ISO 10272 Detection and enumeration of

Campylobacter in food and animal feeding stuffs

- Revision -

Enne de Boer

AHG Campylobacter

Revision EN ISO 10272-1:2006 & ISO/TS 10272-2:2006

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ISO/TC 34/SC 9 meeting in Valencia (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.

Ad Hoc Group Campylobacter

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Belgium: Nadine Botteldoorn

France: Marie-José Laisney

Germany: Kerstin Stingl

Italy: Vincenza Prencipe, Elisabetta Di Giannatale

Netherlands: Wilma Jacobs-Reitsma, Ida Jongenburger,

Enne de Boer

Sweden: Ingrid Hansson

UK: Janet Corry, Mary Howell, Ana Vidal, John Rodgers

Ad Hoc Group Campylobacter

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17/18 April 2012 NVWA Utrecht (NL)

Ad Hoc Group Campylobacter - proposals

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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 (2010): OK

Ad Hoc Group Campylobacter - proposals

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Enrichment

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 2nd medium

Ad Hoc Group Campylobacter - proposals

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Enrichment

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

Ad Hoc Group Campylobacter - proposals

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Enrichment

Proposal: choice of two enrichment procedures depending on the matrix

ISO/TC 34/SC 9 meeting, Buenos Aires (2010): OK

Ad Hoc Group Campylobacter - proposals

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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 (2010): Make further investigation concerning the interest of using two different media for the isolation step

Ad Hoc Group Campylobacter - proposals

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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

Ad Hoc Group Campylobacter - proposals

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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): OK

Ad Hoc Group Campylobacter - proposals

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Identification tests (optional)

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.

Ad Hoc Group Campylobacter CEN/TC 275/WG 6 meeting, 2011,Bournemouth, UK

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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

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.

Microaerobic incubation

Earlier proposal:

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.

Final proposal:

Delete the note, as it is impossible to standardize microaerobic incubation by using bottles with reduced headspace.

Questions from CEN/TC 275/WG 6

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- Optimum incubation time for Preston broth – 24 h

- Ratio of headspace to enrichment volume – no mention of the use 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 Campylobacter species – not clear at the moment

- Should skin or pieces of meat be separated from enrichment broth by the use of filter bags – better in e.g. ISO 6887

ISO 10272-4: Samples from primary production (from animals and their environment)

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)

Samples from primary production: - Detection by

direct plating (e.g. caecal samples) acc. ISO 10272-1B ISO 10272-1A or ISO 10272-1B (depending on contaminating microflora)

- Enumeration using ISO 10272-2

ISO 10272-1: detection proposal

- Is there a need for a separate part 4 for the analysis of samples from primary production?

- The parallel use of enrichment in Preston broth and direct plating as described in ISO 10272-1B is not very practical

ISO 10272-1: detection proposal

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ISO 10272-1A: Detection of Campylobacter by enrichment in products with low numbers of campylobacters and low level of background microflora and/or with stressed campylobacters, e.g. cooked or frozen products

ISO 10272-1B: Detection of Campylobacter by enrichment in products with low numbers of campylobacters and high level of background microflora , e.g. raw meats or raw milk

ISO 10272-1C: Detection of Campylobacter by direct plating in products with high numbers of campylobacters, e.g. faeces, poultry caecal contents or raw poultry meat.

ISO 10272-1A: detection proposal

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ISO 10272-1B: detection proposal

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ISO 10272-1C: detection proposal

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ISO 10272-1C: detection proposal

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Direct plating on selective agar

-For caecal of faecal samples use a loop or a sterile swab to bring some of the well-mixed sample material onto the first half of a mCCDA plate. Use another loop to streak out on the second half 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, and either streak the plate using a loop, or dispense a suitable volume and spread it over the mCCDA plate.

Further development of revised ISO 10272-1 + 2

The 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 the options 1A, 1B and 1C of ISO 10272-1

- to launch a NWIP for a full EN ISO standard (not TS) for ISO 10272-2

The voting period (3 months) will start in November 2012.

Any technical comments should be submitted at this stage.

Validation of revised ISO 10272-1 + 2

If the NWIP will be approved, then validation by interlaboratory studies can start (CEN mandate M/381)

In the current planning the first validation study will be in early spring 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 attention

enne.de.boer@vwa.nl