Microsoft word - equivalence of iso and nmkl o157 methods.doc

Comparison of two methods for the detection of Escherichia coli serogroup O157 in foods and
feeding stuffs: ISO 16654:2001: Microbiology of food and animal feeding stuffs – Horizontal
method for the detection of Escherichia coli O157 and Nordic Committee on Food Analysis:
NMKL No 164, 2. Ed. 2005: Escherichia coli O157. Detection in food and feeding stuffs.
Jeppe Boel
Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V,
Denmark.
E-mail: jeb@dvfv.dk.
Date: 28 November 2006.

At the ISO/TC34 SC9 meeting in Prague in June 2006 it was decided in resolution N° 297 under
“NMKL – Cooperation agreement between NMKL and ISO/TC34 SC9” that “Jeppe Boel has to
check whether the NMKL method is equivalent to the ISO method”. The following document has
been written in response to this request.
ISO and NMKL have both published culture based reference methods for the detection of
Escherichia coli O157 in food and feeding stuffs. The two methods are very similar and both
methods relies on the same principles: 1: Enrichment in modified tryptic soy broth supplemented
with novobiocin (mTSBn), 2: separation and concentration by means of immunomagnetic particles
coated with antibodies to E. coli O157 (immuno-magnetic separation – IMS), 3: Isolation by
subculture onto two selective indicative isolation media (cefixime potassium supplemented Sorbitol
MacConkey (SMAC) agar as mandatory isolation media and the other isolation media is free of
choice), and 4: Confirmation of the E. coli O157. However there are also minor differences in the
methods. In Table 1 a list of important similarities and differences between the two methods are
given.
The NMKL method has been evaluated in a collaborative study (Normark, C (2005): Collaborative
study of method for detection of Escherichia coli O157 in food - NMKL No. 164, 1999.
SLV rapport 2/2005 (National Food Administration, Uppsala, Sweden)). The ISO method has not
been collaboratively evaluated. If the two methods are considered equivalent, this would mean, that
the collaborative validation of the NMKL method also could be considered as documentation of the
ISO method.
Table 1 Comparison of NMKL and ISO methods for the detection of Eschericia coli O157 in food and feed.

Step ISO
Comments
x g or x ml to 9 x ml of enrichment broth. 25 grams of food to 225 ml of enrichment broth. Incubate 6 hours at 41.5°C and further 12-18 hours Incubate the samples at 41.5±0.5°C for 6-8 hours and/or 18-24 hours for 6-8 h and/or 18-24 hours. IMS should be carried out after 6 hours and again, if IMS should be carried out after 6-8 h and again after necessary, after 12 hours to 18 hours of incubation Method relies on non-specified (commercially) Method relies on non-specified (commercially) problematic because of differences in the performance of different “bead systems” and the quality of the applied antibodies. In the Nordic countries most laboratories uses beads from Dynal (see www.invitrogene.com) Comment on the ISO method: when is it “necessary” to use IMS after both 6 and 18-24 hours of incubation. The affinity purified IMS bead-bacteria complex The affinity purified IMS bead-bacteria complex (2x50µl) is seeded on to mandatory media (CT-SMAC) (2x50µl) is seeded on to mandatory media (CT-SMAC) SMAC agar is very similar and a second isolation medium free of choice). and a second isolation medium free of choice). potassium tellurite concentration of 2.5 mg/l. potassium tellurite concentration of 2.5 mg/l. The bacteria-bead complex is streaked out with a The bacteria-bead complex is streaked out with a cotton swab on half of the plate and on the remaining cottonswab is that it helps “breaking down” aggregates of E. coli O157, thereby facilitating the distribution of the target organism on the agar plates. But it is also clear that a cotton swab absorbs some of the liquid (and bacteria), and thus reduces the seeded volume. CT-SMAC: 37°C for 18-24 hours, other isolation 37°C for 18-24 hours for both isolation media media at its recommended temperature and specified Description of the morphology of the typical sorbitol Description of the morphology of the typical sorbitol negative E. coli O157 on CT-SMAC. Other media negative E. coli O157 on CT-SMAC. Other media Up to five typical colonies from each of the isolation Test typical colonies (no number given). Seed onto media are seeded on to non-selective agar. The non-selective agar (e.g. blood agar). Colonies are tested procedure colonies are tested for indole formation in for indole formation in tryptone tryptophane media. tryptone/tryptophan medium. Indole forming isolates Indole forming isolates are investigated serologically are investigated serologically by slide agglutionation. by slide agglutionation. It is stated, that it is also possible to use slide agglutination tests on colonies that Further characterization: Send strain to reference Further characterization: Send strain to reference laboratory Control strain: Non VT producing E. coli O157 Control strain: Non VT producing E. coli O157, ATCC Recommends that spiked samples are used to obtain 43888. routine in reading the selective indicative agar plate. Recommends that spiked samples are used to obtain routine in reading the selective indicative agar plate.

Source: http://www.nmkl.org/Publikasjoner/Sammenlikning/Equivalence%20of%20ISO%20and%20NMKL%20O157%20methods.pdf