Revision ISO 10272 Detection and enumeration of ... - SVA

ISO 10272Detection and enumeration ofCampylobacterin food and animal feeding stuffs- Revision -Enne de BoerAHG Campylobacter

Revision EN ISO 10272-1:2006 & ISO/TS 10272-2:2006ISO/TC 34/SC 9 meeting in Valencia (2009):1. SC9 members decided to launch a revision of EN ISO10272-1:2006 to improve the enrichment and confirmationsteps and microaerobic incubation conditions.2. SC9 members decided to transform ISO/TS 10272-2:2006into an EN ISO standard including a modification ofconfirmation steps.2

Ad Hoc Group CampylobacterBelgium: Nadine BotteldoornFrance: Marie-José LaisneyGermany: Kerstin StinglItaly: Vincenza Prencipe, Elisabetta Di GiannataleNetherlands: Wilma Jacobs-Reitsma, Ida Jongenburger,Enne de BoerSweden: Ingrid HanssonUK: Janet Corry, Mary Howell, Ana Vidal, John Rodgers3

Ad Hoc Group Campylobacter17/18 April 2012NVWAUtrecht (NL)4

EnrichmentAd Hoc Group Campylobacter - proposalsProposal: choice of two enrichment procedures depending on thematrixISO 10272-1A: detection of Campylobacter in foods with lowbackground count of non-campylobacters and/or with stressedcampylobacterse.g. cooked or frozen products contaminated with CampylobacterProcedure: enrichment in Bolton broth, microaerobic incubation 4-6 h at 37ºC, 40-48 h at 41,5ºC;isolation on mCCDA and a 2 nd medium6

EnrichmentAd Hoc Group Campylobacter - proposalsProposal: choice of two enrichment procedures depending on thematrixISO 10272-1B: detection of Campylobacter in foods with highbackground count of non-campylobacterse.g. raw chicken, raw meats, raw milkProcedure:1) enrichment in Preston broth, microaerobic incubation 24 h at41,5ºC; isolation on mCCDA2) direct plating from sample homogenate on mCCDA7

EnrichmentAd Hoc Group Campylobacter - proposalsProposal: choice of two enrichment procedures depending on thematrixISO/TC 34/SC 9 meeting, Buenos Aires (2010): OK8

Ad Hoc Group Campylobacter - proposalsIsolation mediumISO 10272-1:2006 - It is preferable to take a second isolationmedium that is based on a principle different from mCCDA.Examples of isolation media to be used are Skirrow agar,Karmali agar and Preston agarMost of the current available alternatives for mCCDA are notprincipally different, and the use of these media does not havean added value in most cases.ISO/TC 34/SC 9 meeting, Buenos Aires (2010): Make furtherinvestigation concerning the interest of using two differentmedia for the isolation step9

Ad Hoc Group Campylobacter - proposalsEnumeration (ISO 10272-2)Plating of initial suspension and decimal dilutions according to ISO7218:2007, so not in duplicateRevise the counting of low numbers. When low counts areexpected: 1 ml of initial suspension on a large or 3 small agardishes, in duplicate10

Ad Hoc Group Campylobacter - proposalsConfirmation tests- Delete: “Aerobic growth at 41,5ºC”, as some campylobactersshow limited growth at 41,5ºC- Change “Microaerobic growth at 25ºC” in “Aerobic growth at25ºC”- Microscopic examination can be done directly from colonies onblood agar or mCCDA; suspension in Brucella broth is notneededISO/TC 34/SC 9 meeting, Buenos Aires (June 2010): OK11

Ad Hoc Group Campylobacter - proposalsIdentification tests (optional)Delete the antibiotic sensitivity tests for nalidixic acid andcephalothin, because of increased resistance of some species forthese antibioticsAdd a recommendation to use alternative confirmation tests, suchas PCR, immunological tests, microarray, etc.12

Ad Hoc Group CampylobacterCEN/TC 275/WG 6 meeting, 2011,Bournemouth, UKCEN/TC275/WG6 asked the project leader to prepare the finaldrafts for parts 1 and 2 for the next CEN/TC275/WG6 meetingand 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 Campylobacterspecies- Should skin or pieces of meat be separated from enrichmentbroth by the use of filter bags13

Microaerobic incubationmicroaerobic atmosphere withoxygen content 5 % 2 %carbon dioxide 10 % 3 %optional hydrogen ≤10 %with the balance nitrogenISO 10272-1:20066.14NOTE 2As an alternative to incubation in a microaerobic atmosphere, theenrichment can be incubated in screw-capped bottles or flasks filledwith enrichment broth, leaving a headspace of less than 2 cm, andtightly closing the caps.

Earlier proposal:Microaerobic incubationNOTE 2 As an alternative to incubation in a microaerobicatmosphere, the enrichment can be done in tightly closed containersfilled with enrichment broth, leaving a headspace of about 20% of thetotal volume of the container.Proposed text:NOTE 2As an alternative to incubation in a microaerobic atmosphere, theenrichment can be done in tightly closed containers filled withenrichment broth and having a reduced headspace.This alternative should only be applied, when there is sufficientevidence of the creation of correct microaerobic conditions.Final proposal:Delete the note, as it is impossible to standardize microaerobic incubationby using bottles with reduced headspace.

Questions from CEN/TC 275/WG 6- Optimum incubation time for Preston broth – 24 h- Ratio of headspace to enrichment volume – no mention of theuse of reduced headspace- Addition of blood (0, 1 or 5%) to enrichment media – Bolton +5% blood + FBP; Preston broth + 5% blood- Do Preston or Bolton broth select for different Campylobacterspecies – not clear at the moment- Should skin or pieces of meat be separated from enrichmentbroth by the use of filter bags – better in e.g. ISO 688716

ISO 10272-4: Samples from primary production(from animals and their environment)For the development of part 4 of EN ISO 10272, CEN/TC275/WG6asked the Project Leader to liaise with TAG5 (meeting AHVLA,20/21 October 2011)Samples from primary production:- Detection bydirect plating (e.g. caecal samples) acc. ISO 10272-1BISO 10272-1A or ISO 10272-1B (depending oncontaminating microflora)- Enumeration using ISO 10272-2

ISO 10272-1: detectionproposal- Is there a need for a separate part 4 for the analysis ofsamples from primary production?- The parallel use of enrichment in Preston broth and directplating as described in ISO 10272-1B is not very practical

ISO 10272-1: detectionproposalISO 10272-1A: Detection of Campylobacter by enrichment inproducts with low numbers of campylobacters and low level ofbackground microflora and/or with stressed campylobacters, e.g.cooked or frozen productsISO 10272-1B: Detection of Campylobacter by enrichment inproducts with low numbers of campylobacters and high level ofbackground microflora , e.g. raw meats or raw milkISO 10272-1C: Detection of Campylobacter by direct plating inproducts with high numbers of campylobacters, e.g. faeces,poultry caecal contents or raw poultry meat.19

20ISO 10272-1A: detectionproposal

21ISO 10272-1B: detectionproposal

22ISO 10272-1C: detectionproposal

Direct plating on selective agarISO 10272-1C: detectionproposal-For caecal of faecal samples use a loop or a sterile swab tobring some of the well-mixed sample material onto the first halfof a mCCDA plate. Use another loop to streak out on the secondhalf of the plate.-For all other samples, add an appropriate amount of liquid (e.g.MRD or Preston broth), for example 1 to 1 w/w, mix well, andeither streak the plate using a loop, or dispense a suitablevolume and spread it over the mCCDA plate.23

Further development of revised ISO 10272-1 + 2The drafts have been discussed during CEN/TC275/WG6 meeting,Brussels (June 2012) and it was decided- to launch a NWIP (new work item proposal) vote for theoptions 1A, 1B and 1C of ISO 10272-1- to launch a NWIP for a full EN ISO standard (not TS) for ISO10272-2The voting period (3 months) will start in November 2012.Any technical comments should be submitted at this stage.

Validation of revised ISO 10272-1 + 2If the NWIP will be approved, then validation by interlaboratorystudies can start (CEN mandate M/381)In the current planning the first validation study will be in earlyspring 2013 and will be on ISO 10272-2 (enumeration)Projectleaders: Enne de Boer (part 1), Wilma Jacobs (part 2)in collaboration with EURL Campylobacter

Thank you for your attentionenne.de.boer@vwa.nl