ISO 10272 – Horizontal method for detection and ... · PDF fileRevision of ISO 10272 part 1 and 2 Detection and enumeration of Campylobacter Enne de Boer Food and Consumer Product

Revision of ISO 10272 part 1 and 2Detection and enumeration of Campylobacter

Enne de Boer

Food and Consumer Product Safety Authority (VWA)

The Netherlands

Revision ISO 10272, Uppsala, 2009 2

Revision of EN ISO 10272-1:2006 and 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.



Resolution 396 taken at the ISO/TC 34/SC 9 meeting in Valencia (May 2009):

3. Enne de Boer (NL) is nominated as project leader and Ingrid Hansson

(CRL Campylobacter) as co-project leader. The secretariat is asked to

launch a call for experts before June 2009 to constitute the ad hoc group

and is asked to launch an enquiry in parallel to collect proposals and data

for the revision of part 1. The ad hoc group will meet in October 2009

before or after the CRL Campylobacter workshop.



Ad Hoc Group Campylobacter

Belgium: Nadine Botteldoorn

France: Marie-José Laisney

Italy: Vincenza Prencipe

Netherlands: Wilma Jacobs-Reitsma, Enne de Boer

Spain: Belen Moreno

Sweden: Ingrid Hansson

Thailand: Sasitorn Kanarat, Narit Lerkmoung

UK: Clare Aldus, Janet Corry, Alastair Thomas, Bob Madden


EN ISO 10272-1

Campylobacter

microorganisms forming characteristic

colonies on solid selective media when

incubated microaerobically at 41,5ºC

but not at 25ºC, 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


EN ISO 10272-1

ISO 10272-1, first edition, 2006

Microbiology of food and animal

feeding stuffs – Horizontal method

for detection and enumeration of

Campylobacter spp.

Part 1: Detection method

Enrichment: Bolton broth

4-6h 37ºC, 40-48h 41,5ºC

microaerobic

Plating:

-mCCD agar

-2nd medium of own choice


ISO 10272 – considerations for revision- enrichment medium -

ISO 10272-1 is not optimal for the detection of Campylobacter in all

matrices

Bolton broth is not sufficient selective for several matrices, e.g. chicken

products

The incubation conditions (time/temperature) are not optimal for all

matrices


Thermotolerant Campylobacter- enrichment media -

broth selective agents

Preston polymyxin B, rifampicin, trimethoprim, cycloheximide

Bolton cefoperazone (20 mg/L), vancomycin, trimethoprim, amphotericin B

Park & Sanders cefoperazone (32 mg/L), vancomycin, trimethoprim, cycloheximide

Exeter cefoperazone (15 mg/L), polymyxin B, vancomycin, amphotericin B

Hunt & Radle cefoperazone (15 mg/L), trimethoprim, vancomycin, cycloheximide


Thermotolerant Campylobacter- enrichment -

The choice of the enrichment medium will depend on

-matrix to be tested

-microflora present

- numbers and state of the campylobacters

- types and numbers of other microflora

-incubation temperature and period

-choice of isolation medium


Thermotolerant Campylobacter- enrichment -

No single enrichment medium is optimal for the detection of Campylobacter

in all types of food

Especially the contamination with other microflora in the product to be

analyzed will determine the preferred enrichment medium

Bolton broth and Preston broth seem to be the most suitable media for the

detection of Campylobacter in foods

Preston broth is more selective than Bolton broth


ISO 10272-1 – revision- enrichment -

ISO 17995:2005 – Water quality – Detection and enumeration of

thermotolerant Campylobacter species

Parallel enrichment in Preston broth and Bolton broth

NOTE If the available sample size is limited, the most appropriate

enrichment broth should be used. For waters with expected high

background count, it is more appropriate to use Preston broth, and for

clean waters or where the background count is likely to be low Bolton broth

is more appropriate


ISO 10272-1 – revision- enrichment -

Proposal:

ISO 10272-1A: detection of Campylobacter in foods with low background

count of non-campylobacters and/or with stressed campylobacters

ISO 10272-1B: detection of Campylobacter in foods with high background

count of non-campylobacters


ISO 10272 – proposal for revision- enrichment -

ISO 10272-1A: detection of Campylobacter in foods with low background

count and/or with stressed campylobacters

e.g. cooked or frozen products contaminated with Campylobacter

Bolton broth, incubation 4-6h 37ºC, 40-48h 41,5ºC, microaerobic

Isolation on mCCD agar and a 2nd agarmedium


ISO 10272 – proposal for revision- enrichment -

ISO 10272-1B: detection of Campylobacter in foods with high background

count

e.g. raw chicken products, raw meats, raw milk

- Preston broth, incubation 24h at 41,5ºC, microaerobic; isolation on

mCCDA

- direct plating from sample homogenate on mCCD agar


ISO 10272 – considerations for revision- isolation medium -

-There is no major disagreement on the use of mCCD agar as the first

selective isolation medium

-The second medium should be based on a principle different from

mCCD agar to increase the chance of detecting campylobacters

-Most of the current available alternatives for mCCD agar are not

principally different, and the use of these media does not have an added

value in most cases

-Combine mCCD agar with a medium not containing cefoperazone,

such as Butzler agar or Preston agar


Campylobacter- isolation media -

agar medium blood/charcoal antibiotics

Skirrow horse blood Van, Trim, Polym

Butzler horse blood Bac, Col, Cez, Nov, Cycl

Preston horse blood Polym, Rif, Trim, Cycl

mCCD charcoal Cefop, AmB

Karmali charcoal Cefop, Van, Cycl

Abeyta-Hunt-Bark - Cefop, Rif, AmB

Campy Food ID horse serum Cefop, Van, AmB

Brilliance Campylobacter - ?


ISO 10272 – revision- microaerobic incubation -

Microaerobic atmosphere – different options

-a cabinet which automatically controls the gaseous atmosphere

-filling of jars with the appropriate gas mixture

-use of jars and gas-generating kits



small headspace in srew-capped bottles

The exact atmosphere developing in

a closed bottle will be a consequence

of many factors such as the chemical

nature of the sample, the shape of the

bottle, the depth of headspace and the

metabolic activity of the microflora present

Moran et al, LAM 48 (2009) 628-632



Addition of blood, charcoal, haemin and FBP (ferro sulfate, sodium

meta bisulfite, sodium pyruvate) will neutralise the toxic effect of

oxygen, hydrogen peroxide and superoxide ions


ISO/TS 10272-2

ISO/TS 10272-2, first edition, 2006

Microbiology of food and animal feeding stuffs – Horizontal method

for detection and enumeration of Campylobacter spp.

Part 2: Colony-count technique

Colony counting on mCCD agar


ISO 10272-2 – revision

Plating of initial suspension and decimal dilutions according to ISO

7218:2007, so not in duplicate

When low counts expected: 1 ml of initial suspension on a large or 3

small agar dishes, in duplicate

dxxxdxx

aN

nn VV 22111,0


ISO 10272 - confirmation -

Confirmation tests value

- Morphology/Motility +++

- Oxidase +++

- Microaerobic growth at 25ºC ++ (?)

- Aerobic growth at 41,5ºC ++ (?)



Microscopic examination

ISO 10272-1, 9.4.1.1: Suspend one colony from the Columbia

blood agar plate in 1 ml of Brucella broth and examine for

morphology and motility using a microscope

Proposal: microscopy directly from the selective plate


ISO 10272 - confirmation: aerobic growth -

Photos: Wilma Jacobs-Reitsma


ISO 10272 - confirmation: aerobic growth -

Proposal: delete ‘Aerobic growth at 41,5ºC’ as a confirmation test


ISO 10272 - identification -

Identification of species value

- Catalase +

- Hippurate +++

- Indoxyl acetate +++

- Nalidixic acid -

- Cephalothin +

Proposal: delete the antibiotic sensitivity tests for nalidixic acid and cephalothin



Other tests may be useful for

confirmation and identification

- latex agglutination tests

Microscreen (Lucron),

INDX-Campy (Biotrading),

Dryspot Campylobacter Test (Oxoid),

BBL-Campyslide (BD)

……………………


ISO 10272 - identification -

Other tests for confirmation and identification

PCR

microarray

O.B.I.S. campy kit (Gram-lysis and L-alanyl aminopeptidse) (Oxoid)

immunological tests

……………………………………


ISO 10272 – revision- need for additional data -

Request for results of comparative methodology studies

Please provide us with data of earlier performed studies

Call for participation to a comparative study to be organized by the

Ad Hoc Working Group in the next half year


ISO 10272

Thank you for your attention and your

contribution to a better standard method

ISO 10272!

[email protected]

Documents

ISO 10272 – Horizontal method for detection and ... · PDF fileRevision of ISO 10272 part 1 and 2 Detection and enumeration of Campylobacter Enne de Boer Food and Consumer Product