Overview of standard methods development of ISO and CEN

Alexandre LECLERCQ Convenor of CEN TC275/WG6 Food Microbiology

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CRITERIA / STANDARD
(Codex alimentarius, CAC/GL 21 of 1997)

Micro-organisms of interest - interest for the product ; Sampling plan; Usable detection and/or quantification methods Satisfactory microbiological limits for the food at the critical point of the food chain ; The number of analytical units allowing a conformity compared to these limits ; The food to which it applies Stages of the food chain to which it applies ; All measures to be taken when it is not satisfied.
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Target: Which Microorganisms ?

Prions ? Virus Bacteriophages Bacteria/ Food Human/animal Spores GMO ?

Insects

Fungi Water
Environnement Protozoa Parasites

Metabolites (Fungi, bact.)

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Toxins (Algae, Fungi, bact.)

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Toxicology ?

Example: Bacteria
Pathogen
1 microorganism

Bacteria resistant to antibiotics Opportunistic Deterioration flora Bioindicator flora Sanitary Technology Probiotics Floral with technological interest / Starter Flora of Ali-terrorism
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1 Population, 1 ecology

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The target becomes more and more complex

Example of the producing E coli of Shiga-like toxins

Serotype detection
Interesting public health: as EHEC Interesting veterinary sector

Detection of virulence genes

VT/eaeA/HlyA
Test in Vivo / cellular culture

Immunological detection of verotoxins!

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Viable but not cultivable

Log CFU Colony viability

Survival Culture Enumeration

Time

Molecular biology / Cytometry / Microscopy

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PROCESS: ASSAY

Sample collection Personnal Manutention Preparation Premises Equipment

Management Quality System

Working conditions

Calibration
Methods ??

ANALYSES
Reagents Results Report

Quality Control

Measurement uncertainty

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Recording

Validation

Audit

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Standardized methods
Reference text Elaborated by qualified body, with consensus among all the interested parties Continuous, repetitive and voluntary application Speak the same language
Distinction between full standard and Technical specification
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Standardize or Harmonize: Why ?

Avoid disparate methods of microbiological analysis Facilitate exchange of food products Improve reliability of microbiological analysis Relevant to general development of quality assurance

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International Standardization (1947)

ISO (50 Standardization bodies) ISO standard: optional integration in national collection, revision each 5 years TC34: Agricultural food products SubCommittee 9: Microbiology: horizontal methods Chairman: B. LOMBARD, Secretariat: C. DE LAMOTTE

Vienna agreement: ISO standards

CEN standards

Scope: Standardization in the field of human and animal foodstuffs, covering the food chain from primary production to consumption, a well as animal and vegetable propagation materials, in particualr, but not limited to, terminology, sampling, methods of test and analysis, product specifications, food and feed safety and quality of management and requirements for packaging, storage and transportation. excluded: products covered by ISO/TC54 Essential oils and ISO/TC93 (Starch)

Mission: Standard horizontalisation

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Functioning
ISO CASCO/BT ISO TC34

SC9

WG1
Meat and meat products

WG2 Statistics

WG3 Method Validation

WG4
Proficiency testing

WG5
Culture media

WG6
Parasites

WG7
Microbiological examinations

Project group
Probiotics

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European Standardization (1985)

CEN (Bruxelles, 19 Standardization Bodies), TC275: Food Analysis / WG6 Microbial Contaminant
Chairman: A. LECLERCQ, Secretariat: C. DE LAMOTTE

EN Standard: Obligatory integration in national collection Pre-established recognition of standards ( by ISO)

Scope: Standardization of horizontal microbiological methods for all food and animal feeding stuff analysis and for any other sample that can be the source of microbiological contamination of food products
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Functioning
CEN BT
TC 327 Probiotics

TC 275

TC 302 MILK
ISO TC34/SC9

WG6 EU PROJECT
AdHoc group Ad(Hoc group C. sakazakii AdHoc group L. monocytogenes
Histamine, B. cereus toxins, Staph. toxins

TAG1

TAG2

TAG3 PCR

TAG4 Virus

TAG5
Primary

TAG6

TAG7

Adhoc Group STEC

Media Validation

Sampling Parasites production techniques

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Alternative method # Full standard?

For a given scope, it shall estimate the same scope analyte than the one measured by the corresponding reference method, and shall meet the following requirements:
rapid analysis and/or response easy carrying and/or automation analytical performances (accuracy, sensitivity..)
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Standard and Outbreak

(ISO TC34/SC9 and CENTC275/WG6)

Possible Introduction of the following text as a note in the scope of the standards on pathogen detection For epidemiological purposes or during outbreak investigations, other methods may need to be used in addition to this standard. A standard could not cover all matrices and all target microorganisms!

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Microbiology of food and animal feeding stuffs

Microbiology of the food chain

According to the DG SANCO agreement, the scope of WG6 is enlarged to include enlarged bottled water and water used in food processing only when standardized water standardized microbiology methods do not exist.
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General requirements and guidance for microbiological examinations

Amemdment of EN ISO 7218 published in 2007 (ISO TC34/SC9/WG7, A. Leclercq)
General requirements and guidance/options intended for three main uses: implementation of ISO/TC 34/SC 9 or ISO/TC 34/SC 5 standards for detection or enumeration of microorganisms, named hereafter specific standards; good laboratory practice for food microbiological laboratories (the purpose is not to detail them in this International Standard, manuals are available for that purpose); guidance for accreditation of food microbiological laboratories (this International Standard describes the technical requirements according to Annex B of ISO/IEC 17025:2005 for the accreditation of a microbiological laboratory by national organizations). covers examination for bacteria, yeasts and moulds and can be used if supplemented with specific guidance for prions, parasites and viruses. It does not cover the examination for toxins or other metabolites (e.g. amines) from microorganisms. applies to the microbiology of food, animal feeding stuffs, the food production environment and the primary production environment. The purpose of this International Standard is to help to ensure the validity of food microbiology examinations, to assist in ensuring that the general techniques used for conducting these examinations are the same in all laboratories, to help achieve homogeneous results in different laboratories, and to contribute towards the safety of the laboratory personnel by preventing risks of infection. IAFP, Berlin, 2009 17/50

Microorganisms

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Salmonella: Revision of EN ISO 6579

(ISO TC34/SC9) Part 1: Detection and AmdA1 (primary production samples)
deal with the detection of S. Typhi and S. Paratyphi in a normative annex considering the use of the SC enrichment broth launch a trial comparing enrichment in the BAM/USP formulation of tetrathionate broth and in MKTTn. collect data comparing the use of RVS and MSRV for food analysis XLD should be retained as the mandatory isolation medium usefulness of some biochemical tests

Part 2: Enumeration
a new MPN method, able to analyze simultaneously a lot of samples at a lower cost. This technique is based on the ability of the MSRV medium to select and enriched motile Salmonella that may be further isolated on chromogenic medium

Part 3: Serotyping
Based on Kauffman whites scheme
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Campylobacter
(CEN TC275/WG6)

Revision of EN ISO 10272-1:2006 (detection Campylobacter) and of ISO/TS 10272-2:2006 (enumeration Campylobacter Colony-count technique). To improve the enrichment and confirmation steps and microaerobic incubation conditions

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Listeria spp. detection and enumeration

(CEN TC275/WG6/adhoc group, R. Beumer)

Revision of EN ISO 11290

L. monocytogenes Listeria spp. and L. monocytogenes

Simplification of identification

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Revision EN ISO 11290: Identification Scheme

Presumptive L.monocytogenes ALOA or other chromogenic medium Gram stain (optional) Haemolysis CAMP (if necessary) Rhamnose Xylose Other presumptive Listeria species Gram stain Haemolysis CAMP (if necessary) VP test Further tests if speciation required (rhamnose, xylose, mannitol) Gram stain Haemolysis CAMP (if necessary) VP test Further tests if speciation required (rhamnose, xylose, mannitol)

Second medium if not chromogenic

Gram stain Haemolysis CAMP (if necessary) RhamnoseXylose

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VTEC non-O157
(CEN TC275/WG6/adhoc group, S. Morabito (CRL)) o Project technical specification
o CEN ISO/WD TS 13136 Microbiology of food and animal feeding stuffs -- Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to O157, O111, O26, O103 and O145 serogroups -- Qualitative Real-time polymerase chain reaction (PCR)-based method

o First detection of stx,eae genes and after RT-PCR for serotypes followed by isolation of colonies o Problems of patents: RT-PCR for serogroup detection

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o A new need: An European standard for stx characterization by PCR or RT-PCR IAFP, Berlin, 2009

Bacillus cereus / Bacillus anthracis

Revision of EN ISO 7932:2004 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of presumptive Bacillus cereus - Colonycount technique at 30 C Investigation of chromogenic media Investigation of the need of a Bacillus anthracis standard
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Enterobacter sakazakii
(CEN TC275/WG6/adhoc Group, H. Joosten & C. Iversen)

Draft: Horizontal method for the detection of Cronobacter spp. (Enterobacter sakazakii)
Relative poor performance with environmental samples Recovery some sensitive strains from some milk powders

Enrichment broth is a Cronobacter screening broth:

Need to investigate sub cultures of negative broths and the possibility of recoloration of the broth

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Brochothrix thermosphacta
(ISO TC34/SC9/WG2 (meat and meat products), E. De Boer)

Possible Revision of ISO 13722 In parallel with NMKL

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Bioindicator - Coliforms: Evolution of methods

Acid production from lactose (new definition)

Gaz and acid production dacides from lactose (old definition) Escherichia coli

Enzymatic methods (new definition)

Escherichia coli Klebsiella Enterobacter Citrobacter Yersinia Serratia Hafnia In green, coliforms present in environment and human faeces
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Klebsiella Enterobacter Citrobacter

Pantoea Kluyvera

Underlined, coliforms from environment

Escherichia coli Klebsiella Enterobacter Citrobacter Yersinia Serratia Hafnia Pantoea Kluyvera Cedeca Ewingella Moellerella Leclercia Canella Yokenella IAFP, Berlin, 2009

Enterobacteriaceae: Bioindicator
(ISO TC34/SC9, E. de Boer)

Enterobacteriaceae Detection and enumeration (ISO 21528:2004) Revise part 1 of ISO 21528 by omitting the EE broth enrichment step and to modify the confirmation step (fermentation of glucose has been changed in the

the use of O/F medium).

Establish an amendment to part 2 of ISO 21528 to modify the confirmation step in order to develop the 2 parts at the same time
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Virus
(CEN TC275/WG6/TAG4, R. Rangdale (CRL))

Draft of a full standard: Horizontal method for detection of hepatitis A virus and norovirus in food using real-time RT-PCR- Part 1: Method for quantitative determination Norovirus genogroups I and II New target matrices: According to regulation EC 178/2002, bottled water and water used in the food industry is a food.
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Parasites
(ISO TC34/SC9/WG6 and CEN TC275/WG6/TAG7, P. Rossi (CRL))

Work at ISO level (call of experts):

Cryptosporidium Giardia

work at CEN level: Standardize both

the artificial digestion microscopic method for the detection of Trichinella in muscles and serological methods for detection of antibodies to Trichinella.

Problem of reference material and antigens.

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Yeast and moulds

(ISO TC34/SC9, A. Leclercq)

aw 0.95 et >0.6 Media DG18 ISO 21527-2

aw >0.95 Media DRBC ISO 21527-1

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METHODS

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TAG 6 "Sampling techniques"

(CEN TC275/WG6/TAG6, M. Poumeyrol)

Revision of EN ISO series : Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Agreement with the development standards about sampling techniques.
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General requirements relating to Molecular methods for detection and quantification of microorganisms
(CEN TC275/WG6/TAG3, K. Berghof)
Standards: General rules for PCR and validation of thermocycler New project standards for Real Time - PCR

polymerase chain reaction (PCR) for the detection of food-borne pathogens -- General requirements and definitions

EN ISO/DIS 22119 Microbiology of food and animal feeding stuffs -- Real-time

EN ISO/DIS 22118 Microbiology of food and animal feeding stuffs -- Polymerase chain reaction (PCR) for the detection of food-borne pathogens -- Performance characteristics of molecular detection methods
The performance characteristics of molecular methods shall be established according to the procedures of EN ISO 16140 if applicable

DNA Biochips? New part of Scope: Primary production stage

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CEN ISO/TS PCR detection for specific pathogens

(CEN TC275/WG6/TAG3, K. Berghof)
Detection of botulinum neurotoxin producing Clostridia First draft Real-Time PCR: Detection of neurotoxin genes type A, B, E and F

Pathogenic Vibrio parahemolyticus Detection and enumeration on account of poor performance of cultural methods ISO 21871 and 21872 toxR gene sequence R72H and hemolysin genes TDH and TRH

Yersinia enterocolitica and pseudotuberculosis Investigation on enrichment

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ELISA / Immunological methods

(CEN TC275/WG6/Adhoc group, J. Hennekinne?)

Necessity of General rules and guidelines Necessity for detection of staphylococcal enterotoxins

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Primary production stage

(CEN TC275/WG6/TAG5, R. Danguy des dserts)
Scope: From primary production (e.g. farm, natural environment) to slaughter/harvest . The border would be live animals in the slaughter house Samples taken after slaughter to indicate microbiological status of live animals are included Samples taken to indicate hugiene of meat are excluded Work = 3 projects: EN ISO/WD 6887-6 Preparation of test samples, initial suspension and decimal dilutions for microbiological examination -- Part 6: Specific rules for the preparation of samples taken at the primary production stage
EN ISO/WD 13307 Primary production stage -- Sampling techniques EN ISO/WD 10272-4 Primary production stage Detection and enumeration of Campylobacter in samples from primary production
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New area have to be investigated: Fish and shellfish products

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Quality management

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Culture media
(ISO/TC34/SC9/WG5 and CEN TC275/WG6/TAG1, M. Greenwood) Revised EN ISO 11133-1 had been published in February 2009 again as a TS: Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part 1 : general guidelines on quality assurance for the preparation of culture media in the laboratory. Joint standard water and food 2 parts of ISO 11133 will become 1 standard pr EN ISO 11133 (Preparation, production, storage and performance testing of culture media) Joint standard water and food Scope extended to reagents and confirmatory media

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Validation
( ISO/TC34 SC9/WG3 and CEN TC275/WG6/TAG2, P. Int Veld) - PG1: Terminology - PG2: Validation of proprietary methods (quantitative and qualitative methods) - PG3: Intermediate validation - PG4: Method verification - PG5: In-house validation (against and without a reference method) - PG6: Technical requirements for establishment/revision of standardized methods
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Validation
( ISO/TC34 SC9/WG3 and CEN TC275/WG6/TAG2, P. Int Veld)
Work First draft: NWIP EN ISO 16140-1 Terminology of Method Validation NWIP EN ISO 16140-2 Protocol for the validation of alternative method Part 2 : Protocol for the validation of propietary methods against a reference method Link with ISO TC34/SC9/WG2 Statistics Revision of EN ISO 16140:2003 Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods
Circulation of the draft of project of Amendment A1 of EN ISO 16410:2003 which correct errors in clause 6.3. Extension of its scope to PCR and Immunological methods

EN ISO standards perhaps recognised by AOAC (Paul WEHLING, AOAC, Chairman Statistical Committee, new member of WG2. A full international validation process of microbiological alternative method EN ISO 16140 and the life of reference method? IAFP, Berlin, 2009

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New concept of standards

(i.e. chemistry)

Reference methods

Validation, Equivalence

Alternative methods

Performance criteria without description of method

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European Mandate by EC for Validation of reference methods: 15 METHODS

1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) foodtechnique Detection of food-borne viruses in food based on PCR technique Detection and quantification of histamine Detection of staphylococcal enterotoxins for dairy products and other matrices Detection of Bacillus cereus enterotoxins in food (emetic and diarrheal toxin) diarrheal Detection of Enterobacter sakazakii 21872EN 21872-1 Horizontal method for the detection of pathogenic Vibrio Part 1: Detection of Vibrio parahaemolyticus and 21872EN 21872-2 Horizontal method for the detection of pathogenic Vibrio Part 2: Detection of species other than Vibrio parahaemolyticus and Vibrio cholerae Enterobacteriaceae Horizontal methods for the detection and enumeration of Enterobacteriaceae Part 1: Detection and enumeration by pre-enrichment MPN technique with pre-enrichment 11290EN ISO 11290-1 Horizontal method for the detection and enumeration of Listeria monocytogenes Part 1: Detection method 11290EN ISO 11290-2 Horizontal method for the detection and enumeration of Listeria monocytogenes Part 2: Enumeration metho O157 EN ISO 16654 Horizontal method for the detection of E. coli O157 10271EN ISO 10271-1 Horizontal method for the detection and enumeration of Campylobacter spp. Part 1: Detection method method 10272EN ISO 10272-2 Horizontal method for the detection and enumeration of Campylobacter spp. Part 2: Colony count technique technique EN ISO 6579/FDAmd 1 Horizontal method for the detection of Salmonella spp. Amendment 1 Annex D: Detection of production Salmonella spp. in animal faeces and in samples from the primary production stage EN ISO 10273 Horizontal method for the detection of presumptive pathogenic Yersinia enterocolitica

Vibrio cholerae

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Proficiency testing
(ISO TC34/SC9/WG4, S. Passmore)

Final vote: CEN ISO/TS 22117 Proficiency testing Request for ILAC and EA recommending them to recognise this TS (Technical Specification) as technical requirements for proficiency testing organisation in the field of food microbiology (DIS ISO 17043).
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Measurement uncertainty
(ISO TC34/SC9/WG2, B. Lombard)

ISO/TS 19036: measurement uncertainty

Amendment A1: low number

New Work:
uncertainty of sampling in food microbiology, maximum allowable values for measurement uncertainty

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News works or standards projects

Iso level: New project group: Detection of contaminants in starter cultures and probiotics (projecleader, S. Zuber, Switzerland) Iso level: CEN ISO/TS 10272 3 Horizontal method for the detection and enumeration of Campylobacter spp - Part 3 Semi-quantitative method Iso level: ISO TS 2871 & 2 for vibrio: correction of the volume of culture transferred from first enrichment to second enrichment. Iso level: Comparison of ISO 7251 to ISO 16649-3 Horizontal method for the detection and enumeration of presumptive Escherichia coli - Most probable number technique ISo level: Horizontal method for the enumeration of microorganisms : EN ISO 4833 Poured plate colony count technique at 30 C Surface colony-count technique at 30 C Iso level: Enumeration of Alicyclobacillus acidoterrestris CEN level: Enterotoxin Bacillus cereus (P. int veld) CEN level: Standardization of antimicrobial susceptibility analysis of foodborne pathogens CEN level: Detection and quantification of histamine
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Future for standardization of food microbiology

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Cover the food chain Integrate new technology Improve the publication delay of standards More flexible Avoid to put method inside regulation Financial support for ring trials ! Description of method or standard based only on performance criteria?
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Acknowlegments

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