Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Standardisation in (food) microbiology
Sense and nonsense in standardisation (2)
Rijkelt Beumer
1
Standardisation: horizontal or vertical
* the perennial question
why are we here?
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Standardisation: horizontal or vertical
horizontal methods
* do not need necessarily to be the most rapid or sensitive
* using a more sensitive method:
* does not change the incidence of problems
* does not protect consumers
* can hamper international trade
3
Standardisation: horizontal or vertical
what are vertical methods used for?
* to overcome problems associated with a particular
commodity
* for practicability
* by trade association members (industry) for compliance
or for internal monitoring of HACCP
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2 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
There is only one true vertical method
* i.e. a procedural modification that cannot be
incorporated into, or replace a procedure in the
international standard
* industry based method for HACCP verification and
hygiene monitoring not meeting the
sensitivity/reproducibility requirements of the
reference method
5
Problem of ‘uncertainty of measurement’
*
*
*
*
*
*
repeatability (same situation)
reproducibility (various situations)
recovery
matrices
reference samples
artificial and/or naturally contaminated samples, level of
contamination
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3 (21)
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Uncertainty of measurement
* microbiologists and statisticians not necessarily have the
same opinion
* big difference between detection of chemical compounds
and microorganisms (not always understood by
statisticians)
7
Standardisation in (food) microbiology
not always the best method is used:
EN ISO 6579:2002 - Horizontal method for the detection
of Salmonella spp.
* pre-enrichment: buffered peptone water (16-20h 37ºC)
* selective enrichment
* RVS-broth (24h 41,5°C)
* MK tetrathionate novobiocin broth (24h 37°C)
* isolation: Xylose lysine deoxycholate (XLD) agar and
second medium: free choice
8
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Standardisation in (food) microbiology
* In the USA MK tetrathionate and Selenite Cysteine
broth were used for the detection of Salmonella
(AOAC method)
* RVS and Selenite Broth were used in the ISO method
* Selenite Broth too dangerous to use, so a good chance to
replace it by RVS in the AOAC protocol
9
Standardisation in (food) microbiology
* only one enrichment broth, RVS, would be sufficient
* due to ‘politics’we still have two: MK and RVS
* however, the best method would be: BPW and MSRV
10
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Standardisation in (food) microbiology
* via a back door MSRV will be incorporated in ISO 6579
for the detection of Salmonella in poultry faeces,
feathers, etc
11
Standardisation in (food) microbiology
* after several years, MSRV will probably be the golden
standard.
* still big differences between ISO, AOAC, IDF, AFNOR,
NORDVAL, in particular for validation of rapid methods
* there was a tri-partite agreement between ISO, AOAC
and IDF, since 2004 only between ISO and AOAC,
however, not all problems can be solved
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Validation of rapid methods
problem of the manufacturer
* one validation should be sufficient
* protocols are different
* AOAC and MICROVAL: pilot study (2009) one validation
* AFNOR (and probably NORDVAL) use rather ‘easy’
protocols
13
Validation of rapid methods
* Microval, result of Eureka (initiative Unilever Research Lab
project for microbiological validation (NEN)
* Founding fathers: AFNOR, NEN, Nestle and Unilever
* 7 EU countries and 21 partners involved
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Validation of rapid methods
Principles of the Microval certification (1)
* The first principle of the certification is to perform a
method comparison study of the alternative method
against a suitable reference method,
* followed by an interlaboratory method performance,
study of both the alternative and the reference method.
15
Validation of rapid methods
Principles of the Microval certification (2)
* The second principle is that the quality organisation of
the manufacturer where the materials are produced
must be in conform to ISO 9001/ISO 13485
* The third principle is that a regular control of the
quality of the certified methods, which is made after
the certification is granted
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8 (21)
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Validation of rapid methods
* ISO 16140 and AOAC Microbiology Guidelines, although
not identical, are very similar
Both require:
* Methods comparison study
* Inclusivity/exclusivity data
* Inter-laboratory collaborative study
* Expert review
ISO reference methods for validations approved by
AOAC and MICROVAL (not yet AFNOR)
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Steps in validation of official methods
*
*
*
*
*
*
*
*
*
selection of procedure
ruggedness testing
pre-collaborative study
Preparation and clearance of the collaborative study
protocol
selection collaborators; sample preparation;
sample analysis; submission of data;
statistical analysis and manuscript preparation
recommendation for first action (max 2 years)
recommendation for final action (indefinite)
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9 (21)
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Steps in validation of official methods
validation of rapid methods: need to be consistent
* artificially (<80%) or naturally contaminated samples
* validation study protocols (min # food types 20, 3
inocula levels: uninoculated, low and high level)
* source of inoculum strains
* number of inoculum strains
* comparison of test method with reference method
19
ISO is International
it must take into account all of the 157 member countries
member body, correspondent member, subscriber member
it cannot work to the detriment of the
less sophisticated countries, but the more sophisticated
countries must not suffer either
we can have several methods, but they must be equally
reproducible and equally sensitive
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21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
Discrimination
* regulatory methods
* compliance with regulations, international affair
* horizontal commodity-wide, e.g. E. coli in foods (meat
products?)
* regulatory methods of local significance
* national affair, local product specific
* vertical, e.g. E. coli in merquez (sausage)
* investigatory methods
* compliance with HACCP
* vertical: trade affair, factory, line, product specific,
e.g. E. coli in environmental sample of specific meat
product line or factory
21
Weaknesses
* slowness: up to 3 years to publish a standard method
* reviewed only every 5 years
and the treadmill begins again
* not (yet) properly validated (being addressed)
* technically/editorially poor
* language barriers contribute to time problem
* do not keep up with technical developments
* do not propose alternatives
* bound by bureaucracy and democracy
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11 (21)
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A whole is only as good as its parts
* ISO and CEN depend upon national membership
* representatives must have up-to-date experience
* Members must not lose sight of the reasons for
standardisation and harmonisation
23
A survey of standards (1)
Bacillus cereus
EN ISO 7932
General guidance for the enumeration of presumptive
Bacillus cereus – cct at 30°C
ISO 21871
Horizontal method for the enumeration of low numbers of
Bacillus cereus – MPN technique
Brochothrix thermosphacta
ISO 13722
meat and meat products –enumeration of Brochothrix
thermosphacta - cct
24
12 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (2)
Campylobacter
ISO 10272-1
Detection of Campylobacter spp
ISO TS 10272-2:2006
Enumeration of Campylobacter spp
ISO CD 10272-3
Semi-quantative detection of Campylobacter
25
A survey of standards (3)
Coliforms
ISO 4831
Horizontal method for the detection and enumeration of
coliforms –MPN technique
ISO 4832
Horizontal method for the detection and enumeration of
coliforms – cct
26
13 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (4)
Clostridium perfringens
ISO 7937
Horizontal method for enumeration of Clostridium
perfringens –cct
Enterobacter sakazakii
ISO TS 22964: 2006
milk and milk products – detection of Enterob. sakazakii
ISO TS 22964
method for environmental samples
27
A survey of standards (5)
Enterobacteriaceae
ISO 21528
Horizontal method for detection and enumeration of
Enterobacteriaceae
– part 1: detection and enumeration by MPN technique with
pre-enrichment
- part 2: cct
Confirmation (n=60)
glucose agar OF medium microtiter plate
24h
37
60
60
48h
60
60
60
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14 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (6)
Lactic acid bacteria
ISO 15214
Horizontal method for the isolation of mesophilic lactic
acid bacteria – cct
Listeria monocytogenes
ISO 11290
Horizontal method for the detection and enumeration of
Listeria monocytogenes
part 1: detection method
part 2: enumeration method
amendment 1: ALOA
29
A survey of standards (7)
Pseudomonas
ISO 13720
meat and meat products – enumeration of Pseudomonas spp
revision 2007:
cephalotin instead of cephaloridine
glucose test optional?
Salmonella
EN ISO 6579
Horizontal method for the detection of Salmonella spp
amendment 1,annex D: Detection of Salmonella spp in animal
faeces and in samples from the primary production stage
30
15 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (8)
Salmonella enumeration
in preparation
proposal
part 1: low numbers, MPN
part 2: high numbers,direct plating
Shigella
pr EN ISO 21567
Horizontal method for the detection of Shigella spp
31
A survey of standards (9)
Staphylococcus aureus
ISO 6888-1
Horizontal method for the enumeration of coagulasepositive staphylococci (Staphylococcus aureus and other
species)
part 1: cct with confirmation
part 2: cct without confirmation
part 3: MPN technique for low numbers
32
16 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (10)
Total aerobic count
ISO 4833
Horizontal method for the enumeration of microorganisms
– cct at 30°C
Vibrio (pathogenic spp)
ISO TS 21872
Horizontal method for the detection of presumptive
pathogenic Vibrio by digestive tract
excl. V. cholerae, V. fluvalialis, V. hollisae, V. mimicus, and
V. parahaemolyticus.
33
A survey of standards (11)
Vibrio parahaemolyticus
ISO 8914
General guidance for the detection of Vibrio
parahaemolyticus
Yeasts and Moulds
ISO 7954 –cct technique, medium with chloramphenicol
ISO 13681
meat and meat products – enumeration of yeasts and
moulds –cct using medium with oxytetracyclin/gentamycin
34
17 (21)
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Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (12)
Yeasts and moulds
ISO FDIS 21527
Horizontal method for the enumeration of yeasts and
moulds
part 1: products with aw > 0.95
part 2: products with aw 0.95
35
A survey of standards (13)
Yersinia enterocolitica
ISO 10273
general guidance for the detection of presumptive
pathogenic Yersinia enterocolitica
Preparation and testing of culture media
EN ISO TS 11133-1
Guidelines on preparation and production of culture media
part 1: general guidelines on preparation and production of
culture media in the laboratory
part 2: practical guidelines of performance testing of
culture media
36
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21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (14)
EN ISO 6887
Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination
ISO 6887-1
General rules for the preparation of the initial suspension
and decimal dilutions
EN ISO 6887-2
Specific rules for meat and meat products
EN ISO 6887-3
Specific rules for fish and fish products
EN ISO 6887-4
Specific rules for products other than milk and milk
37
products, meat and meat products, fish and fishery products
A survey of standards (15)
Surface sampling
ISO 18593
Horizontal method for the enumeration of aerobic bacteria
from surfaces using contact plate and swab methods
Carcass sampling
ISO 17604
Carcass sampling for microbiological analysis
annex D: sampling of poultry carcasses
38
19 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (16)
Standardisation PCR techniques
ISO 22174
PCR – General requirements and conditions
ISO TS 20836
Performance testing for thermal cyclers
EN ISO 20837
PCR – Requirements for sample preparation for qualitative
detection
EN ISO 20838
PCR – Requirements for amplification and detection
for qualitative methods
39
A survey of standards (17)
PCR standards under construction:
- real time PCR
- performance criteria for PCR
PCR detection of:
- Clostridium botulinum
- Vibrio (pathogenic spp)
- Yersinia enterocolitica
40
20 (21)
21)
Presentation at the 3rd SAFOODNET seminar
St Olav’
Olav’s Hotel,
Hotel, Tallinn,
Tallinn, Estonia;
Estonia; May 4-6, 2009
A survey of standards (18)
Other activities
- sampling techniques
- working group on statistics
- STEC, other than O157
- Mycobacterium paratuberculosis
- viruses
- parasites
Primary production, microbiological status of cattle:
faeces, shoes, boots, dust, insects, rodents, crates,
feathers, tonsils, lymph nodes etc.
41
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