ISO 10272 – Horizontal method for detection and ... · PDF fileRevision of ISO 10272...
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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.
Revision ISO 10272, Uppsala, 2009 3
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):
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.
Revision ISO 10272, Uppsala, 2009 4
Revision of EN ISO 10272-1:2006 and ISO/TS 10272-2:2006
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
Revision ISO 10272, Uppsala, 2009 5
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
Revision ISO 10272, Uppsala, 2009 6
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
Revision ISO 10272, Uppsala, 2009 7
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
Revision ISO 10272, Uppsala, 2009 8
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
Revision ISO 10272, Uppsala, 2009 9
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
Revision ISO 10272, Uppsala, 2009 10
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
Revision ISO 10272, Uppsala, 2009 11
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
Revision ISO 10272, Uppsala, 2009 12
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
Revision ISO 10272, Uppsala, 2009 13
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
Revision ISO 10272, Uppsala, 2009 14
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
Revision ISO 10272, Uppsala, 2009 15
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
Revision ISO 10272, Uppsala, 2009 16
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 - ?
Revision ISO 10272, Uppsala, 2009 17
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
Revision ISO 10272, Uppsala, 2009 18
ISO 10272 – revision- microaerobic incubation -
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
Revision ISO 10272, Uppsala, 2009 19
ISO 10272 – revision- microaerobic incubation -
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
Revision ISO 10272, Uppsala, 2009 20
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
Revision ISO 10272, Uppsala, 2009 21
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
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Revision ISO 10272, Uppsala, 2009 22
ISO 10272 - confirmation -
Confirmation tests value
- Morphology/Motility +++
- Oxidase +++
- Microaerobic growth at 25ºC ++ (?)
- Aerobic growth at 41,5ºC ++ (?)
Revision ISO 10272, Uppsala, 2009 23
ISO 10272 - confirmation -
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
Revision ISO 10272, Uppsala, 2009 24
ISO 10272 - confirmation: aerobic growth -
Photos: Wilma Jacobs-Reitsma
Revision ISO 10272, Uppsala, 2009 25
ISO 10272 - confirmation: aerobic growth -
Proposal: delete ‘Aerobic growth at 41,5ºC’ as a confirmation test
Revision ISO 10272, Uppsala, 2009 26
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
Revision ISO 10272, Uppsala, 2009 27
ISO 10272 - confirmation -
Other tests may be useful for
confirmation and identification
- latex agglutination tests
Microscreen (Lucron),
INDX-Campy (Biotrading),
Dryspot Campylobacter Test (Oxoid),
BBL-Campyslide (BD)
……………………
Revision ISO 10272, Uppsala, 2009 28
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
……………………………………
Revision ISO 10272, Uppsala, 2009 29
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
Revision ISO 10272, Uppsala, 2009 30
ISO 10272
Thank you for your attention and your
contribution to a better standard method
ISO 10272!