ISO 10272 revision and further development Enne de Boer on behalf of the working group EURL - congratulations with the first 5 years and the approval! EURL Campylobacter 6th Workshop Uppsala, 3-5 October 2011
Revision EN ISO 10272-1:2006 & ISO/TS 10272-2:2006 Resolution 396 taken at the ISO/TC 34/SC 9 meeting in Valencia (May 2009): 1. SC9 members decided to launch a revision of EN ISO 10272-1:2006 to improve the enrichment and confirmation steps and microaerobic incubation conditions. 2. SC9 members decided to transform ISO/TS 10272-2:2006 into an EN ISO standard including a modification of confirmation steps. 2
Revision EN ISO 10272-1:2006 & ISO/TS 10272-2:2006 Ad Hoc Group Campylobacter Belgium: Nadine Botteldoorn France: Marie-José Laisney Italy: Vincenza Prencipe, Elisabetta Di Giannatale Netherlands: Wilma Jacobs-Reitsma, Enne de Boer Sweden: Ingrid Hansson UK: Janet Corry, John Rodgers 3st meeting, 3 October 2011, SVA, Uppsala 3
Ad Hoc Group Campylobacter - proposals Title and definition Campylobacter microorganisms forming characteristic colonies on solid selective media when incubated microaerobically at 41,5ºC and which possess the characteristic motility and biochemical and growth properties described when the tests are conducted in accordance with this part of ISO 10272 Proposal: maintain as in current standard ISO/TC 34/SC 9 meeting, Buenos Aires (June 2010) Resolution 448-1: OK 4
Enrichment Ad Hoc Group Campylobacter - proposals Proposal: choice of two enrichment procedures depending on the matrix ISO 10272-1A: detection of Campylobacter in foods with low background count of non-campylobacters and/or with stressed campylobacters e.g. cooked or frozen products contaminated with Campylobacter Procedure: enrichment in Bolton broth, microaerobic incubation 4-6 h at 37ºC, 40-48 h at 41,5ºC; isolation on mccda and a 2 nd medium 5
Enrichment Ad Hoc Group Campylobacter - proposals Proposal: choice of two enrichment procedures depending on the matrix ISO 10272-1B: detection of Campylobacter in foods with high background count of non-campylobacters e.g. raw chicken, raw meats, raw milk Procedure: 1) enrichment in Preston broth, microaerobic incubation 24 h at 41,5ºC; isolation on mccda 2) direct plating from sample homogenate on mccda 6
Enrichment Ad Hoc Group Campylobacter - proposals Proposal: choice of two enrichment procedures depending on the matrix ISO/TC 34/SC 9 meeting, Buenos Aires (June 2010) Resolution 448-2: OK 7
Ad Hoc Group Campylobacter - proposals Isolation medium ISO 10272-1:2006 - It is preferable to take a second isolation medium that is based on a principle different from mccda. Examples of isolation media to be used are Skirrow agar, Karmali agar and Preston agar Most of the current available alternatives for mccda are not principally different, and the use of these media does not have an added value in most cases. ISO/TC 34/SC 9 meeting, Buenos Aires (June 2010) Resolution 448-3: Make further investigation concerning the interest of using two different media for the isolation step 8
Ad Hoc Group Campylobacter - proposals Enumeration (ISO 10272-2) Plating of initial suspension and decimal dilutions according to ISO 7218:2007, so not in duplicate Revise the counting of low numbers. When low counts are expected: 1 ml of initial suspension on a large or 3 small agar dishes, in duplicate 9
Ad Hoc Group Campylobacter - proposals Confirmation tests - Delete: Aerobic growth at 41,5ºC, as some campylobacters show limited growth at 41,5ºC - Change Microaerobic growth at 25ºC in Aerobic growth at 25ºC - Microscopic examination can be done directly from colonies on blood agar or mccda; suspension in Brucella broth is not needed ISO/TC 34/SC 9 meeting, Buenos Aires (June 2010) Resolution 448-4: OK 10
Ad Hoc Group Campylobacter - proposals Identification tests Delete the antibiotic sensitivity tests for nalidixic acid and cephalothin, because of increased resistance of some species for these antibiotics Add a recommendation to use alternative confirmation tests, such as PCR, immunological tests, microarray, etc. 11
Ad Hoc Group Campylobacter CEN/TC 275/WG 6 meeting, June 2011,Bournemouth, UK Resolution N 265-1 CEN/TC275/WG6 asked the project leader to prepare the final drafts for parts 1 and 2 for the next CEN/TC275/WG6 meeting and to reply to the following questions: - Optimum incubation time for Preston broth - Ratio of headspace to enrichment volume - Addition of blood (0, 1 or 5%) to enrichment media - Do Preston or Bolton broth select for different Campylobacter species - Should skin or pieces of meat be separated from enrichment broth by the use of filter bags 12
Detection of Campylobacter observations Humphrey (UK) comparison of Bolton and Exeter broth for isolation of Campylobacter from bootsocks Conclusion: Exeter broth performs slightly better than Bolton broth Less overgrowth of isolation plates after Exeter enrichment John Rodgers (UK) Exeter broth better than Bolton broth and Preston broth for detection of Campylobacter in caecal samples. Exeter broth is very good for C. jejuni, but less good for C. coli. Deletion of polymyxin B resulted in better detection of C. coli. 13
Detection of Campylobacter observations Conclusion WG Not to include Exeter broth in the standard - Only experiences from some countries (UK) - Changing formulations 14
Detection of Campylobacter observations Moran et al. (UK) propose a modified Bolton broth with added potassium clavulanate to eliminate ESBLs for the detection of Campylobacter in raw chicken Lett. Appl. Microbiol. (2011), 52(6):614-618 Needs further evaluation by other laboratories 15
Observations: Proficiency Test 8 EURL Campylobacter Sample BB 48h, mccda PB 24h, mccda +/other +/other C. coli 34/- 31/- E. coli -/31 -/19 C. coli 34/- 32/- C. lari 34/- 31/- C. coli + E. coli 29/5 32/1 C. jejuni 34/- 33/- C. coli 34/- 28/- C. jejuni + E. coli 26/8 32/-
Observations: Proficiency Test 8 EURL Campylobacter Bolton broth more sensitive (higher number of Campylobacter-positive samples) than Preston broth Preston broth is clearly more selective than Bolton broth; higher sensitivity in the presence of contaminants (e.g. E. coli)
Detection of Campylobacter observations Wilma Jacobs-Reitsma (NL) Dutch standard for detection of Campylobacter in skin and meat of broilers Enrichment in Preston broth for 24 h Preston broth without blood, including FBP-supplement
Detection of Campylobacter observations Janet Corry (UK) Detection of Campylobacter in neck flaps Prepare a 1:1 suspension Homogenize in a Pulsifier Enrichment in Preston broth for 24h 24h or 48h incubation not significantly different Microaerobic incubation in stomacher bags much better than aerobic incubation
Detection of Campylobacter observations Marie-José Laisney (F) Detection of Campylobacter in chicken products Enrichment in Preston broth better than in Bolton broth 24h or 48h incubation not significantly different No difference in ratio C. jejuni/c. coli After incubation for 48h: shift to more C. coli Butzler agar was the best isolation medium
Microaerobic incubation microaerobic atmosphere with oxygen content 5 % 2 % carbon dioxide 10 % 3 % optional hydrogen 10 % with the balance nitrogen ISO 10272-1:2006 6.14 NOTE 2 As an alternative to incubation in a microaerobic atmosphere, the enrichment can be incubated in screw-capped bottles or flasks filled with enrichment broth, leaving a headspace of less than 2 cm, and tightly closing the caps.
Earlier proposal: Microaerobic incubation NOTE 2 As an alternative to incubation in a microaerobic atmosphere, the enrichment can be done in tightly closed containers filled with enrichment broth, leaving a headspace of about 20% of the total volume of the container. Proposed text: NOTE 2 As an alternative to incubation in a microaerobic atmosphere, the enrichment can be done in tightly closed containers filled with enrichment broth and having a reduced headspace. This alternative should only be applied, when there is sufficient evidence of the creation of correct microaerobic conditions.
Direct plating in part 1B Should parallel direct plating together with enrichment in Preston broth be maintained?
Choice between ISO 10272-1A and ISO 10272-1B 1A low background count of non-campylobacters and/or stressed campylobacters 1B high background count of non-campylobacters Not always easy to make a choice between the use of 1A or 1B (e.g. frozen chicken meat, vegetables)
Questions from CEN/TC 275/WG 6 - Optimum incubation time for Preston broth 24 h - Ratio of headspace to enrichment volume no mention of ratio; leaving option to use reduced headspace, but with validation - Addition of blood (0, 1 or 5%) to enrichment media Bolton + 5% blood; Preston broth w/o blood + FBP supplement - Do Preston or Bolton broth select for different Campylobacter species not clear at the moment - Should skin or pieces of meat be separated from enrichment broth by the use of filter bags to be advised 25
ISO 10272-4: Samples from primary production (from animals and their environment) Resolution N 265-2: For the development of part 4 of EN ISO 10272, CEN/TC275/WG6 asked the Project Leader to liaise with TAG5 (meeting AHVLA, 20/21 October 2011) Proposal of WG: Technical Specification - Detection by direct plating (e.g. caecal samples) ISO 10272-1A or ISO 10272-1B (depending on contaminating microflora) - Enumeration using ISO 10272-2
Further development of revised ISO 10272-1 + 2 Establishment of draft standards by the working group in April 2012 Discussion and agreement (?) during CEN/TC275/WG6 meeting, Brussels (June 2012) Start of validation by interlaboratory studies (CEN mandate M/381) in autumn 2012/winter 2013