prEN ISO 13136-2

SLOVENSKI STANDARD
01-april-2024
Mikrobiologija v prehranski verigi - Odkrivanje prisotnosti, izolacija in
karakterizacija Escherichia coli (STEC), ki proizvaja Shiga toksin - 2. del:
Horizontalna metoda za karakterizacijo izolatov Escherichia coli (STEC), ki
proizvaja Shiga toksin (ISO/DIS 13136-1:2024)
Microbiology of the food chain - Detection, isolation and characterization of Shiga toxin-
producing Escherichia coli (STEC) - Part 2: Horizontal method for the characterization of
Shiga toxin-producing Escherichia coli (STEC) isolates (ISO/DIS 13136-2:2024)
Mikrobiologie der Lebensmittelkette - Nachweis, Isolierung und Charakterisierung von
Shiga-Toxin bildenden Escherichia coli (STEC) - Teil 2: Horizontales Verfahren zur
Charakterisierung von Shiga-Toxin bildenden Escherichia coli (STEC) Isolaten (ISO/DIS
13136-2:2024)
Microbiologie de la chaîne alimentaire - Détection, isolement et caractérisation des
Escherichia coli producteurs de Shigatoxines (STEC) - Partie 2: Méthode horizontale
pour la caractérisation d’isolats des Escherichia coli producteurs de Shigatoxines
(STEC) (STEC) (ISO/DIS 13136-2:2024)
Ta slovenski standard je istoveten z: prEN ISO 13136-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 13136-2
ISO/TC 34/SC 9
Microbiology of the food
Secretariat: AFNOR
chain — Detection, isolation and
Voting begins on:
characterization of Shiga toxin-
2024-02-28
producing Escherichia coli (STEC) —
Voting terminates on:
2024-05-22
Part 2:
Horizontal method for the
characterization of Shiga toxin-
producing Escherichia coli (STEC)
isolates
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
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Reference number
ISO/DIS 13136-2:2024(en)
DRAFT
ISO/DIS 13136-2:2024(en)
International
Standard
ISO/DIS 13136-2
ISO/TC 34/SC 9
Microbiology of the food
Secretariat: AFNOR
chain — Detection, isolation and
Voting begins on:
characterization of Shiga toxin-
producing Escherichia coli (STEC) —
Voting terminates on:
Part 2:
Horizontal method for the
characterization of Shiga toxin-
producing Escherichia coli (STEC)
isolates
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2024
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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Published in Switzerland Reference number
ISO/DIS 13136-2:2024(en)
ii
ISO/DIS 13136-2:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure . 5
9.1 Subtyping of stx genes .5
9.1.1 Template preparation .5
9.1.2 Setting up the PCR reaction .5
9.1.3 Agarose gel electrophoresis .5
9.2 Testing for the presence of eae and aggR genes .6
9.2.1 Template preparation .6
9.2.2 Real-time PCR amplification .6
9.2.3 Interpretation of Real-time PCR results .6
9.2.4 Internal Amplification Control for Real-time PCR . .6
9.3 Determination of the serogroups O157, O26, O103, O145, O111, O45 and O121 by Real-
time PCR amplification of O-associated genes .7
10 Expression of the results. . 7
11 Test Report . 7
12 Quality assurance . 8
Annex A (informative) Outline of the procedure . 9
Annex B (normative) Culture media and reagents . 10
Annex C (informative) Setting up the PCR reactions for stx genes subtyping .12
Annex D (informative) Primers and probes for the Real-time PCR assays for the detection of
eae and aggR genes .15
Annex E (informative) Primers and probes for the Real-time PCR assays for the detection of
O-associated genes . . 16
Bibliography .18

iii
ISO/DIS 13136-2:2024(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent
rights identified during the development of the document will be in the Introduction and/or on the ISO list of
patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in collaboration with
the European Committee for Standardization (CEN) Technical Committee CEN/TC 463, Microbiology of
the food chain, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).
This second edition, together with the ISO 13136 Part 1, cancels and replaces the first edition
(ISO/TS 13136:2012), which has been technically revised.
The main changes compared to the previous edition are as follows:
— Structure of the standard (Full Standard rather than Technical Specification, split in Part 1 and Part 2)
— Description of the characterization of STEC isolates, in terms of determining the presence of a set of
virulence genes and serogroups, and subtyping of stx genes
A list of all parts in the ISO 13136 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/DIS 13136-2:2024(en)
Introduction
Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens causing severe disease, including
haemorrhagic colitis (HC) and the haemolytic uremic syndrome (HUS).
The Stx are the definative virulence factor of STEC, and belong to a heterogenous family of AB toxins,
including two antigenically distinct types, Stx1 and Stx2. STEC harbor stx1 and/or stx2 genes. Large
variability in the stx gene sequences has been described and both stx1 and stx2 can be divided into several
subtypes, with some of these subtypes having a greater association with the most severe forms of STEC
infection (References [1, 2]). More than 180 serogroups of E. coli have been identified (Reference [3])), and a
small group of these serogroups have been historically associated with STEC strains of major public health
concern, being frequently isolated from patients with the most severe forms of infection (Reference [4]).
Nevertheless, the range of STEC serogroups associated with human disease is much broader and continuously
evolving, and in more recent scientific literature all STEC isolates are considered human pathogens, with the
potential to cause at least diarrhoea (Reference [5]).
Beside the serogroup, other markers of STEC associated with HC and HUS cases have been described, such
as the locus of enterocyte effacement (LEE) pathogenicity island (indicated by the presence of the eae gene,
encoding the adhesin intimin) or, more recently, the determinants of the enteroaggregative adhesion typically
present in Enteroaggreagative E. coli (References [5, 6]). Such features, and particularly the eae gene, are
now regarded as aggravating factors for STEC infections, but may not be essential for the occurrence of
severe diseases (Reference [5]). Additionally, greater diversity in the genetic features associated with STEC
pathogenicity continues to be described, such as strains carrying virulence factors associated with more
than one E. coli pathotype, referred to as hybrid or cross-pathotypestrains (References [7-9]), highlighting
the heterogeneity of this E. coli pathogroup and the need for a more detailed characterization of the isolates.
The present standard method describes the characterization of STEC isolates, obtained either by the
application of ISO 13136-1, or any other method. This characterization includes the determination of the
following features:
The subtype of Stx- encoding genes
— The presence of the genes eae and aggR that are involved in the colonization of hosts by STEC.
1)
— Serogroup
The following nomenclature has been adopted in this standard method:
stx: Shiga toxin encoding gene (synonymous with vtx);
Stx: Shiga toxin (synonymous with VT);
STEC: Shiga toxin-producing Escherichia coli (synonymous with VTEC: Verocytotoxin-producing Escherichia
coli), E. coli strains possessing Stx-coding genes
eae: gene encoding the adhesin intimin, causing the ‘Attaching and Effacing’ adhesion;
aggR: gene encoding the transcriptional activator AggR, activating several factors involved in
enteroaggregative adhesion;
stx1a, stx1c, stx1d: genes encoding the three subtypes of stx1, respectively;
stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g: genes encoding subtypes of stx2.
The technical changes listed in the Foreword, introduced in this document compared to ISO/TS 13136:2012,
are considered as major (see ISO 17468). These technical changes have a major impact on the performance
characteristics of the method.
1) The serogroups concerned by the methodology specified in this standard are O157, O26, O111, O145 and O103
(historically termed as top-5 serogroups), O45 and O121 (which, together with the aforementioned five serogroups, are
often isolated from patients with STEC infection in the United States).

v
DRAFT International Standard ISO/DIS 13136-2:2024(en)
Microbiology of the food chain — Detection, isolation and
characterization of Shiga toxin-producing Escherichia coli
(STEC) —
Part 2:
Horizontal method for the characterization of Shiga toxin-
producing Escherichia coli (STEC) isolates
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting and characterizing STEC are only undertaken in properly equipped laboratories, by skilled
microbiologists, and that great care is taken in the disposal of all materials that have potentially
come into contact with bacterial cultures. Persons using this document should be familiar with
normal laboratory practice. This document does not purport to address all the safety aspects
associated with its use. It is the responsibility of the user to establish appropriate safety and health
practices, as well as working in compliance with national and international regulations concerning
the handling and containment of biological agents.
1 Scope
This document is applicable to pure cultures of STEC. The present standard describes the characterization
of STEC strains, isolated from any source. In particular, the characterization of STEC strains described here
regards:
— Subtyping of the Stx-encoding genes
— Determination of the presence of the eae gene
— Determination of the presence of the aggR gene
— Identification of the presence of genes associated with the following serogroups: O157, O26, O111, O103,
O145, O121 and O45.
The full characterization of the isolated STEC strains is achieved by performing all the modules described
here. The characterization scheme is not sequential, and the different modules can be implemented
separately based on specific needs (e.g. Regulatory needs, Competent Authority’s requests, clients’ request).
Alternative methods may be used, including Whole Genome Sequencing (WGS), provided these are validated
according to the most relevant part of reference standard series ISO 16140.
2 Normative references
The following documents are referred to in the text in such a way that some (or all) of their content
constitutes requirements of this document. The latest edition of the referenced document (including any
amendments) applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO/DIS 13136-2:2024(en)
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 22119, Microbiology of food and animal feeding stuffs — Real-time polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection
of food-borne pathogens — General requirements and definitions
ISO 22118, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection
and quantification of food-borne pathogens — Performance characteristics
ISO 23418:2022, Microbiology of the food chain — Whole genome sequencing for typing and genomic
characterization of bacteria — General requirements and guidance
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
For the purposes of this document, the following terms and definitions apply.
3.1
Shiga toxin-producing Escherichia coli (STEC)
E. coli strain possessing one or multiple Stx-encoding genes (stx)
3.2
stx1 and stx2
Genes encoding Stx1 and Stx2, respectively
3.3
eae
Gene encoding the adhesin intimin, located on the Locus of enterocyte effacement and consisting in a key
factor for the ‘Attaching and Effacing’ colonization mechanism
3.4
aggR
Gene encoding the transcriptional activator AggR, activating several factors involved in the enteroaggregative
adhesion characteristic of enteroaggregative E. coli (EAEC). EAEC are a pathogroup of diarrheagenic E. coli,
and hybrid strains of EAEC/STEC have been reported in association with severe disease, such as the STEC
O104:H4 responsible for a large outbreak occurring in the EU in 2011.
3.5
stx genes subtypes
The two antigenically distinct types of Stx, Stx1 and Stx2, are each divided into different subtypes, namely
three subtypes for Stx1 (Stx1a, Stx1c and Stx1d) and at least seven subtypes for Stx2 (Stx2a, Stx2b, Stx2c,
Stx2d, Stx2e, Stx2f and Stx2g) (Refrence [1]). New Stx subtypes are continuously identified, including Stx1e,
Stx2h, Stx2i, Stx2j, Stx2k, Stx2l, Stx2m, and Stx2o (References [10-14]). The present standard regards the
determination of three stx1 genes subtypes (stx1a, stx1c and stx1d) and the seven stx2 genes subtypes stx2a
to stx2g.
3.6
O-antigen
Serogroups or “O” antigens are identified by numbers, counting from 1 to 188 at the time of the preparation
of this document, but the serogroups’ list is evolving constantly.

ISO/DIS 13136-2:2024(en)
4 Principle
The method specified comprises the following three characterization modules:
— Stx-encoding gene subtyping (determination of the presence of stx1a, stx1c, stx1d; stx2a, stx2b, stx2c,
stx2d, stx2e, stx2f, stx2g) – based on PCR
— Determination of the presence of eae, involved in the attaching and effacing adhesion – based on real-
time PCR
— Determination of the presence of aggR, involved in the Enteroaggregative E. coli colonization – based on
eal-time PCR
— Determination of the presence of genes associated with O157, O26, O111, O103, O145, O45 and O121
serogroups – based on real-time PCR
In detail, DNA is extracted from a single colony and analysed by PCR or real-time PCR, depending on the assay
performed. The full characterization of a STEC isolate is achieved by performing all the assays, nonetheless
the different determinations are not all normative and may be carried out separately in response to specific
needs, i.e. on the basis of the clients’ requests (including Competent Authority or on the basis of regulatory
needs).
A diagram of the different modules of the procedure is provided in Annex A.
Alternative methodological approaches different from those described in this document may include slide-
or microtiter-agglutination for serogroup-determination or Whole genome sequencing (WGS) for the
extensive strains’ characterization. If WGS is applied, the ‘ISO 23418:2022 Microbiology of the food chain —
Whole genome sequencing for typing and genomic characterization of bacteria — General requirements and
guidance’ shall be applied for obtaining the STEC strains’ WGS data and ensuing subsequent bioinformatic
analysis.
The WGS-based determinations of the presence of the virulence genes, as well as stx subtyping and
identification of serogroup, shall be done by analysing the sequences obtained. Different tools are available
for the analysis of STEC genome sequences, either as commercial software or through public webservers
(e.g., https:// aries .iss .it and https:// cge .cbs .dtu .dk/ services). Irrespective of the software used, the log
files related to the analyses carried out (containing, for instance, information on the virulence genes
or serogroups-associated genes and the databases used) should be stored as part of the raw data. When
performing WGS, it is recommended to re-test the colony inoculated for the DNA purification for the presence
of stx, as stx-phages may be lost during culturing.
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218. The composition of culture media and
reagents and their preparation are specified in Annex B. For performance testing of culture media, see
Table B1 and follow the procedures in accordance with ISO 11133.
6 Equipment and consumables
Disposable labware or plasticware is an acceptable alternative to reusable glassware if it has suitable
specifications. Usual microbiological laboratory equipment (see ISO 7218) are needed and, in particular, the
following:
6.1 Water bath or heating block, capable of being maintained at temperatures up to 100 °C.
6.2 Incubator, capable of being maintained at 37 °C ± 1 °C.
6.3 Nucleic acid extraction apparatus. Appropriate equipment according to the method adopted (if
needed).
ISO/DIS 13136-2:2024(en)
[7]
6.4 Pipettes of several capacities (between 1 µl and 1 000 µl, ISO 7550 ).
6.5 Thin-walled real-time PCR microtubes, multi-well PCR microplates or other suitable light
transparent disposable plasticware.
6.6 Real-time PCR apparatus (detection of eae and aggR, detection of serogroups) Thermal cycler with a
detector for fluorescence emission following 5’ nuclease PCR assay. Several brands of apparatus are available
and can be chosen according to the laboratory policies.
6.7 Thermal cycler (stx subtyping) and PCR microtubes. Several brands of equipment are available and
can be chosen according to the policy of the laboratory.
6.9 Microcentrifuge or Plate spinner, in case 96-well plate is used.
6.10 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). As specified in ISO 7218.
6.11 Drying cabinet or ventilated oven, capable of being maintained between 25 °C and 50 °C, or a
laminar airflow cabinet for plates drying (if needed).
6.12 Refrigerator (for storage of prepared media), capable of operating at 5 °C ± 3 °C.
6.13 Freezer (for storage of DNA and PCR reagents), capable of operating at -18 °C ± 3 °C.
6.14 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be
made which are accurate to ±0,1 pH unit.
6.15 Sterile tubes, bottles or flasks with caps, of appropriate capacity.
Bottles or flasks with non-toxic metallic or plastic screwcaps may be used.
6.16 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
6.17 Water bath, capable of operating at 47 °C to 60 °C.
6.18 Laminar flow hood
6.19 Cabinet for PCR
6.20 Electrophoresis equipment
7 Sampling
Sampling is not specified in this document. This document is applicable to STEC isolated from any sources
and obtained by the application of ISO 13136-1 or other methods.
8 Preparation of test sample
The procedure consists in the characterization of isolated STEC strains. The sample preparation consists
in the purification of the nucleic acid for the following characterization, or for some applications (e.g.
serotyping through agglutination) in the strain itself. The preparation of the test sample is described in the
specific characterization modules included in this document.

ISO/DIS 13136-2:2024(en)
9 Procedure
9.1 Subtyping of stx genes
This procedure is applied to determine the stx gene subtypes in isolated E. coli strains, in which the presence
of stx1 and/or stx2 has already been detected, e.g. though the procedure described in ISO 13136-1. The
identification of the stx subtypes is performed by specific PCR reactions, using primers designed from
analyses of stx sequences (Reference [1]) and reported in Table C1 in Annex C.
The method is composed of the following steps:
— Template preparation
— Setting up the PCR reaction
— Determination of the PCR results by agarose gel electrophoresis
9.1.1 Template preparation
A single bacterial colony of the STEC strain is picked from a solid medium (e.g. nutrient agar or chromogenic
agar), inoculated in TSB and incubated at 37 °C for 18 h ± 3 h. A volume of 25 µl of the overnight culture is
added to 975 µl of molecular-grade sterile water in a microcentrifuge tube. The tube is placed in boiling
water for 15 min, then centrifuged at 18 000 g for 5 min. Following the transfer of the supernatant to a clean
microfuge tube, it can be directly used as a template for PCR or stored at -20 °C for further analyses.
9.1.2 Setting up the PCR reaction
PCR assays are set up in a total volume of 25 µl for triplex PCR (for the detection of stx1 subtypes) and 20 µl
for single PCR (for stx2 subtyping) assays, as described in Table C1 o
...