European Union Reference Laboratory for Salmonella Vol. 22 No. 1 March 2016 ISSN 2211-6877 Continuation of Newsletter Community Reference Laboratory for Salmonella ISSN 1572-3836
Page 2 of 47 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: e-mail: +31 30 274 3537 (Kirsten Mooijman) +31 30 274 4290 (Wilma Jacobs) kirsten.mooijman@rivm.nl wilma.jacobs@rivm.nl
Page 3 of 47 Contents Editorial Note... 4 Contribution of the EURL-Salmonella... 6 From the Literature... 25
Page 4 of 47 Editorial Note Bilthoven, 4 April 2016 Dear colleagues, In the first quarter of this year several activities were performed in relation to EURL-Salmonella interlaboratory comparison studies. In January 2016, a follow-up study of the interlaboratory comparison study on detection of Salmonella in whole liquid egg was organised for one NRL-Salmonella. Also in January 2016, the serotyping results of the 20 th interlaboratory comparison study on typing of Salmonella were analysed. Early February the laboratories received their own results as well as the interim summary report containing the results of all participants. This interim summary report can also be found at the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid= rivmp:304857&versionid=&subobjectname= All but one NRL-Salmonella found good results in this study. Currently we are preparing the follow-up study for this NRL. The results of the study on PFGE typing are still under analysis and will soon be reported. In February 2016, the 19 th interlaboratory comparison study on the detection of Salmonella in samples from the primary production stage (boot socks) was organised. The results are currently under analysis and will soon be reported to the participants. As you may have noticed, we are also quite busy with the organisation of the EURL-Salmonella workshop of 2016. We have received all registration forms and recently we have informed our contact in St. Malo (France) with the list of participants and requests for reservation of hotels. We have also started with drafting the programme of the workshop. As soon as this is worked out in more detail the participants will be informed. Earlier this year the link to the White-Kauffmann-Le Minor scheme was changed. All NRLs-Salmonella were informed by e-mail in February 2016. The new link is the following: https://www.pasteur.fr/sites/www.pasteur.fr/files/wklm_en.pdf By the end of March we have sent the annual report of the activities of EURL- Salmonella performed in 2015 to EC DG-Sante. For your information this report is also included in this newsletter. In the first quarter of this year several reports on activities of the EURL- Salmonella were published. These reports are no longer distributed on paper, but are available through the EURL-Salmonella/RIVM website. The link for downloading is given for each report. Mooijman, K.A. The 20 th EURL-Salmonella workshop 28 and 29 May 2015, Berlin, Germany. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: 2015-0083 (February 2016). http://www.rivm.nl/bibliotheek/rapporten/2015-0083.pdf Kuijpers A.F.A., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study animal feed III (2014) - Detection of Salmonella in chicken feed. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: 2015-0080 (March 2016). http://www.rivm.nl/bibliotheek/rapporten/2015-0080.pdf
Page 5 of 47 Jacobs-Reitsma, W.F., Maas, H.M.E., de Pinna, E., Mensink, M.E. and Mooijman, K.A. Nineteenth EURL-Salmonella interlaboratory comparison study (2014) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM report no.: 2015-0081 (March 2016). http://www.rivm.nl/bibliotheek/rapporten/2015-0081.pdf Best wishes, Kirsten Mooijman Head EURL-Salmonella
Page 6 of 47 Contribution of the EURL-Salmonella Technical report on activities of the European Union Reference Laboratory for Salmonella in 2015 K.A. Mooijman 22 March 2016 National Institute for Public Health and the Environment (RIVM) Centre for Zoonoses and Environmental microbiology (cz&o) Letter-report 019/2016 Z&O Mo/km RIVM project-number: E/114506/15 Contact: K.A. Mooijman; kirsten.mooijman@rivm.nl RIVM Z&O Head EURL-Salmonella P.O. Box 1 3720 BA Bilthoven The Netherlands Introduction The work plan of EURL-Salmonella for the year under review, 2015, was submitted to the European Commission in August 2014 and slightly amended in October 2014. This report details consequential activities of the EURL-Salmonella according to the agreed work plan. General In 2015, 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 supporting activities 4. Giving assistance to the Commission and ad hoc activities 5. Communication 6. Training 7. Molecular typing of Salmonella spp. 8. Missions Deliverables Reports In 2015, the following reports were published: Kuijpers A.F.A. and Mooijman, K.A. EU Interlaboratory comparison study Primary Production XVII (2014) - Detection of Salmonella in chicken faeces. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: 2014-011. The interim summary of this interlaboratory comparison study was published in April 2014 and is available through the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:24 7023&versionid=&subobjectname=. The final report was published in September 2015 and is available through the EURL-Salmonella website: http://www.rivm.nl/bibliotheek/rapporten/2014-0011.pdf
Page 7 of 47 Early 2016, the following reports were published: Mooijman, K.A. The 20 th EURL-Salmonella workshop 28 and 29 May 2015, Berlin, Germany. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: 2015-0083 (January 2016). The presentations were published on the EURL-Salmonella website on 2 June 2015: http://www.eurlsalmonella.eu/workshops/workshop_2015 The draft report was sent to DG-Sanco in October 2015. The final report was published in January 2016 and is available through the EURL-Salmonella website: http://www.rivm.nl/bibliotheek/rapporten/2015-0083.pdf Kuijpers A.F.A., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study animal feed III (2014) - Detection of Salmonella in chicken feed. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: 2015-0080. The interim summary of this interlaboratory comparison study was published in November 2014 and is available through the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:26 4448&versionid=&subobjectname=. The final report was published in March 2016 and is available at the following link: http://www.rivm.nl/bibliotheek/rapporten/2015-0080.pdf Jacobs-Reitsma, W.F., Maas, H.M.E., de Pinna, E., Mensink, M.E. and Mooijman, K.A. Nineteenth EURL-Salmonella interlaboratory comparison study (2014) on typing of Salmonella spp. RIVM report no.: 2015-0081. The interim summary of this study was published in February 2015 and is available through the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:27 1634&versionid=&subobjectname=. The final report was published in March 2016 and is available at the following link: http://www.rivm.nl/bibliotheek/rapporten/2015-0081.pdf The following reports are under preparation: Pol-Hofstad, I.E. and Mooijman, K.A. EU Interlaboratory comparison study Primary Production XVIII (2015) - Detection of Salmonella in pig faeces. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: 2015-0082. The interim summary of this interlaboratory comparison study was published in May 2015 and is available through the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:28 0206&versionid=&subobjectname=. The final report is under preparation. Kuijpers A.F.A., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food VII (2015) - Detection of Salmonella in whole liquid chicken egg. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: 2016-0042. The interim summary of this interlaboratory comparison study was published in November 2015 and is available through the EURL-Salmonella website: http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:29 3851&versionid=&subobjectname=. The report is under preparation. ISO and CEN A consolidated report of 8 EURLs (coordinated by the EURL-Salmonella) on the meetings of ISO/TC34/SC9 and CEN/TC275/WG6 held in Delft, the Netherlands on 22-26 June 2015, was sent to DG-Sanco on 4 August 2015. ISO 6579-1: Detection of Salmonella The voting on ISO/FDIS 6579-1 was launched on 12 November 2015 and finished on 12 January 2016. A second Working Draft of ISO 16140-6 was prepared and sent to the members of ISO- WG3 for comments in September 2015. The title of the document is: Microbiology of the food chain - Method validation Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing. The Committee Draft (CD) voting was launched on 11 February 2016.
Page 8 of 47 A second Working Draft of a guidance document for the drafting of CEN/ISO standards for microbiology of the food chain was prepared in September 2015 and sent to the members of the ISO ad hoc group for comments. A third Working Draft was sent for comments to the project leaders of ISO/TC34/SC9 and CEN/TC275/WG6 in December 2015. The title of the document is: Microbiology of the food chain Template and guidance for drafting ISO/CEN Standard methods.' 1. Interlaboratory comparison studies General Since 2013, the matrices under analyses for the different interlaboratory comparison studies are artificially contaminated with a diluted culture of a Salmonella serovar at the laboratory of the EURL-Salmonella. These types of samples mimic well real-life samples and are easy in use for the NRLs for Salmonella. For the set-up of the studies the directions of CEN ISO/TS 22117 are followed, which indicates that for comparative tests of qualitative methods each participant should test at least 18 samples in total. These 18 samples consist of six replicates of three different levels of the target strain: blank (matrix) samples; low level (matrix) samples (close to the detection limit of the method); high level (matrix) samples (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 tested negative. For the reporting of the results of the interlaboratory comparison studies by the NRLs- Salmonella, web based test reports are used. In the course of 2015 these test reports were further improved. The number of questions on general laboratory information was reduced as this information can be requested at the NRL in case of deviating results. For the reporting of the results details per medium combination was no longer requested as this is not done for routine analysis. Reporting if a sample was found positive or negative for Salmonella, independent on the medium combination used, was considered sufficient. Another technical improvement of the web-based test reports was introduced by giving the possibility to save the reported data page by page instead of only after completing the full report. Details on the performance of third countries in the EURL-Salmonella interlaboratory comparison studies are reported annually to DG-Sante. In 2015 this report was prepared and sent to DG-Sante in September. Follow-up studies from interlaboratory comparison studies organised in 2014 In October 2014, the third interlaboratory comparison study on the detection of Salmonella in animal feed samples was organised. In this study, 34 NRLs for Salmonella participated: 30 NRLs from the (28) EU Member States and 4 NRLs from third countries (member countries of the European Free Trade Association, an EU candidate Member State and one NRL from a non-european country). Each NRL analysed in total 21 samples: 18 samples of each 25 g chicken feed artificially contaminated with three different levels (blank, low and high level) of Salmonella Senftenberg (SSE) and 3 control samples. The used contamination levels of the diluted cultures were: 20 cfu/sample and 61 cfu/sample, resulting in respectively 2 MPN/sample and 11 MPN/sample at the day of the study. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in RVS and MKTTn). Additionally it was requested to also use Annex D of ISO 6579:2007 (with selective enrichment on MSRV). Two NRLs scored below the level of good performance in relation to their control samples. Both NRLs made transcription errors in reporting of the results and their results were indicated as moderate performance. One NRL made the same kind of transcription error for the third time in a row in interlaboratory comparison studies on detection of Salmonella in food or animal feed samples. This laboratory was contacted for a follow-up study in combination with a visit of two staff members of the EURL-Salmonella in February 2015. During a two-day visit, the procedures of the NRL were
Page 9 of 47 checked for possible (technical) problems to explain the deviating results. At the end of the visit, a report containing observations and recommendations for possible improvements was drafted by the staff members of the EURL-Salmonella and discussed with the staff members of the NRL. The deviating results of this laboratory seem mainly to have been caused by problems with reporting the results through the web-based test reports. Due to problems with internet, reported data were lost several times because the form was not completed before the internet stopped working. During the follow-up studies no deviating results were found and no problems with reporting were faced. The results of the follow-up study fulfilled the criteria of good performance. A report with all information about the performance in the food/animal feed studies of 2011, 2013, 2014 and the visit of the EURL-Salmonella in 2015 was sent to EC, DG-Sante in April 2015. It was concluded that no further actions were needed. As a conclusion of this problem with reporting of the results, the EURL-Salmonella further improved the web-based reporting form so that it would be possible to save reported data at every page instead of only after completing the full report. In November 2014 the 19 th interlaboratory comparison study on typing of Salmonella spp. was organised. This study contained a compulsory part on serotyping and an optional part on phage typing and an optional part on PFGE (Pulsed Field Gel Electrophoresis) typing. The typing study was organised in collaboration (under a subcontract) with the Public Health England (PHE) in London, United Kingdom, for the part on phage typing. This 2014 study was the last study including a part on phage typing. At the workshop of 2014 it has been decided to stop the organisation of this part of the interlaboratory comparison studies, due to the low number of participants. In total 35 NRLs for Salmonella participated: 29 NRLs from the (28) EU Member States and 6 NRLs from third countries (member countries of the European Free Trade Association and from (potential) EU candidate Member States). All (35) NRLs participated in the serotyping part of the study. For this, 20 different serovars of Salmonella enteric subsp. enterica had to be analysed. Additionally a 21 st strain of another Salmonella enterica subspecies was added for serotyping. The serotyping of this latter strain was optional, and the results were not taken into account for the evaluation of the performance of the NRL. Seven NRLs performed phage typing of 10 different strains of Salmonella Typhimurium and of 10 different strains of Salmonella Enteritidis. Eighteen NRLs performed PFGE typing on 10 different Salmonella strains. The NRLs reported the results of the serotyping and phage typing before early December 2014. The PFGE results were reported separately before the end of December 2014. Two laboratories (of which one from a non EU-MS) did not fulfil the criteria of good performance for the serotyping and a follow-up study for these laboratories was organised in March 2015. The NRL from the non EU-MS was not able to participate in the follow-up study due to lack of resources. The NRL of the EU-MS did participate in the follow-up study and found good performance. The results of all laboratories and for all methods (serotyping, phage typing and PFGE) were presented at the EURL-Salmonella workshop in May 2015 and the full report of this study is likely to be published in early 2016 (see Introduction ). Interlaboratory comparison studies on detection of Salmonella organised in 2015 In March 2015, the 18 th interlaboratory comparison study on the detection of Salmonella in samples from the primary production stage was organised. In this study, 36 NRLs for Salmonella participated: 30 NRLs from the (28) EU Member States and 6 NRLs from third countries (member countries of the European Free Trade Association, (potential) EU candidate Member States and one NRL from a non-european country). Each NRL had to analyse a total of 21 samples: 18 samples of each 25 g pig faeces artificially contaminated with three different levels (blank, low and high level) of monophasic Salmonella Typhimurium (monostm) and 3 control samples. Pre-studies at the EURL-Salmonella showed that moulds and yeasts grew at the surface of the pig faeces during storage at 5 C or 10 C. To prevent growth of yeast and moulds, the pig faeces samples were stored at -20 C. Pre-tests showed that Salmonella was susceptible towards freezing, but by using higher starting inoculation levels (10 times higher than used in earlier studies), it was expected that the majority of the samples could
Page 10 of 47 still be tested positive for Salmonella. For this purpose the low level samples were inoculated with 84 cfu/25 g and the high level samples were inoculated with 530 cfu/25 g. The prescribed method for analyses was Annex D of ISO 6579 (2007), with selective enrichment on MSRV. All NRLs reported the results before mid-april 2015, after which the analyses of the results were performed. In contrast to the good results in the pre-studies, the results of the participants in the interlaboratory comparison study varied considerably. Of the low contaminated pig faeces samples, only 21% was found positive for Salmonella, while for the high contaminated samples only 58% was found positive where 100% was expected. Due to the large variation in results it was not possible to evaluate the performance of the laboratories. An interim summary report of the study was sent to the participants in May 2015 and the results were also presented at the EURL-Salmonella workshop in May 2015. The final report is under preparation (see Introduction ). In September 2015, the seventh EURL-Salmonella interlaboratory comparison study on the detection of Salmonella in a food matrix was organised. In this study, 36 NRLs for Salmonella participated: 30 NRLs from the (28) EU Member States and 6 NRLs from third countries (EU candidate MS or potential EU candidate MS, members of the European Free Trade Association (EFTA) and a non-european countries). Each NRL analysed in total 20 samples: 18 samples of each 25 g whole liquid egg artificially contaminated with three different levels (blank, low and high level) of Salmonella Enteritidis (SE) and 2 control samples. The used contamination levels of the diluted cultures were: 20 cfu/sample and 100 cfu/sample. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in RVS and MKTTn). For this study it was anticipated that the Final Draft International Standard (FDIS) version of ISO 6579-1 was published in fall 2015. This latter document describes the (final) updated technical steps for the detection of Salmonella in food, animal feed and samples from the primary production stage. An important change in this document, compared to the 2002 version of ISO 6579, is the possibility to choose between RVS and MSRV for the selective enrichment of Salmonella from food and animal feed samples. For that reason, this choice was already introduced in the current study, meaning that additional to MKTTn, either RVS or MSRV could be used for selective enrichment. It was also allowed to use all three selective enrichment media. For the reporting of the results, the participants were asked to report what would have been reported in case these samples would have been routine samples, meaning that the indication positive (1) or negative (0) per sample (after confirmation) was sufficient (irrespective of the combination of selective enrichment medium and isolation medium). All NRLs reported the results by mid-october 2015. In November 2015, the participants received information on their performance as well as an interim summary report including the results of all participants. One NRL (EU-MS) scored below the level of good performance as they tested two blank samples false positive for Salmonella. This laboratory performed a follow-up study in January 2016 and scored eventually a good performance. Interlaboratory comparison studies on typing of Salmonella organised in 2015 In October/November 2015 the 20 th interlaboratory comparison study on typing of Salmonella spp. was organised. This study contained a compulsory part on serotyping and an optional part on PFGE (Pulsed Field Gel Electrophoresis) typing. In total 34 NRLs for Salmonella participated: 29 NRLs from the (28) EU Member States and 5 NRLs from third countries (EU candidate MS or potential EU candidate MS and member countries of the European Free Trade Association (EFTA)). All (34) NRLs participated in the serotyping part of the study. For this, 20 different serovars of Salmonella enteric subsp. enterica had to be analysed. Additionally a 21 st strain of an uncommon Salmonella serovar was added for serotyping. The serotyping of this latter serovar was optional, and the results were not taken into account for the evaluation of the performance of the NRL. Eighteen NRLs participated in the PFGE part of the study. For this, 10 different Salmonella strains had to be analysed. The Salmonella strains for this part of the study were selected in close cooperation with the Statens Serum Institute (SSI; Copenhagen, Denmark), who
Page 11 of 47 organises EQA schemes for PFGE typing for the laboratories analysing human samples of the ECDC-FWD network. The NRLs reported the results of the serotyping before early December 2015. The PFGE results were reported separately by December 2015/January 2016. The analysis of the serotyping results were performed in January 2016 after which the NRLs received their own results and an interim summary report, including the results of all laboratories in February 2016. One laboratory (EU-MS) did not fulfil the criteria of good performance for the serotyping and a follow-up study for this laboratory is planned in March/April 2016. The results of the study on PFGE typing are still under analysis. 2. Workshop On 28 and 29 May 2015, the annual EURL-Salmonella workshop was organised in Berlin, Germany, with the help of the NRL-Salmonella in Germany. A total of 48 participants were present at the workshop: 33 participants from the 28 EU-MS 3 participants from the EFTA countries 4 participants from EU candidate MSs or potential EU candidate MSs 3 participants from EURL-Salmonella 3 guest speakers 1 participant from EFSA 1 participant from DG-Sante Shortly before the workshop, 2 participants from 2 EU MS cancelled their participation due to lack of staff and due to illness. Presentations were given on the following subjects: Results of the interlaboratory comparison studies as organised by the EURL- Salmonella since the previous workshop (May 2014); Proposals for new interlaboratory comparison studies; The EU Salmonella monitoring data on food-borne outbreaks and antimicrobial resistance of 2013, published by EFSA; Information on the activities by EFSA for the databases for molecular typing data; Molecular typing of Salmonella; Information on Salmonella outbreaks; Information on activities in ISO and CEN related to Salmonella, including standardisation of PCR method(s) for identification of monophasic Salmonella Typhimurium; Activities of 5 NRLs to fulfil their tasks and duties; Work-programme of the EURL-Salmonella for the coming year. During the workshop an evaluation form about the workshop was distributed and the participants were requested to complete it (anonymously). The evaluation form was handed over to 47 participants of the workshop and 43 completed forms were returned, being a response of 91,5%. From the answers of the respondents, it could be concluded that the participants were in general satisfied about the workshop. The scientific programme was considered as interesting. More details on the presentations, discussion and evaluation of the workshop is summarised in the report of the workshop. The draft version of this report was sent to EC DG-Sante in October 2015. All presentations were placed on the EURL-Salmonella website (http://www.eurlsalmonella.eu/workshops/workshop_2015) on 2 June 2015. The final report was published in January 2016 (see Introduction ). 3. Supporting activities Activities in ISO and CEN EURL-Salmonella is involved (as project leader/convenor or as member of (working) groups) in several activities of ISO and CEN. More specific in:
Page 12 of 47 - 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 Microbiology of the food chain. Both groups organised their annual meeting in Delft, the Netherlands from 22 to 26 June 2015. Kirsten Mooijman and Wilma Jacobs of the EURL-Salmonella are (co-)project leaders of groups in CEN and ISO dealing with methods for Salmonella, validation of typing methods and drafting of a guidance document for drafting ISO/CEN standard methods. Wilma and Kirsten presented the progress of these groups at the plenary meeting of SC9 and of WG6. A consolidated report of 8 EURLs (coordinated by the EURL-Salmonella) on the meetings was sent to DG-Sante on 4 August 2015 (see Introduction ). EN ISO 6579-1: Detection of Salmonella (activity in CEN/TC275/WG6) Progress of the work since the previous plenary meeting in Washington, USA, June 2014: 2014, 5 June 5 November pren/iso-dis voting. 2014, December/ 2015, January: reviewing of comments to DIS 6579-1 and drafting replies to the remarks on ISO/DIS 6579-1 2015, 27 and 28 January: meeting of CEN-TAG8 to discuss the comments on DIS 6579-1 and to update the document. 2015, February/March: collecting additional information from TAG8 for improving the document. 2015, 2 April: updated version of DIS 6579-1 (related to comments of DIS voting) sent to TAG8 for (final) check until 1 May 2015. 2015, May: document updated for FDIS voting. 2015, 22 May: draft FDIS 6579-1 sent to secretariat CEN/TC275/WG6 (Gwenola Hardouin) to launch the FDIS vote (and sent to TAG8). 2015, 23 June: presentation of progress TAG8 at annual meeting of CEN/TC275/WG6 discussion on a few items (e.g. standard text for test portion, title of clause 6, annexes B and C). 2015, 1 August: document slightly amended by Gwenola and sent to ISO Central Secretariat (ISO/CS) to start the FDIS voting. 2015, 12 November 2015 12 January 2016 FDIS voting. 2016, 14 January results FDIS voting: 100% (20) positive in CEN, 96% positive in ISO (1 negative, 24 positive), with some editorial comments. 2016, February: drafting replies to (editorial) comments. 2016, February/March preparation of written consultation at SC9 & WG6 for agreement on the addition of information concerning minor revisions & change reference to Annex F (preparation dairy products) At the meeting in Delft, Kirsten informed the members of WG6 on the progress with drafting part 1 of EN ISO 6579 on Detection of Salmonella. From June to November 2014 the CEN enquiry/dis voting of EN ISO 6579-1 took place, with the following results: ISO: 25 approvals and 2 disapprovals (Canada and France) CEN: 22 approvals and 1 disapproval (France) Overall the voting result was positive, with in total 25 pages of comments. On 27 and 28 January 2015, TAG8 met in Brussels to discuss the comments and to update the document. The main comments for disapproval of pren DIS 6579-1 as well as the replies from TAG8 were presented at the meeting of WG6. Comment from Canada: Change incubation temperature of MKTTn from 37 C to 41,5 C (or 42,5 C). Reply of CEN-TAG 8: Not accepted. Incubation of MKTTn at 37 C has been decided for the current version of ISO 6579: 2002. Many (European) laboratories have experiences (for many types of samples) with the use of MKTTn, incubated at 37 C, for more than 13 years. Furthermore, validation data have been obtained with MKTTn incubated at 37 C in 2000 (summarised in Annex C.1 of ISO 6579-1).
Page 13 of 47 Comment 1 from France: According to a French study (performed by NRL-Salmonella France) many more samples from the primary production stage will be found positive (approximately 23 %) if additional to selective enrichment on MSRV (at 41,5 C) also selective enrichment in MKTTn broth is performed, incubated at 41,5 C. Reply of CEN-TAG 8: TAG 8 has discussed this information in detail and decided (together with France) not to change the procedure, because there seem to be too many factors involved influencing the data. For example: in the French study a relatively large amount of samples contained lactose positive Salmonella (approx. 20%), which detection was dependent on the chosen isolation medium. It was therefore decided to add only the following informative note to 9.3.3 (selective enrichment pps): NOTE - Sensitivity may be improved by using a second selective enrichment procedure, e.g. MKTTn broth incubated at 41,5 C for 24 h. At the meeting of WG6 it was stressed that it is very important that the results of the French study will be published, preferable before the end of 2015 so that a reference to this publication can still be included in the final publication of EN ISO 6579-1 (at the note of clause 9.3.3). As an option for a (relatively) fast publication of the results of the study it was suggested to publish it in the Newsletter of the EURL-Salmonella, so that a link can be added to the Bibliography of EN ISO 6579-1. Finally, the study was published in the Newsletter of the EURL-Salmonella of December 2015 (http://www.eurlsalmonella.eu/dsresource?type=pdf&disposition=inline&objectid=rivmp:2 99850&versionid=&subobjectname=). Comment 2 from France: Serological testing should become optional instead of mandatory after biochemical testing. Reply of CEN-TAG 8: Partly accepted. Limited serological testing is required (up to group O and group H), especially as the number of mandatory biochemical tests is limited (only tests for Triple sugar/iron (TSI), ureum and L-Lysine decarboxylation (LDC)). After the TAG8 meeting in January 2015, the document was updated to include the comments from the CEN enquiry/dis voting. Next, the amended document was sent to the members of TAG8 for a final check in April 2015. After this, a few additional comments were received from some members of TAG8 which were introduced in the final draft version of EN ISO 6579-1. By 22 May 2015 the final draft document was sent to the secretariat of WG6 (Gwenola Hardouin) to launch the FDIS voting (launched 12 November 2015). Barbara Gerten indicated that as the final draft document now includes all amendments as requested by ISO/TC34/SC5 (IDF milk and milk products ), SC5 agreed to withdraw the vertical ISO 6785 ( Detection of Salmonella in milk and milk products ) as soon as ISO 6579-1 is published. Standardisation of PCR method(s) for identification of monophasic Salmonella Typhimurium At the annual meeting of CEN/TC275/WG6 in 2014 it was agreed to start working on standardisation of PCR methods for identification of monophasic Salmonella Typhimurium (monostm). It was suggested to draft this standard procedure(s) as a new Annex to ISO/TR 6579-3 ( Serotyping of Salmonella ). As ISO/TR 6579-3 concerns a guidance document, meaning that the method is informative and not normative, also this annex will become a guidance protocol and will not become normative. The work will be done in CEN- TAG3 (project leader Burkhard Malorny of NRL-Salmonella Germany) in close cooperation with CEN-TAG8 (ISO 6579-1 Detection of Salmonella) and ISO-WG10 (ISO/TR 6579-3 Serotyping of Salmonella), for which both the project leader is Kirsten Mooijman (EURL- Salmonella). In fall 2014, the EURL-Salmonella sent a call for available methods to the members of CEN-TAG8, ISO-WG10 and (some) NRLs for Salmonella. The results of this inventory were sent to Burkhard for presentation at the meeting of CEN-TAG3 in November 2014. As a result of this meeting, additional information on details of the methods was asked by the EURL at CEN-TAG8, ISO-WG10 and the NRLs for Salmonella. A summary of all collected
Page 14 of 47 information was presented at the next meeting of CEN-TAG3 in April 2015, at the EURL- Salmonella workshop in May 2015 and at the annual meeting of CEN/TC275/WG6 in June 2015. At the annual meeting of CEN-WG6 it was agreed to continue with the technical work by CEN-TAG3 and to move the work to ISO-WG10 as soon as the procedure has been worked out in more detail. Conclusion of the discussions at the different meetings were: - Focus on protocols for identifying monostm lacking the second phase (1,4,[5],12:i:-) - Standardise more than one protocol: a gel based PCR and a Real time PCR and if possible, a gel based PCR using the Real time PCR reagents; - Determine the performance characteristics of the different protocols with a standard set of strains. In fall 2015, a work-plan was made (by Kirsten and Burkhard) for the next steps in the standardisation process and this was presented (and agreed) at the meeting of CEN-TAG3 in November 2015. It was agreed that early 2016, Burkhard would start drafting the protocols and Kirsten would make an inventory among the NRLs-Salmonella and the members of CEN-TAG8 and ISO-WG10 asking for: - their interest in reviewing the draft protocols; - their interest in participation in the verification study for determining the performance characteristics of the different protocols; - their availability of (interesting) strains for the verification study. ISO/TC34/SC9 WG3 on validation of microbiological methods In 2014 a New Work Item Proposal (NWIP) was launched for a procedure for validation of proprietary alternative confirmation/typing methods. This procedure will become a part of the ISO 16140 series on validation of microbiological methods. Such a procedure is not yet available but is highly needed, especially to validate alternative methods for (sub-) typing of Salmonella. For that reason, Wilma Jacobs and Kirsten Mooijman of EURL-Salmonella have become respectively project leader and co-project leader for this subject. In 2015, two working draft versions of the ISO standard have been prepared and discussed in the drafting group and in ISO-WG3 at meetings in January, April and October 2015. The comments to the second working draft were reviewed and the document was updated after which the voting of the Committee Draft (CD) version was launched in February 2016. ISO/TC34/SC9 Ad hoc group on template and guidance for drafting microbiological ISO/CEN standard methods At the plenary meeting of ISO/TC34/SC9 in Washington in June 2014, it was decided to raise an ad hoc group for drafting a guidance document for the drafting of microbiological ISO/CEN standards. This document is intended to (further) harmonise the content and layout of standards for microbiology of the food chain. Kirsten Mooijman and Wilma Jacobs were asked to become respectively project leader and co-project leader of this group because of their extensive experiences with drafting ISO/CEN documents. The document will become an internal guidance document for convenors and project leaders of SC9 and WG6. In December 2014 a first working draft (WD1) was sent to the ad hoc group for comments. Some comments were discussed at the annual meeting of ISO-SC9 and CEN- WG6 in June 2015. In September a second working draft was prepared, including the comments to WD1, and sent to the ad hoc group. Comments to WD2 were used to prepare a third working draft, which was sent to the project leaders of ISO-SC9 and CEN-WG6 in December 2015. Comments to WD3 will be used to prepare a fourth working draft (WD4) in spring 2016 and will be discussed at the annual meeting of SC9 and WG6 in May 2016. ISO/TC34/SC9 ad hoc group on Harmonization of incubation temperatures During the drafting of CEN ISO/TR 6579-3 (serotyping of Salmonella) and EN ISO 6579-1 (detection of Salmonella), it was discussed whether the temperature range for incubation of non-selective media could be made broader (34-38 C, instead of 37 C ± 1 C). This to (i) be able to use less stringent incubators and (ii) to have better harmonisation with methods used in (e.g.) USA and Canada. At the annual meeting in 2013, the broadening of the temperature ranges for incubation of non-selective media for culturing different bacteria (not only Salmonella) was agreed. However, after this agreement an additional question was raised during the drafting of pren DIS 6579-1, whether this broader
Page 15 of 47 temperature range can be used for the incubation of selective media as well. As this question did not only relate to the culturing of Salmonella, but may be relevant to other bacteria as well, it was agreed at the annual meeting in 2014, to raise an ad hoc group which will have a closer look at data from predictive microbiology from databases. The project leader from France presented the results at the annual meeting of SC9 in 2015 and it was agreed to draft a protocol to ask the members to perform some additional experiments. The help of the EURL-Salmonella was asked with the drafting (and testing) of the protocol. Several versions of the draft protocol were exchanged between the project leader in France and the EURL-Salmonella. In November 2015 a final draft version of the protocol was sent to the ad hoc group. Some first experiments following this protocol were performed at the laboratory of the EURL-Salmonella in January 2016. As a result of these first experiments it was decided to amend the protocol (and reporting form) again before sending it to the members of ISO-SC9 and CEN-WG6. ISO/34/SC9 WG4 Revision of ISO/TS 22117 Proficiency testing (PT) ISO/TS 22117 was published in 2010 and it was decided in 2014 to revise the document for several reasons (e.g. to make it a full standard, to include PT schemes for viruses, parasites, primary production, yeasts and moulds, and molecular methods). The involvement of the EURLs in this working group is considered important as they have much experience with the organisation of PT schemes. In October 2015 a meeting of WG4 was organized in London, UK to make a start with the revision of the document. Kirsten Mooijman of the EURL-Salmonella participated in this meeting. ISO/TC34/SC9 WG8 Revision of ISO 6887 Sample preparation series From November 2013 to April 2014, the DIS voting of four parts of ISO 6887 took place. Kirsten Mooijman is partly involved in drafting of the documents, especially in relation to pooling and compositing of samples (part 1) and for sample preparation of specific products like cocoa and acid and acidifying products. The preparation of the latter products is currently described in ISO 6579 (for detection of Salmonella) and is moved to the new part 4 of ISO 6887. After the DIS voting, further input was given for updating the different parts of ISO 6887 in 2014 and 2015. It is expected that the FDIS versions of the four parts will be launched early 2016. CEN/TC275/WG6 TAG 9 on pre-enrichment In 2012 a Technical Advisory Group (TAG) was raised in CEN on the improvement of the pre-enrichment step to enhance the recovery of Gram-negative bacteria. For the detection of several Gram-negative bacteria like Salmonella, Cronobacter, STEC and Enterobacteriaceae a pre-enrichment step is included in the procedure. As convenor of the two groups for Salmonella, Kirsten Mooijman has become member of this TAG 9. In 2014 no activities were performed, due to lack of a project leader. At the annual meeting of CEN-WG6 in June 2015 a new project leader was appointed and a first teleconference was organised in December 2015 to restart the activities. Other standardisation activities Within AOAC, an ISPAM (International Stakeholder Panel on Alternative Methods) working group on Harmonization of Salmonella methods was raised in January 2015. This group was formed to determine how and if the US and ISO reference methods for Salmonella can be harmonised. Members of this group are, amongst others, representatives from AOAC, FDA, USDA and Health Canada. Kirsten Mooijman of the EURL-Salmonella also participates in this working group as representative for the ISO working groups on Salmonella. The meetings of the group concern mainly teleconferences and most contacts are through e- mail. The group has organised four teleconferences in 2015. At first an overview was made of the different steps of four US methods and the ISO 6579 method. It was agreed to first collect available validation data of the methods and to determine if there are matrices for which the US and ISO Salmonella methods are statistically equivalent using the calculation tools of ISO 16140-2. In a few validation studies performed in the past, US methods as well as the ISO method have been tested. An attempt was made to use the raw data of these studies for comparing the methods following ISO 16140-2. Conclusions were most of the time hard to draw due to the difference in set-up of the studies and/or due to lack of data. It still needs to be decided what next steps can be done.
Page 16 of 47 Samples for interlaboratory comparison studies Samples for interlaboratory comparison study primary production, March 2015 For this study it was decided to use pig faeces as a matrix. In pre-studies, it was found that moulds and yeasts grew at the surface of the pig faeces when stored at 5 C or at 10 C. Unfortunately it was not possible to switch to chicken faeces for this study, due to an outbreak of Avian influenza in the Netherlands in Autumn 2014. Therefore, as an alternative, the pig faeces samples were stored at -20 C to prevent growth of yeasts and moulds. Samples of each 25 g were artificially contaminated with a diluted culture of monophasic Salmonella Typhimurium of pig origin. In the pre-studies the stability of the artificially contaminated samples (17 cfu/25 g and 70 cfu/25 g) were tested during a combination of long storage at -20 C (approximately 4 weeks) and short storage at 5 C (3-6 days), to mimic the conditions of the samples during transport and storage for the interlaboratory comparison study as much as possible. These pre-studies showed that still 2-3 of the 6 low contaminated samples and 5-6 of the 6 high contaminated samples could be tested positive for Salmonella after these storage conditions. Based on these results it was decided to use similar storage conditions for the samples of the interlaboratory comparison study, but to use somewhat higher contamination levels (to be sure that Salmonella could still be detected after storage): 84 cfu/25 g and 530 cfu/25 g (Table 1). The amount of background flora in the pig faeces was determined shortly after arrival at the laboratory of the EURL and in the week of the interlaboratory comparison study (after storage at -20 C). For this the total aerobic count (following ISO 4833-1) as well as the number of Enterobacteriaceae (following ISO 21528-2) was tested. The initial amount of background flora in the pig faeces was high and remained relatively high after storage at - 20 C (see Table 2). In the week of the study, the number of Salmonella in the samples after inoculation was determined by using an MPN format (Most Probable Number) of Annex D of ISO 6579. The MPN results are summarised in Table 1. Despite the good results of the pre-tests, Salmonella was unexpectedly more sensitive to freezing in the samples of the interlaboratory comparison study. The MPN results showed that in the low level faeces samples, no Salmonella could be detected, while in the high level samples only 0,7 cfu/25 g could be found (Table 1). This was confirmed by the results obtained by the participants. The sensitivity rates of the high and low level contaminated pig faeces samples were very low, especially for the low contaminated samples. Because of the instability of the Salmonella concentration in the samples it was not possible to evaluate the performance of the laboratories in this study. Table 1 Inoculum levels and MPN results of the artificially contaminated pig faeces samples used in the EURL-Salmonella PPS interlaboratory study, organised in March 2015 Date of testing Low level monophasic S. Typhimurium cfu/25 g (95% CI) High level monophasic S. Typhimurium cfu/25 g (95% CI) 12 February 2015 84 530 Inoculum of chicken faeces 16 March 2015 (date of the study) (storage for 5 weeks at -20 C, 1 day at 5 C and 6 days at -20 C) MPN of inoculated pig faeces (95 % confidence limit) 0 (0 0,7) 0,7 (0,2 2,2)
Page 17 of 47 Table 2 Amount of background flora in the pig faeces, tested immediately after receipt and during the interlaboratory comparison study in March 2015 (after storage at -20 C) Date of testing Number of aerobic bacteria cfu/g Number of Enterobacteriaceae cfu/g 9 February 2015 1.5 x 10 9 4.7 x 10 6 16 March 2015, after storage at -20 C 1.2 x 10 7 1.3 x 10 3 Samples for interlaboratory comparison study Food, September 2015 For the study on detection of Salmonella in a food matrix in 2015 it was decided to evaluate the possibility to use whole liquid egg as matrix. This matrix was not used before in an interlaboratory comparison study of the EURL-Salmonella, therefore it was necessary to perform some experiments on the stability of Salmonella, as well as on the background flora in the egg samples after storage. Samples of pasteurised whole liquid egg of 25 g each were artificially contaminated with a diluted culture of Salmonella Enteritidis (originating from chicken products) at a low and a high level (10-20 cfu/25 g and 50-100 cfu/25 g). The samples were stored at +5 C and at +10 C for several weeks, to test the stability of the contaminated samples. Furthermore, like for the former studies, also the amount of background flora was tested. After two weeks of storage at both temperatures, all samples were still positive for Salmonella and the amount of background flora slightly increased. Eventually, the following samples with a diluted culture of Salmonella Enteritidis were prepared at the EURL-Salmonella for the interlaboratory comparison study on detection of Salmonella in whole liquid egg: Portions of 25 g (pasteurised) whole liquid egg were each added to a plastic bag. Each sample was, individually, artificially contaminated with a diluted culture of: - Salmonella Enteritidis at a contamination level of approximately 20 cfu/25 g, or - Salmonella Enteritidis at a contamination level of approximately 100 cfu/25 g. In the week of the study, the number of Salmonella in the samples was determined by using an MPN format (Most Probable Number) of Annex D of ISO 6579. The MPN results are summarised in Table 3. The amount of background flora in the whole liquid egg was determined shortly after arrival at the laboratory of the EURL and in the week of the interlaboratory comparison study (after storage at +5 C). The results are summarized in Table 4. Table 3 Inoculum levels and MPN results of the artificially contaminated whole liquid egg samples used in the EURL-Salmonella Food interlaboratory study, organised in September 2015 Date of testing 10 September 2015 Inoculum of whole liquid egg 21 September 2015 (date study) MPN of egg, inoculated with S. Enteritidis (95 % confidence limit) after 2 weeks of storage at +5 C Low level S. Enteritidis cfu/25 g (95% CI) 21 101 35 (11-110) >65 High level S. Enteritidis cfu/25 g (95% CI) Table 4 Amount of background flora in the whole liquid egg, tested immediately after receipt and during the interlaboratory comparison study in September 2015 (after storage at +5 C) Date of testing Number of aerobic bacteria cfu/g 8 September 2015 1 x 10 1 <10 Number of Enterobacteriaceae cfu/g 22 September 2015 (date study), after 2 weeks of storage at +5 C 5 x 10 3 <10
Page 18 of 47 Samples for interlaboratory comparison study primary production, February 2016 For the study on detection of Salmonella in samples from the primary production stage (pps) to be organised in spring 2016, it was decided to use again boot socks to which chicken faeces is adhered. This type of samples has also been used in the interlaboratory comparison study for pps in 2013. In this latter study the samples were artificially contaminated with Salmonella Typhimurium (ATCC 14028), resulting in sufficient stable materials for the interlaboratory comparison study. As the results of the pps study of 2015 could not be used to analyse the performance of the laboratories it was considered important for the 2016 study to choose samples which have proven to be stable in the past. The pre-studies performed in fall 2015, showed that the low contaminated (approximately 10 cfu/25 g) as well as the high contaminated (approximately 65 cfu/25 g) samples could still be tested positive (5/5) after 2 weeks of storage at +5 C, as well as at +10 C. These results give confidence in the interlaboratory comparison study for pps in 2016. 4. Giving assistance to the Commission and ad hoc activities Several questions were received from several parties (European Commission DG-Sante, NRLs-Salmonella, European Food Safety Authority - EFSA, European Centre for Disease Prevention and Control ECDC, and other institutes inside and outside the EU) on the following subjects (list not exhaustive): - methods (qualitative and quantitative, classical, molecular); - validation of (alternative) methods, determining performance characteristics; - confirmation and typing of Salmonella (biochemical, serotyping, molecular typing); - pooling of samples; - ISO and CEN activities; - artificial contamination of samples; - information on thermologgers for mailing samples; - confirmation of strains (biochemically/serotyping) of NRLs-Salmonella; - determining distinction between vaccine strains and wild strains; - requests from NRLs-Salmonella for specific serotypes for use in experiments/quality control; - costs of molecular typing of Salmonella; - comments on the revised draft EC Regulation 2073/2005; - comments on draft OIE Terrestrial manual 2016, chapter on Salmonella. On average the EURL-Salmonella received 5-10 questions every week, varying from simple to complex. All questions were answered as quickly as possible. Depending on the complexity of the questions, answers could be given immediately by the experts of the EURL-Salmonella, or further information was gained from other experts (inside or outside the RIVM) or from literature. Regularly the EURL receives requests from laboratories for participation in the comparative tests and/or in the EURL workshops or trainings. If these questions come from non-nrl laboratories, most of the time the EURL rejects these requests because of lack of capacity. During the workshop in May 2014 an NRL-Salmonella indicated to face a problem in their country with the interpretation of several EU regulations in relation to monophasic Salmonella Typhimurium. In these regulations, the O:1 in the antigenic formula of this serotype is not underlined (1,4,[5],12:i:-). In the relevant country this has been interpreted as meaning that no measures are needed in case O:1 is not found. EC DG- Sante indicated that this concerns an editorial error and that the legislation is intended for all variants of monophasic Salmonella Typhimurium. To correct this, EC DG-Sante has drafted a letter, for which the EURL has given technical advice to the content in 2014. In 2015 this information was summarised (by DG-Sante) in corrigenda to the relevant Regulations. By the end of 2014 the EFSA working group on the collection of molecular typing data from food and animal isolates finished its activities. For the continuation of the activities in relation to the EFSA database for molecular typing data, a steering committee was raised. Members of this steering committee are from EFSA, ECDC, EURLs for Salmonella, STEC,