to obtain the desired contamination level. Next gelatine capsules are filled with the mixed powder to obtain the final reference materials and these materials are stored at -20 C. The following reference materials (capsules), to artificially contaminate a matrix, have been used in the bacteriological detection studies since fall 2008: Salmonella Typhimurium at a contamination level of approximately 5 cfu and 50 cfu per capsule (STM5 and STM50) and Salmonella Enteritidis at a contamination level of approximately 20 cfu and 100 cfu per capsule (SE20 and SE100). In October 2009 an interlaboratory comparison studies on the bacteriological detection of Salmonella spp. in minced chicken meat was organised. In this study, 32 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 3 NRLs from member countries of the European Free Trade Association and 1 NRL from a EU candidate member state. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in Rappaport Vassiliadis broth with Soya (RVS) and in Muller Kauffmann Tetrathionatenovobiocin broth (MKTTn)). Additional it was requested to also use Annex D of ISO 6579:2007 (with selective enrichment on Modified semi-solid Rappaport Vassiliadis (MSRV) agar). In November 2009 an interim summary report of the study was sent to the participants. The results of only one NRL did not fulfil the criteria of good performance (one false positive blank control sample and a too low number of positive results with the artificially contaminated chicken meat sample with low level Salmonella Enteritidis). It was observed that this NRL has had similar problems in two earlier interlaboratory comparison studies. After this last study, the NRL had taken several (extra) actions in trying to improve their performance: cleaning and disinfection to prevent cross contamination, change from plastic bags to jars for pre-enrichment in BPW, extra controls to check the performance of the media, etc. To check the implementation of these measures and to see whether extra advices could be given, a follow-up in combination with a visit of two staff members of the CRL was organised in January The results of the follow up study fulfilled the criteria of good performance, albeit at the lower end for the sensitivity results. The NRL agreed to take additional actions to try to further improve their performance. The report of the visit and the results of the follow-up study have been reported to the relevant NRL and to DG-Sanco in February The conclusions of this follow-up were included in the full report of the study, which was published in September 2010 (see Introduction). 4

8 In December 2009 the 14 th interlaboratory comparison study on typing of Salmonella spp. was organised for the NRLs-Salmonella. In parallel a typing study for the laboratories of the Food- and Waterborne Diseases and Zoonoses (FWD) surveillance network Europe, under contract with the ECDC, was organised. The typing study was organised in collaboration with the Health Protection Agency (HPA, London, United Kingdom), for phage typing. In total 31 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 2 NRLs from member countries of the European Free Trade Association and 1 NRL from an EU candidate member state. Each NRL performed the serotyping of 20 different Salmonella serovars and 5 NRLs performed the phage typing of 10 different strains of Salmonella Typhimurium and 10 different strains of Salmonella Enteritidis. The results were sent to the CRL by the end of December 2009/early January The analysis of the results was performed in January/February Eight laboratories did not fulfil the criteria for good performance for the serotyping and performed a follow-up study in April For one of these laboratories this was a repeated poor performance. Therefore it was decided to visit this laboratory to give a training at the spot during the follow-up study. After the follow-up study all participating laboratories fulfilled the criteria of good performance. For the first time also an interim summary report was prepared for this interlaboratory comparison study on typing, which was published in May The results were presented at the CRL-Salmonella workshop in June The final report of the study is still under preparation (see Introduction). In March 2010 the 13 th interlaboratory comparison study on the detection of Salmonella spp. in a veterinary matrix (chicken faeces) was organised. In this study, 33 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 3 NRLs from member countries of the European Free Trade Association and, on request of DG-Sanco, 2 NRLs from third countries (non-europe). The prescribed method for analyses was Annex D of ISO 6579 (2007), with selective enrichment on Modified semi-solid Rappaport Vassiliadis (MSRV) agar. In April 2010 an interim summary report of the study was sent to the participants and the results were presented at the CRL-Salmonella workshop in June In the study additional control samples were introduced. Chicken faeces mixed with an antibiotic (gentamicin) for which Salmonella Enteritidis (SE) was sensitive had to be tested in the presence of SE20 capsules. The laboratories were not aware of this deviating matrix. In total four of these control samples had to be tested. Tests performed at the laboratory of the CRL showed that occasionally a sample could still be tested positive for Salmonella but finding more than 2/4 samples positive in one laboratory was quite unlikely and might indicate that no faeces was added to the SE capsules. One NRL found 4/4 of the antibiotic control samples positive for the prescribed procedure. The NRL performed several additional tests to try to find an explanation for these results. They indicated to have followed the protocol. If the laboratory indeed followed the protocol correctly, the only explanation could be that they added the faeces without antibiotics to the specific SE control samples. It was not considered necessary to organise a follow-up study for this laboratory, although their results were indicated as moderate. Another participant had problems with reconstitution of some capsules so that they missed some positive samples. This result was also indicated as moderate and no follow-up was considered necessary as all other samples were analysed correctly. Furthermore the laboratory has always shown correct results during earlier studies. The final report of the study was published in December 2010 (see Introduction). In September 2010 the fourth interlaboratory comparison studies on the bacteriological detection of Salmonella spp. in a food sample was organised. At the workshop it was discussed that the NRLs as well as the CRL prefer to introduce samples in the interlaboratory comparison studies of which treatment is more closely related to real life samples. For the current samples the reference materials (capsules) first need to be reconstituted at 37 C for 45 min, after which the matrix can be added. This procedure enhances the risk of cross contamination. Furthermore, for routine analyses of food samples it is common practice to mix the food sample with BPW by placing the sample in a stomacher or pulsifier for 1 min. However, with the current samples of the 5

9 interlaboratory comparison studies it is preferred not to do so. Therefore alternative samples have been tested at the laboratory of the CRL (use of lenticules, see 3. Research ). The tests with lenticules were not yet finalised before the interlaboratory comparison study of September, so that for this study still capsules were used as reference materials. The only improvement to mimic better the routine samples, the amount of matrix to be tested was enhanced (from 10 g to 25 g). As matrix a mixture of minced pork and minced beef was used which had to be mixed with low and high level reference materials. In this study only reference materials containing Salmonella Typhimurium were used, as this serovar is more related to meat products from pigs and cattle than Salmonella Enteritidis. The NRLs had to test 24 (blind) capsules with matrix (8 containing STM50, 8 containing STM5 and 8 being blank capsules) and 5 (blind) control samples without matrix (1 capsule containing STM50, 3 capsules containing STM 5 and 1 blank capsule). In the study, 31 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States and 3 NRLs from member countries of the European Free Trade Association. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in RVS and MKTTn). Additional it was requested to also use Annex D of ISO 6579:2007 (with selective enrichment on MSRV). In November 2010 an interim summary report of the study was sent to the participants. The results of two NRLs did not fulfil the criteria of good performance (false positive blank results). A follow up study was organised for these laboratories in January 2011, in which they both scored good performance. The final report is under preparation. In November 2010 the 15 th interlaboratory comparison study on typing of Salmonella spp. was organised for the NRLs-Salmonella. In parallel a third typing study for the laboratories of the Food- and Waterborne Diseases and Zoonoses (FWD) surveillance network Europe, under contract with the ECDC, was organised. The typing study was organised in collaboration with the Health Protection Agency (HPA, London, United Kingdom), for phage typing. In total 33 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 2 NRLs from member countries of the European Free Trade Association and 3 NRLs from EU candidate member states. Each NRL performed the serotyping of 20 different Salmonella serovars and 7 NRLs performed the phage typing of 10 different strains of Salmonella Typhimurium and 10 different strains of Salmonella Enteritidis. The results were sent to the CRL before the end of December The analysis of the results was performed in January 2011 after which the NRLs received their own results. Four laboratories did not fulfil the criteria for good performance for the serotyping. A follow-up is planned to be organised in March It is also planned to prepare an interim summary report in March 2011, containing the results of all laboratories. 2. Workshop On 27 June 2010, the annual CRL-Salmonella workshop was organised in Saint Malo, France. The location was chosen to make it possible to organise the workshop in conjunction with the International Symposium on Salmonella and Salmonellosis (I3S), which was organised in Saint Malo from 28 to 30 June Each NRL was represented by, at least, one participant. Two Member States were not able to participate in the workshop: Cyprus and Malta. The NRL of Cyprus indicated not to be able to come due to the fact that at the time of the workshop their laboratory activities were assessed for accreditation and they could not miss any staff at this audit. Malta indicated personal reasons for not being present at the workshop. From the EFTA countries, Switzerland and Iceland did not participate due to respectively personal and financial reasons. Candidate country Former Yugoslav Republic of Macedonia neither participated as they could not receive a financial contribution for participation in the workshop from TAIEX. In total 38 participants were present at the workshop, of which several for their own costs. The workshop lasted only one day and the presentations were focussed on the results of the CRL interlaboratory comparison studies, in which the NRLs-Salmonella had participated. Plans for the coming year were also presented. Besides the activities of the 6

10 CRL, other subjects included: Community summary report on Zoonoses (EFSA 2008), overview of the baseline studies on Salmonella and information on the standardisation of methods at International (ISO) and European (CEN) level. The draft report of the workshop was sent to EC DG-Sanco in July All presentations were placed on the CRL-Salmonella website ( one week after the workshop. The final report was published in January 2011 (see Introduction). 3. Research Activities in ISO and CEN CRL-Salmonella is involved (as project leader or as member of working groups) in several activities of ISO and CEN. More specific in: - ISO/TC34/SC9: International Standardisation Organisation, Technical Committee 34 on Food products, Subcommittee 9 Microbiology. - CEN/TC275/WG6: European Committee for Standardisation, Technical Committee 275 for Food analysis Horizontal methods, Working Group 6 for Microbial contaminants. Both groups organised its annual meeting in Buenos Aires, Argentina from 31 May until 4 June Kirsten Mooijman of the CRL-Salmonella is convenor of three groups in ISO dealing with methods for Salmonella. She presented the progress of these groups at the plenary meeting SC9 and WG6. In 2009 it was decided to revise EN ISO 6579:2002 on detection of Salmonella spp. in food and animal feed. By that time it was also agreed to split EN ISO 6579 into 3 parts to deal with detection (part 1), enumeration (part 2) and serotyping (part 3) of Salmonella spp. under one EN ISO number. ISO : Detection of Salmonella At the ISO/TC34/SC9 meeting in Valencia in May 2009, it was decided to raise a working group (WG9) to deal with the revision of ISO Kirsten Mooijman was appointed as convenor of this working group. At this 2009 meeting, a resolution was taken (no. 395) in which several items for revision of the ISO document for detection of Salmonella has been indicated. The items of Resolution 395 were dealt with item by item during the meeting at Buenos Aires: 1. Description of the detection of S. Typhi and S. Paratyphi in a normative annex of ISO 6579, considering the use of the Selenite Cystine (SC) enrichment broth: A text proposal has been made for this annex and it is suggested by WG9 to include the detection of S. Gallinarum (biovars gallinarum and pullorum) as well. However, it was argued by SC9 that these latter Salmonella strains are not related to human health and are only pathogenic for animals. It was agreed that this will be checked with the WHO reference centre for Salmonella. Additional, the secretariat of SC9 will ask OIE whether they would agree with inclusion of the detection of S. Gallinarum in an annex of the amended ISO The annex will become normative and in the full text of ISO 6579 it should be clearly indicated when this annex will be used. 2. To launch a trial comparing selective enrichment in the BAM/USP formulation of tetrathionate broth and in MKTTn (ISO6579 formulation). Kirsten Mooijman will prepare a protocol and will provide this to the SC9 secretariat. The secretariat will send this protocol to the SC9 members: The protocol was prepared and sent to the members in August In total 5 datasets were received, all showing similar results. In summary MKTTn (ISO formulation) gave the best results. WG9 therefore suggested to retain MKTTn in the amended ISO SC9 agreed. 3. The SC9 secretariat will launch an enquiry for data comparing the use of RVS and MSRV for food analysis. WG9 will also perform a literature review on this subject: In total 7 datasets were received in which MSRV was compared to ISO 6579 (or to only RVS). In one study the comparison was done between MSRV and ISO 6785 (for milk and milk products, selective enrichment in SC and RVS). All studies showed similar 7

11 results: MSRV gave equal or better results than ISO WG9 therefore suggests to allow the choice of subculturing to either RVS or MSRV (both to be incubated at 41,5 C). It was discussed whether such a choice would be allowed. There were no strong feelings not to allow this as both media are very similar. Furthermore, the situation is comparable to the isolation step where one medium is prescribed and the second one is free for choice. It was agreed that WG9 will continue with this suggested choice and wait for further reactions as soon as a draft is distributed. 4. Postpone the discussion about a further 24h incubation of the selective enrichment media until further information about the choice of the selective enrichment media is available: this will be dealt with later. 5. Retain XLD as the mandatory isolation medium. Clearer direction on suitable media for the second plate should be given in the document: a draft proposal for this has been made in the first draft document. France suggested to replace or give the choice for XLT4 instead of XLD in case of analysing dirty samples like primary production samples. This was further discussed in a meeting of WG9 organised in October By then it became clear that XLT4 is especially of advantage when a culture is plated out from MKTTn. However, in ISO 6579 (current Annex D) only MSRV is used for selective enrichment of primary production samples. Experiments in France had shown that for isolation from MSRV no difference was observed between XLD and XLT4. It was therefore again concluded to retain XLD as the mandatory isolation medium. To the draft amended version of ISO 6579 tables were added in an annex to clarify on what selective principles several different isolation media are based. With this information it may be more easy for a laboratory to choose a second isolation medium which will give additional information to XLD. 6. The SC9 secretariat will launch an enquiry to collect data to support the possibility of refrigerating BPW and/or selective enrichment media before subculture: a call was launched in April Some data have become available and it seems to be no problem to store cultured BPW or selective enrichment media for 72 h in the refrigerator. This information will be added in a note to the amended version of ISO Make the plating stage less prescriptive: a text proposal has been made for the first draft. 8. Make the confirmation stage less prescriptive in terms of number of colonies to be confirmed: a text proposal has been made for the first draft. 9. The non-selective medium for purification of colonies should be left to choice: a text proposal has been made for the first draft. 10. Include a note to allow parallel biochemical testing and purity check: a text proposal has been made for the first draft. Furthermore, it was mentioned that it is important that the text is clarified on serological confirmation in ISO and serotyping in ISO Additional, it was stated that it should not be prescribed in a standardised method that isolates have to be further typed at a reference laboratory. This text need to be amended accordingly in ISO Investigate the usefulness of some biochemical tests: In fall 2010 some information was received which indicated that it may not always be necessary to perform all biochemical tests to confirm Salmonella. It was decided still to retain all tests in the document, but to indicate some tests to be optional. A first amended draft text of ISO was sent to WG9 in May The document was further discussed together with additional information at a meeting of WG9 organised in October At this meeting further agreements were made on drafting ISO It was agreed that several members of WG9 would send contributions for the amendment of the document to Kirsten Mooijman by the end of Kirsten will than prepare a new draft proposal of ISO early This new draft proposal may next be sent to the plenary ISO group SC9 for a first vote. At the plenary meeting of SC9 in Buenos Aires and at the last meeting of WG9 it was agreed that Kirsten will draft a report in which all information and research results collected for revision of ISO will be summarised. As soon as this report will be finalised it will be made available to SC9 and also to the NRLs for Salmonella. A first draft 8

12 version of this report was prepared by Kirsten, but as further information is still collected and additional research for improving ISO is still done, the finalisation of the report awaits this additional information and results. At the SC9 meeting in Buenos Aires it was indicated that the work of WG9 will be moved from ISO/TC34/SC9 to CEN/TC275/WG6. This is necessary as the validation of Annex D of ISO 6579 is part of the CEN mandate and in 2006 it has been agreed that work in relation with the CEN mandate will be performed in CEN. Therefore, ISO-WG9 will become dormant and a new group will be raised at CEN level. ISO : NWIP/TS Enumeration of Salmonella by a mini-mpn technique The finalisation of the draft ISO TS document was delayed due to the fact that it was necessary to wait for the information from the ISO working group on statistics (WG2) on the MPN tool. In February 2010 the amended document was sent to the secretariat of SC9. The voting of the document has not been launched yet as it is necessary to have a resolution of CEN to publish the document as a Technical Specification (TS). For this it is necessary to write a justification letter why the document should be published as a TS and not as a full standard. At ISO level it has been discussed whether it would be easier/quicker still to publish the document as a full standard. However, if the document will be published as a full standard it will be necessary to include validation data. Some data are available, but it will take time to collect them and to check whether they fulfil the criteria for inclusion in a standard. If the data do not fulfil the criteria it will further delay the publication of the standard. Therefore, it was decided still to publish the document as a TS and administrative details will be sorted out with CEN. Unfortunately the document was still not published by the end of It is not clear why no further progress is made in ISO and CEN. ISO : Serotyping method for Salmonella The ISO ad hoc group on serotyping met for the first time on 14 December At this meeting it was agreed to prepare a guidance document for serotyping and therefore to publish the document as a Technical Report (TR). At the SC9 meeting it has been asked whether publication of a TR would not cause problems for CEN. As this was not fully clear, it was agreed that it first need to be checked at CEN (by SC9). The draft guidance document which was distributed with the second enquiry was taken as a basis and at the ad hoc group meeting some amendments and additions were agreed. Members of the ad hoc group provided Kirsten with text proposals by March 2010, after which Kirsten prepared an amended document. This latter document was sent to the members of the ad hoc group in the first week of May 2010 and further comments and contributions were asked. In fall 2010 all comments and contributions were received and it is planned that Kirsten will prepare an updated draft document which may by launched for voting as New Work Item Proposal (NWIP) by the secretariat of ISO/TC34/SC9 in CEN mandate on validation of microbiological methods In January 2006 the EC addressed a mandate to CEN/TC275/WG6 for the validation of 15 microbiological methods. CRL-Salmonella has been assigned as project leader for the validation of Annex D of ISO 6579: 2007 (Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage). A draft proposal was sent to CEN in For the validation study, ISO 16140: 2003 need to be followed, or the new draft of this ISO document. For the validation a ring trial shall be organised. The CRL-Salmonella will request some NRLs-Salmonella to participate in such a ring trial, as they are well experienced with the method. The progress with the full proposal of the 15 project leaders in the CEN mandate was very slow, due to bureaucratic problems and inexperience of AFNOR with such a large mandate. At the meeting in Buenos Aires an update was given. After several (small) amendments of the proposal, done by the secretariat of WG6, the final proposal has been sent to CEN central in May However, by the end of May the proposal was still 9

13 not submitted to the EC, due to the fact that the question on the leadership of the revision on ISO 6579 (ISO or CEN?) first needed to be solved. It was clarified that as it was agreed in 2006 that all work under the CEN mandate should be lead by CEN, the revision of part 1 of ISO 6579 should be moved to CEN. The work for part 2 (enumeration) and part 3 (serotyping) could stay in ISO. After solving this question, CEN central promised to submit the proposal as quickly as possible to the EC. Early 2011 the project leaders were surprised by the information that the contract was signed for the project. Therefore it may be possible that some primary work for the CEN mandate may start in Methods For the revision of ISO 6579 part 1 several activities have been performed: - The yield of pure cultures of different Salmonella serovars, as well as after addition to minced pork was tested in different formulations of tetrathionate broths (formulations according to BAM/USP and ISO 6579). The results of the pure cultures as well as of the artificially contaminated pork were comparable to the results found by other laboratories: MKTTn of ISO 6579 gave the best results (highest yield of Salmonella and lowest yield of the background flora). - Literature has been searched for the possible use of MSRV for the detection of Salmonella spp. in food and animal feed samples. In total 7 (large) datasets were screened, of which one concerned a full validation study according to ISO In these datasets also the results of the interlaboratory comparison studies as organised by the CRL-Salmonella have been used. All results showed that MSRV gave equal or better results than ISO The effect of storage time was tested on several media for the detection of Salmonella. In the current ISO 6579 it is indicated that most media need to be prepared fresh and storage at 5 C is only allowed for a few days. Large batches of ready-to-use selective enrichment media MSRV, MKTTn and RVS and plating out media XLD and Smid2 were stored at 5 C and regularly tested (weekly to monthly) with 5 samples of minced meat (mixed pork and beef), artificially contaminated with Salmonella Enteritidis at a level of approximately 9 cfp/25 g. All samples still show positive results after 6 months of storage of the media, except for MSRV, for which a maximum storage time of 4 weeks was shown (which is a prolongation of the current storage time with 2 weeks). - A first screening of the literature was performed to check the possibility of refrigerating cultured BPW and/or selective enrichment media before subculture. Some information was found which supports the possibility of refrigerating cultured BPW and cultured selective enrichment media up to 72 h. When necessary some additional experiments will be performed at the laboratory of the CRL; - Two times a pooling experiment was performed. For this the so-called dry pooling as well as the wet pooling of samples was tested. The set-up and results are described below. Pooling experiment Tested matrices: minced chicken meat and minced pork/beef. Reference materials: a. For the test with minced chicken meat: Salmonella Enteritidis in skim milk, frozen at - 80 C at a level of approximately 9 cfp/sample. b. For the test with minced pork/beef: Salmonella Enteritidis in lenticules, frozen at -20 C at a level of approximately 5 cfp/sample. Set-up experiment: 3x 25 g of matrix were each artificially contaminated with 1 reference material and stored at 5 C for 3 days; 1. Dry pooling: 25 g artificially contaminated matrix g non-contaminated matrix (=4x 25 g) added to 1125 ml BPW. After normal incubation time (37 C for 18 h) the procedure of ISO 6579 (and Annex D) was followed to inoculate the selective 10

14 enrichment media: 0,1 ml onto MSRV agar; 0,1 ml in 10 ml RVS and 1 ml in 10 ml MKTTn. 2. Wet pooling: 1x 25 g artificially contaminated matrix and 4x 25 g non-contaminated matrix were each added to 225 ml BPW and incubated at 37 C for 18 h. From each cultured BPW, 5 ml was taken and the five BPW cultures were mixed in one tube. Of the 25 ml pooled BPW cultures, 0,1 ml was used to inoculate one plate of MSRV agar; 0,5 ml was used to inoculate 50 ml RVS and 5 ml was used to inoculate 50 ml MKTTn. 3. Control: 1x 25 g artificially contaminated matrix was pre-enriched in 225 ml BPW and tested in the normal way on RVS, MKTTn and MSRV. The dry pooling as well as the wet pooling showed positive results for all tested samples of minced chicken meat and minced pork/beef on all tested media. In July 2010 a draft protocol for the (statistical) set-up of pool experiments has been received from the working group on statistics of ISO. This protocol was used to draft a plan to perform some further pooling experiments. In summary this plan looks as follows: - Two strains of each of the following Salmonella serovars will be tested: S. Typhimurium, S. Enteritidis, monophasic S. Typhimurium: 1,4,[5],12:i:- - Four types of poultry matrices will be tested (skin of chicken and turkey, minced meat of chicken and turkey). Of each type of matrix, 5 different samples will be tested. - Each matrix sample will be contaminated with each of the six strains at a level of approximately 5 cfp/25 g. Prior to this contamination step, each strain will be sublethally injured by following a spiking protocol (e.g. storage of a culture at -20 C for 3 days). - Dry pooling and wet pooling will be tested (see above). For each type of pooling one artificially contaminated sample of 25 g will be mixed with 4 non-contaminated samples of 25 g, either at the sample level (dry pooling) or at the level of the BPW cultures (wet pooling). - The method for analyses will be ISO 6579 and Annex D of ISO In the second half of 2010 it was tested what type of stress would give the highest amount of sublethally injuries to the 6 Salmonella strains as mentioned above. According to a draft protocol of MicroVal Technical Committee of September 2010 injury efficiency is usually evaluated by enumerating the pure culture on a selective and on a non selective agar medium. More than 0,5 log cfp/g difference between the result found on the nonselective medium and the results found on the selective medium is expected after performing a stress protocol. Much differences were seen between the stress protocols but also between the strains. For one Salmonella Enteritidis (SE) strain the highest amount of injury was found after storage at -20 C for 3 days. However, for a second SE strain the highest effect was seen after 15 min at 50 C. Similar results were found for the two monophasic S. Typhimurium strains. The two S. Typhimurium (STM) strains behaved more comparable (highest injuries after storage at 50 C for 15 min). The results are summarised in Annex 1. The information on injury efficiencies will be used for the pooling experiments which are planned to start early PCR technique In 2007 some artificially contaminated samples (chicken faeces, pig faeces, minced meat) were tested for Salmonella using a Real-Time Polymerase Chain Reaction technique. All samples gave similar results with the RT-PCR and with the bacteriological detection method. In 2008 several naturally contaminated samples were obtained, with different contamination levels. These samples were frozen at -20 C and also tested with a Real-Time PCR technique in fall Additional also some fresh artificially contaminated minced meat samples were tested with the bacteriological detection method (ISO 6579) and with the Real-Time PCR technique. The first step in the bacteriological method as well as for the PCR technique was the same. The samples were pre-enriched in BPW at 37 C for 18 hours after which either a selective enrichment 11

15 medium was inoculated (bacteriological detection) or DNA was isolated to be tested with the PCR method. The used PCR technique has been described by Malorny et al, In general comparable results were found between the bacteriological method and the PCR technique. However, some samples containing low numbers of Salmonella in a dirty matrix like chicken faeces gave some unclear results with PCR. It is most likely that the matrix and or the background flora disturb the PCR in these samples. It is planned to perform a few more tests, like dilution of the samples, early Samples for interlaboratory comparison studies Matrix mixed with antibiotic For the current protocol of the interlaboratory comparison studies, the participating laboratories have to mix reference materials (capsules) and matrix in their own laboratories. It has been chosen to perform the studies in this way to better guarantee the contamination level of the reference materials during transport. However, it also has a risk that participants do not add any matrix, which will make the analyses (of only the reference materials) much easier. Therefore, it was decided to include with one of the interlaboratory comparison studies a (blind) check on whether the participants follow the protocol correctly and do add matrix to the reference materials. For this purpose the CRL mixed the matrix with an antibiotic to which the Salmonella strain in the reference materials was sensitive and the background flora in the matrix (preferably) not. If participants would then follow the protocol correctly, the samples would be tested negative for Salmonella. However, if no matrix was added to the reference materials, the results would be positive. Several experiments were needed to be able to prepare such a matrix mixture: - A literature review was done and experts were consulted to find the most optimal choice for the antibiotic and its concentration. - After the choice for the antibiotic was made, several concentrations were tested on the Salmonella reference materials without the addition of matrix. - Experiments were done to find the most optimal way to mix several kilo s of matrix with the antibiotic solution. - Mixtures of matrix with antibiotic were tested with different reference materials at different levels of Salmonella. Also the influence of the antibiotic on the background flora was tested, by checking the number of Enterobacteriaceae and the total amount of aerobic bacteria in the mixed matrix. - The stability of the mixed matrix was also tested, as the antibiotic should still inhibit the growth of Salmonella after mailing the samples and after storage at the participating laboratory. The desired effect was obtained by mixing 1 kg chicken faeces with 1,5 g active Gentamicine. When 25 g of this mixture was added to reference materials containing Salmonella Enteritidis at a level of approximately 20 cfp/capsule, most of the samples were tested negative. Even after storage of the mixed faeces at 5 C for 3 weeks, only 1/25 samples was tested positive. The mixed faeces was used in the interlaboratory comparison study of March 2010 and only a few laboratories found occasionally 1/4 samples positive, which was acceptable, showing that the participants did follow the protocol. Only one laboratory found 4/4 samples positive (see 1. Interlaboratory comparison studies ). Either this was caused by the fact that no mixed matrix was added to the reference materials or by the fact that non-mixed matrix was added. Improve relation with routine samples The current treatment of the samples for the interlaboratory comparison studies deviate from the treatment of routine samples in the participating laboratories. Therefore the following was proposed to improve the relation with real life samples for amount and treatment of the samples: 1 Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A. and Helmuth, R., Diagnostic Real-Time PCR for detection of Salmonella in food. Appl. Env. Microbiol., Vol. 70, 12,

16 Analyses of 25 g matrix instead of 10 g; Optimisation of the procedure for artificial contamination of samples; Make it possible to treat samples similar as routine samples (e.g. use of stomacher). To test the possibilities, several experiments were carried out in which two types of reference materials (capsules and lenticules) were tested. In experiments performed several years ago it was shown that for capsules the best results (highest number of positive samples) were obtained when the capsules first were dissolved in BPW at 37 C for 45 min, after which the matrix was added. Furthermore, it seems to be better not to mix the matrix with the dissolved capsule. Hence, the current experiments with the capsules were limited to tests with higher amounts of matrix (25 g). The lenticules were tested with the different treatments as well. For the tests with the capsules the following was performed: 4 capsules containing Salmonella Typhimurium at a level of approximately 5 cfp/capsule were each reconstituted in 225 ml BPW at 37 C for 45 min. Next to each jar with BPW, 25 g minced pork was added. Next the procedures as described in ISO 6579 (selective enrichment in RVS and MKTTn) and in Annex D of ISO 6579 (selective enrichment on MSRV) were followed. All samples showed to be positive, where the yield of Salmonella in the MKTTn was approximately 10 7 cfp/ml after 24 h of incubation. The yield of the background flora in the same MKTTn cultures varied from 10 4 to 10 8 cfp/ml. Hence, no major problems were expected by enhancing the amount of matrix from 10 g to 25 g in the interlaboratory comparison studies in which capsule reference materials are used. For the tests with lenticules the following was performed: Tested lenticules: Salmonella Typhimurium at a level of approximately 9 cfp/lenticule (STM9) and Salmonella Enteritidis at a level of approximately 7 cfp/ lenticule (STM7). Tested matrices: mince meat (beef or mixed beef/pork) and chicken faeces. Set-up experiments: a. To each of 5 portions of 25 g matrix, 1 lenticule is added and stored at 3 C for 3-4 days. Next, each sample is added to 225 ml BPW. b. 5 portions of 25 g matrix are stored at 3 C for 3-4 days. At the day of analyses, 25 g of matrix together with 1 lenticule is added to 225 ml BPW. After adding the samples to BPW the food samples are mixed in a pulsifier for 1 min. Of the faeces samples 5 x 25 g (+lenticule) is also mixed in a pulsifier and 5x 25 g (+lenticule) is not mixed at all (which is more usual for faeces samples). The food samples have been tested by following ISO 6579 (selective enrichment in RVS and MKTTn) and Annex D of ISO 6579 (selective enrichment on MSRV). The faeces samples were tested by following Annex D of ISO 6579 only. In the minced meat approximately 10 3 cfp/g Enterobacteriaceae and approximately 10 7 cfp/g total aerobic bacteria were present. In the chicken faeces this was respectively 10 cfp/g and 10 7 cfp/g. The STM9 samples were tested positive for all tested matrices and media. The SE7 minced meat samples were all tested positive on all media, although some samples were tested negative with one plating-out medium, but positive with the other plating-out medium. Of the SE7 faeces samples one was tested negative after mixing with the pulsifier. All other samples were found positive. As mixing is not a usual procedure for faecal samples, this result was not considered problematic. Additional to the tests mentioned above, the lenticules were also tested in the set-up of an interlaboratory comparison study. For this the following was tested: - 25 lenticules were each tested with 25 g of chicken faeces (negative for Salmonella). These 25 lenticules existed of 5 lenticules containing Salmonella Enteritidis at a level of approximately 6 cfp/lenticule (SE6), 5 lenticules with SE60, 5 lenticules with Salmonella Typhimurium at a level of approximately 6 cfp/lenticule (STM6), 5 lenticules with STM60 and 5 blank lenticules. It was not necessary to first reconstitute the lenticules before adding the faeces. At first 25 g faeces was added to 225 ml BPW, immediately followed by the addition of a lenticule. The BPW with sample was left at 13

17 room temperature for min to rehydrate the lenticule, followed by the normal procedure of Annex D of ISO lenticules were tested without faeces (control samples), being 2 blank lenticules, 2 lenticules with SE6, 1 lenticule with SE60 and 2 lenticules with STM6. - Method: Annex D of ISO 6579, with selective enrichment on MSRV and plating out on XLD and Brilliance agar. All samples showed the expected results. The results of this test ring trial encourage the use of the lenticules in the full studies. It is planned to use lenticules for the first time in the interlaboratory comparison study for the detection of Salmonella in chicken faeces, which will be organised in February The above mentioned results show that lenticules may be a good alternative to the capsule reference materials. To be sure that abuse temperatures during transport do not negatively influence the number of Salmonella in the lenticules, a challenge test was done. For this test lenticules containing SE75, SE5 and STM65 were stored at 5 C, 22 C and at 30 C. The numbers of Salmonella in the stored lenticules were tested at day 0, after 3 days and after 7 days of storage. The lenticules containing Salmonella Typhimurium showed to be stable at all tested temperatures. The SE5 lenticules were only stored at 22 C, and no effect was seen on the number of SE after one week of storage at this temperature. The SE75 lenticules were stable at 5 C, but showed some decrease when stored at 22 C or at 30 C. However, after a week of storage at these temperatures the numbers were still sufficient high to be well able to detect Salmonella in the samples. The results are summarised in a figure in Annex Giving assistance to the Commission and ad hoc activities Several questions were received from several parties (European Commission, NRLs- Salmonella, other institutes inside and outside the EU) on subjects like methods (qualitative and quantitative, molecular, serological), validation of methods, typing of Salmonella, storage of strains, reference materials, advice on ring trials, sampling, ISO and CEN activities, certain Salmonella serovars, accreditation. When possible, the questions were answered as quickly as possible, or were forwarded to other experts. To give assistance to the Commission, Arjen van de Giessen and Kirsten Mooijman participated in several meetings of working groups of DG-Sanco (working group on Zoonoses and occasionally working group on microbiological criteria) as well as of EFSA. In 2009 the question was raised on the possibility of pooling of samples. Literature was reviewed as well as some experiments were performed, as indicated in clause 3 (Research). The question is still a current topic for the revision of EC Regulation 2073/2005 on the microbiological criteria of Salmonella in poultry meat. A protocol for experiments on pooling of samples has been written and first experiments were performed (see 3. Research). In 2009 it was also discussed how to deal with monophasic Salmonella Typhimurium (STM-like) strains. For this item a working group was raised in EFSA, in which Kirsten Mooijman has participated. The opinion of the EFSA group was published in September In March 2010, DG-Sanco was contacted by ECDC about a Salmonella outbreak investigation. In 2009 and ongoing in 2010 an outbreak of S. Goldcoast in 6 EU countries was seen with pork containing products as possible source. A high proportion of human cases shared indistinguishable PFGE profile and ECDC wanted to check PFGE profiles from animals (especially pigs) and food in the EU. It was therefore requested if it would be possible to use the CRL/NRL-Salmonella network to: - Collect S. Goldcoast isolates to perform PFGE at the CRL - Collect PFGE profiles of S. Goldcoast from NRLs 14

18 Immediately after this request, the CRL-Salmonella contacted the NRLs to ask for available strains/pfge-profiles. Within 2 weeks, replies were received from 22 NRLs indicating an availability of in total 600 isolates of S. Goldcoast. In the following months, several NRLs sent isolates and PFGE profiles to the CRL. Furthermore, at the CRL- Salmonella also several S. Goldcoast isolates were available in stock from, amongst others, baseline surveys. By the end of May, 118 PFGE profiles were available at the CRL, originating from 14 countries, from humans, pigs, cattle and dogs. The results from the different isolates showed very similar PFGE profiles, with only minute differences. Therefore, the information from PFGE for S. Goldcoast seems to be limited. This was further discussed in a teleconference with ECDC and the laboratory in Italy who analysed the human outbreak strains. In fall 2010 the PFGE profiles collected at the CRL and the PFGE profiles of the outbreak investigations were further compared at the CRL and also these results showed a great similarity between the PFGE profiles, confirming that the information from PFGE for S. Goldcoast is limited. This information was communicated to the ECDC. 5. Communication Every three months a newsletter is published through the CRL-Salmonella website. In April 2010, volume 16 no 1 of the newsletter was published, which included information on the workshop of 2010, the technical report of 2009 and the reports of the meetings of ISO/TC34/SC9-WG9 on revision of ISO 6579 and the ad hoc group on serotyping of Salmonella (both met in December 2009). In July 2010, volume 16 no 2 of the newsletter was published, which included the timetable of the interlaboratory comparison studies on the detection of Salmonella spp. in a Food matrix IV. In October 2010, volume 16 no 3 of the newsletter was published, which included the timetable of the fifteenth interlaboratory comparison study on typing of Salmonella (November 2010). Furthermore, the draft working plan of 2011 and the progress report of the activities performed up to August 2010 were published in this newsletter. In January 2011, volume 16 no 4 of the newsletter was published, which included the time table of the interlaboratory comparison studies on the detection of Salmonella spp. in animal faeces XIV (February 2011) Communication with the NRLs is performed as much as possible electronically (through e- mail and through the CRL-Salmonella website). For this purpose one staff member of the CRL takes care of keeping the website up to date ( In November 2010 the CRL was visited by 11 delegates of Mercosur countries: Argentina, Brasil, Uruguay and Paraguay, to receive information on the system of CRL and NRLs in Europe and especially on the activities performed by the CRL-Salmonella. 6. Training Up to August 2009, 5 trainings have been organised. Three trainings were organised on request of 3 NRLs and were given at the laboratory of the CRL and focused on serotyping and/or molecular typing. These trainings were given in January, February and March In each training, 2 staff members of one NRL participated. Two trainings were organised as a follow-up for poor performance of a NRL in more than one interlaboratory comparison study (also see 1. Interlaboratory comparison studies ). Both trainings concerned a visit of 2 staff members of the CRL at the laboratory of the NRL. One visit was organised in January 2010 and focused on detection of Salmonella in food and animal faeces. The other visit was organised in April 2010 and focused on serotyping of Salmonella. 15

19 7. Missions Participation in Working Groups of DG-Sanco and EFSA Arjen van de Giessen and Kirsten Mooijman participated in meetings of working groups of DG-Sanco and EFSA: 20 January 2010: EFSA working group, Parma 12 February 2010: DG-Sanco working group Zoonoses, Brussels 19 March 2010: EFSA working group, Brussels 13 July 2010: DG-Sanco working group microbiological criteria, Brussels 9 September 2010: EFSA working group Parma 29 October 2010: DG-Sanco working group microbiological criteria, Brussels Budget: DG-Sanco or EFSA The following missions in relation with the CRL-Salmonella activities were performed in 2010: January 2010: Visit to a poor performing NRL (detection of Salmonella) Participants: Angelina Kuijpers and Kirsten Mooijman 9-10 February 2010: CRL forum, organised by IRMM, Geel, Belgium Participant: Kirsten Mooijman (presentation given) April 2010: Visit to a poor performing NRL (serotyping of Salmonella) Participants: Henny Maas and Anjo Verbruggen 31 May 4 June 2010: Meetings of CEN/TC275/WG6, ISO/TC34/SC9 (Food microbiology) in Buenos Aires, Argentina. A joint report of several CRLs was sent to DG-Sanco in July Participant: Kirsten Mooijman 27 June 2010: CRL-Salmonella workshop, St. Malo, France Participants: Angelina Kuijpers, Wilma Jacobs and Kirsten Mooijman June 2010: International Symposium on Salmonella and Salmonellosis (I3S), St. Malo, France Participants: Angelina Kuijpers (poster presentation), Wilma Jacobs and Kirsten Mooijman (oral presentation and co-chair of a session) Budget: For Kirsten Mooijman from I3S. For Angelina Kuijpers and Wilma Jacobs: transport from budget workshop CRL, daily allowances and hotel costs from budget RIVM 5 October 2010 Meeting of WG9 of ISO/TC34/SC9 on revision of ISO in Brussels, Belgium Participant: Kirsten Mooijman Mrs. Drs. Kirsten Mooijman Head CRL-Salmonella Bilthoven, 25 March

20 Annex 1 Results stress protocol on the injury efficiency of 6 different Salmonella strains Before stress 15 min 50 C 3 days -20 C 1,5 week 4 C cfp/ml log cfp cfp/ml log cfp cfp/ml log cfp cfp/ml logcfp SE ATCC on NA 8,40E+08 8,92 4,40E+07 7,64 2,30E+06 6,36 5,40E+07 7,73 SE ATCC on XLD 8,20E+08 8,91 1,00E+07 7,00 4,00E+05 5,60 2,50E+07 7,40 Difference logcfp NA-XLD 0,01 0,64 0,76 0,33 SE 532 on NA 7,80E+08 8,89 5,00E+08 8,70 9,00E+05 5,95 2,40E+08 8,38 SE 532 on XLD 8,20E+08 8,91 3,40E+08 8,53 7,30E+05 5,86 1,80E+08 8,26 Difference logcfp NA-XLD -0,02 0,17 0,09 0,12 STM ATCC on NA 1,20E+09 9,08 9,30E+08 8,97 1,90E+07 7,28 6,50E+08 8,81 STM ATCC on XLD 1,10E+09 9,04 3,50E+08 8,54 8,80E+06 6,94 2,90E+08 8,46 Difference logcfp NA-XLD 0,04 0,42 0,33 0,35 STM 756 on NA 7,80E+08 8,89 4,00E+08 8,60 3,50E+06 6,54 2,70E+08 8,43 STM 756 on XLD 6,90E+08 8,84 1,40E+08 8,15 1,80E+06 6,26 1,00E+08 8,00 Difference logcfp NA-XLD 0,05 0,46 0,29 0,43 STM-like S18 on NA 5,80E+08 8,76 2,10E+08 8,32 3,10E+06 6,49 1,50E+08 8,18 STM-like S18 on XLD 5,30E+08 8,72 1,10E+08 8,04 5,60E+05 5,75 1,00E+08 8,00 Difference logcfp NA-XLD 0,04 0,28 0,74 0,18 STM-like LIS on NA 1,10E+09 9,04 3,00E+08 8,48 7,00E+06 6,85 5,40E+08 8,73 STM-like LIS on XLD 9,70E+08 8,99 1,40E+08 8,15 5,80E+06 6,76 1,70E+08 8,23 Difference logcfp NA-XLD 0,05 0,33 0,08 0,50 Tested strains: SE: Salmonella Enteritidis ATCC and Salmonella Enteritidis No 532 isolated from poultry STM: Salmonella Typhimurium ATCC and Salmonella Typhimurium No 756 from poultry STM-like: 4,5,12:i:- No S18 (also used in the interlaboratory comparison study on typing of 2010) and 4,5,12:i:- from LIS (Laboratory for Infectious Diseases and Perinatal Screening of the RIVM). Media: NA: Nutrient Agar (non-selective) XLD: Xylose Desoxycholate agar (XLD) 17