Seminar on Emerging Risk Assessment Approaches for Biopesticides | Session 2: Taxonomy and safety considerations for Bacillus thuringiensis-based pesticides
OECD_ENV
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Oct 30, 2025
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About This Presentation
The seminar aimed to foster dialogue on emerging risk assessment approaches for biopesticides, and to initiate a process for developing recommendations through the exchange of experiences and challenges faced by governments and stakeholders in this area.
Held over three half-days, the seminar focus...
Slide Content
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Seminar on Emerging Risk Assessment Approaches for
Biopesticides
Session 2: Taxonomy and safety considerations for Bacillus
thuringiensis-based pesticides
The 13
th
Expert Group on Biopesticides
February 25
th
, 2025
OECD, Paris
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Seminar on Emerging Risk Assessment Approaches for
Biopesticides
Session 2: Taxonomy and safety considerations for Bacillus
thuringiensis-based pesticides
The 13
th
Expert Group on Biopesticides
February 25
th
, 2025
OECD, Paris
Unclassified / Non classifié
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Background
Bacillus thuringiensis(Bt) has been utilized as a microbial insecticide in OECD
countries for decades and has been regarded as relatively low risk
In recent years and with the help of new genomic tools for identification, the
taxonomy of the Bacillus cereus(Bc) group has been re-examined, questioning
reliance on the presence of Cry protein in distinguishing Bt
Additionally, there is uncertainty with regards to whether pesticidal Btstrains have
been causal agents in foodborne outbreaks
These issues have presented challenges to regulators assessing the safety
of Bt, thereby warranting discussion in the format of this Seminar
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Background
Bacillus thuringiensis(Bt) has been utilized as a microbial insecticide in OECD
countries for decades and has been regarded as relatively low risk
In recent years and with the help of new genomic tools for identification, the
taxonomy of the Bacillus cereus(Bc) group has been re-examined, questioning
reliance on the presence of Cry protein in distinguishing Bt
Additionally, there is uncertainty with regards to whether pesticidal Btstrains have
been causal agents in foodborne outbreaks
These issues have presented challenges to regulators assessing the safety
of Bt, thereby warranting discussion in the format of this Seminar
Unclassified / Non classifié
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Purpose and Scope of session
To exchange informationbetween regulators, researchers, and other stakeholders, on
the evolving taxonomic classification of the Bacillus cereussensu lato group, and factors determining
classification;
potential associations with foodborne disease;
regulatory frameworks where this information has been considered
Toprovide a forum for regulators to discuss implications of such classifications for registered or
prospective microbial pest control agents
Toidentify the challenges when assessing risk of pesticidal Btstrains and possible ways of
managing them
To suggest and discuss optionsof further steps for OECD countries and key stakeholders in
OECD and non-OECD countries to harmonise the risk assessment of Bt-based pesticides
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Purpose and Scope of session
To exchange informationbetween regulators, researchers, and other stakeholders, on
the evolving taxonomic classification of the Bacillus cereussensu lato group, and factors determining
classification;
potential associations with foodborne disease;
regulatory frameworks where this information has been considered
Toprovide a forum for regulators to discuss implications of such classifications for registered or
prospective microbial pest control agents
Toidentify the challenges when assessing risk of pesticidal Btstrains and possible ways of
managing them
To suggest and discuss optionsof further steps for OECD countries and key stakeholders in
OECD and non-OECD countries to harmonise the risk assessment of Bt-based pesticides
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Speakers
Martin Steen MORTENSEN, DTU National Food Institute, Denmark
Angela CATFORD, Health Canada, Canada
Mathilde BONIS, French Agency for Food, Environmental and Occupational Health & Safety-
ANSES, France
Sophia JOHLER, Ludwig Maximilian University of Munich, Germany
José CARVALHO, CertisBiologicals, Germany
Nina JOERGENSEN, FMC
LieveHERMAN, EFSA, Chair of EFSA’s Scientific Panel on Biological Hazards Qualified
Presumption of Safety of Bacillus group, ILVO, Belgium
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Speakers
Martin Steen MORTENSEN, DTU National Food Institute, Denmark
Angela CATFORD, Health Canada, Canada
Mathilde BONIS, French Agency for Food, Environmental and Occupational Health & Safety-
ANSES, France
Sophia JOHLER, Ludwig Maximilian University of Munich, Germany
José CARVALHO, CertisBiologicals, Germany
Nina JOERGENSEN, FMC
LieveHERMAN, EFSA, Chair of EFSA’s Scientific Panel on Biological Hazards Qualified
Presumption of Safety of Bacillus group, ILVO, Belgium
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Bacillus cereus sensu lato
phylogeny
Martin Steen Mortensen
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Bacillus cereus sensu lato
phylogeny
Martin Steen Mortensen
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Agenda
•B. cereus sensu lato members
•Phenotypic classification
•Phylogenetic classification
2
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Agenda
•B. cereus sensu lato members
•Phenotypic classification
•Phylogenetic classification
2
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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B. cereus sensu lato members
3
Carrol LM et al., 2021
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B. cereus sensu lato members
3
Carrol LM et al., 2021
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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B. anthracis
•Pathogenecitydepends on
•Two A–B type toxins
•poly-γ-D-glutamic acid
•Relevant genes carried on two plasmids
B. cereus
•Emetic syndrome
•Cereulide
•Gene carried on a plasmid
•Diarrhoealsyndrome
•Enterotoxins
B. thuringiensis
•Identification and classification
•Insecticidal crystal proteins
•Cry and Cytproteins
•Relevant genes almost exclusively carried on
plasmids
4
Main species
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B. anthracis
•Pathogenecitydepends on
•Two A–B type toxins
•poly-γ-D-glutamic acid
•Relevant genes carried on two plasmids
B. cereus
•Emetic syndrome
•Cereulide
•Gene carried on a plasmid
•Diarrhoealsyndrome
•Enterotoxins
B. thuringiensis
•Identification and classification
•Insecticidal crystal proteins
•Cry and Cytproteins
•Relevant genes almost exclusively carried on
plasmids
4
Main species
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Phylogenetic classification
•5 mayor clades of B. cereus sensu lato
•Phylogeny does not match phenotypes
5
Ehling-Schulz M et al., 2019
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Phylogenetic classification
•5 mayor clades of B. cereus sensu lato
•Phylogeny does not match phenotypes
5
Ehling-Schulz M et al., 2019
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Types of taxonomic issues
•Variable Genomospecies Threshold Problem
•Different ANI-based genomospecies
thresholds for species delineation
•Type Strain Centroid Problem
•Type strains are chosen as the central
point to define clusters
•Sub-optimal Genomospecies Threshold
Problem
•Selected threshold cannot effectively
delineate genomospecies clusters
•Phenotype-Centric Species Delineation
Problem
•Phenotype differs from genomospecies
6
Carrol LM et al., 2021
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Types of taxonomic issues
•Variable Genomospecies Threshold Problem
•Different ANI-based genomospecies
thresholds for species delineation
•Type Strain Centroid Problem
•Type strains are chosen as the central
point to define clusters
•Sub-optimal Genomospecies Threshold
Problem
•Selected threshold cannot effectively
delineate genomospecies clusters
•Phenotype-Centric Species Delineation
Problem
•Phenotype differs from genomospecies
6
Carrol LM et al., 2021
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Taxonomic framework
Taxonomic framework Classification approach (#)Recognized species
BAM Protocol for B. cereusPhenotypic (5)
B. anthracis, B. cereus, B. mycoides, B. thuringiensis, B. weihenstephanensis
NCBI Taxonomy Hybrid (User-Submitted) (22)
B. albus, B. anthracis, B. bingmayongensis, B. cereus, B. clarus, B. cytotoxicus, B. fungorum, B.
gaemokensis, B. luti, B. manliponensis, B. mobilis, B. mycoides, B. nitratireducens, B. pacificus, B.
paramycoides, B. paranthracis, B. proteolyticus, B. pseudomycoides, B. thuringiensis, B. toyonensis, B.
tropicus, B. wiedmannii
GTDB Taxonomy Genomic (40)
B. albus, B. anthracis, B. bingmayongensis, B. bombysepticus, B. cereus, B. cereus_AG, B. cereus_AK, B.
cereus_AQ, B. cereus_AT, B. cereus_AU, B. cereus_AV, B. cereus_AW, B. cereus_AZ, B. cereus_K, B.
cereus_O, B. cereus_S, B. cereus_U, B. cytotoxicus, B. gaemokensis, B. luti, B. manliponensis, B. mobilis, B.
mycoides, B. mycoides_A, B. mycoides_B, B. nitratireducens, B. paramycoides, B. paranthracis, B.
proteolyticus, B. pseudomycoides, B. sp002551815, B. sp002584985, B. sp002746455, B. thuringiensis, B.
thuringiensis_K, B. thuringiensis_N, B. thuringiensis_S, B. toyonensis, B. tropicus, B. wiedmannii
mOTUs/specI Taxonomy Genomic (10)
Cluster 327, Cluster 328, Cluster 329, Cluster 330, Cluster 331, Cluster 332, Cluster 333, Cluster 334, Cluster
335, Cluster 336
B. cereus s.l.
Genomospecies/Subspecies/
Biovar Taxonomy
Hybrid (Standardized) (12)B. bingmayongensis, B. cereus sensu stricto (s.s.), B. clarus, B. cytotoxicus, B. gaemokensis, B. luti, B.
manliponensis, B. mosaicus, B. mycoides, B. paramycoides, B. pseudomycoides, B. toyonensis
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Taxonomic framework
Taxonomic framework Classification approach (#)Recognized species
BAM Protocol for B. cereusPhenotypic (5)
B. anthracis, B. cereus, B. mycoides, B. thuringiensis, B. weihenstephanensis
NCBI Taxonomy Hybrid (User-Submitted) (22)
B. albus, B. anthracis, B. bingmayongensis, B. cereus, B. clarus, B. cytotoxicus, B. fungorum, B.
gaemokensis, B. luti, B. manliponensis, B. mobilis, B. mycoides, B. nitratireducens, B. pacificus, B.
paramycoides, B. paranthracis, B. proteolyticus, B. pseudomycoides, B. thuringiensis, B. toyonensis, B.
tropicus, B. wiedmannii
GTDB Taxonomy Genomic (40)
B. albus, B. anthracis, B. bingmayongensis, B. bombysepticus, B. cereus, B. cereus_AG, B. cereus_AK, B.
cereus_AQ, B. cereus_AT, B. cereus_AU, B. cereus_AV, B. cereus_AW, B. cereus_AZ, B. cereus_K, B.
cereus_O, B. cereus_S, B. cereus_U, B. cytotoxicus, B. gaemokensis, B. luti, B. manliponensis, B. mobilis, B.
mycoides, B. mycoides_A, B. mycoides_B, B. nitratireducens, B. paramycoides, B. paranthracis, B.
proteolyticus, B. pseudomycoides, B. sp002551815, B. sp002584985, B. sp002746455, B. thuringiensis, B.
thuringiensis_K, B. thuringiensis_N, B. thuringiensis_S, B. toyonensis, B. tropicus, B. wiedmannii
mOTUs/specI Taxonomy Genomic (10)
Cluster 327, Cluster 328, Cluster 329, Cluster 330, Cluster 331, Cluster 332, Cluster 333, Cluster 334, Cluster
335, Cluster 336
B. cereus s.l.
Genomospecies/Subspecies/
Biovar Taxonomy
Hybrid (Standardized) (12)B. bingmayongensis, B. cereus sensu stricto (s.s.), B. clarus, B. cytotoxicus, B. gaemokensis, B. luti, B.
manliponensis, B. mosaicus, B. mycoides, B. paramycoides, B. pseudomycoides, B. toyonensis
7
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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Conclusion
•Taxonomic space has undergone many
changes over the past 25 years
•It has ambiguous, unstandardized, and
conflicting nomenclature
•Complicatescommunication between
researchers and stakeholders
•Phylogeny cannot be trusted to
determine phenotypic traits or
pathogenecity
8
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Conclusion
•Taxonomic space has undergone many
changes over the past 25 years
•It has ambiguous, unstandardized, and
conflicting nomenclature
•Complicatescommunication between
researchers and stakeholders
•Phylogeny cannot be trusted to
determine phenotypic traits or
pathogenecity
8
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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9
Tak for idag!
DTU Food
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9
Tak for idag!
DTU Food
DTU FOOD Bacillus cereus sensu lato -phylogeny25-02-2025
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References
•Carroll, L. M.et al.(2021) ‘Keeping up with theBacillus cereusgroup: taxonomy through the genomics era and
beyond’,Critical Reviews in Food Science and Nutrition, 62(28), pp. 7677–7702. doi:
10.1080/10408398.2021.1916735.
•Ehling-Schulz M. et al. (2019) ‘ The Bacillus cereus Group: Bacillus Species with Pathogenic Potential’,
Microbiology Spectrum, volume7, Issue3. doi: 10.1128/microbiolspec.gpp3-0032-2018.
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References
•Carroll, L. M.et al.(2021) ‘Keeping up with theBacillus cereusgroup: taxonomy through the genomics era and
beyond’,Critical Reviews in Food Science and Nutrition, 62(28), pp. 7677–7702. doi:
10.1080/10408398.2021.1916735.
•Ehling-Schulz M. et al. (2019) ‘ The Bacillus cereus Group: Bacillus Species with Pathogenic Potential’,
Microbiology Spectrum, volume7, Issue3. doi: 10.1128/microbiolspec.gpp3-0032-2018.
10
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Food Risk Assessment Experience
with Bacillus cereus
Presentation to: Expert Group on BioPesticides (EGBP) seminar
February 25, 2025
Angela Catford, Section Head Risk Assessment
Bureau of Microbial Hazards, Food and Nutrition Directorate
Health Products and Food Branch, Health Canada
Food Risk Assessment Experience
with Bacillus cereus
Presentation to: Expert Group on BioPesticides (EGBP) seminar
February 25, 2025
Angela Catford, Section Head Risk Assessment
Bureau of Microbial Hazards, Food and Nutrition Directorate
Health Products and Food Branch, Health Canada
Unclassified / Non classifié
Purpose
•To share a Canadian experience in food safety health risk
assessment related to the evolving taxonomic classification of
Bacillus thuringiensis and Bacillus cereus
1
Purpose
•To share a Canadian experience in food safety health risk
assessment related to the evolving taxonomic classification of
Bacillus thuringiensis and Bacillus cereus
1
Unclassified / Non classifié
Bureau of Microbial Hazards
Mission statement:
To enhance the microbiological safety of the Canadian food supply and provide oversight of
novel foods by:
2
Health Products and Food Branch
Food and Nutrition Directorate
Health Canada
Bureau of Microbial Hazards
Microbiology
Evaluation
Division
Microbiology
Research
Division
•developing and communicating policies, guidelines
and standards,
•conducting laboratory research, risk assessments
and pre-market assessments, and
•providing science-based information to the Canadian
public and stakeholders to enable them to make
informed choices
Bureau of Microbial Hazards
Mission statement:
To enhance the microbiological safety of the Canadian food supply and provide oversight of
novel foods by:
2
Health Products and Food Branch
Food and Nutrition Directorate
Health Canada
Bureau of Microbial Hazards
Microbiology
Evaluation
Division
Microbiology
Research
Division
•developing and communicating policies, guidelines
and standards,
•conducting laboratory research, risk assessments
and pre-market assessments, and
•providing science-based information to the Canadian
public and stakeholders to enable them to make
informed choices
Unclassified / Non classifié
Context
Microbiological
Criteria in Canada*
Standards and Guidelines for
Microbiological Safety of Food
- An Interpretive Summary
3
Official Methods for
the Microbiological
Analysis of Foods
MFLP-42, Isolation and
Enumeration of the B. cereus
Group in Foods
*Formally under review:
https://www.canada.ca/en/health-canada/services/food-nutrition/research-programs-analytical-methods/analytical-
methods/compendium-methods/official-methods-microbiological-analysis-foods-compendium-analytical-methods.html
Food Safety
Investigation, Risk
Assessment & Recall
Triggered by:
•Outbreak
•Surveillance, food testing
Context
Microbiological
Criteria in Canada*
Standards and Guidelines for
Microbiological Safety of Food
- An Interpretive Summary
3
Official Methods for
the Microbiological
Analysis of Foods
MFLP-42, Isolation and
Enumeration of the B. cereus
Group in Foods
*Formally under review:
https://www.canada.ca/en/health-canada/services/food-nutrition/research-programs-analytical-methods/analytical-
methods/compendium-methods/official-methods-microbiological-analysis-foods-compendium-analytical-methods.html
Food Safety
Investigation, Risk
Assessment & Recall
Triggered by:
•Outbreak
•Surveillance, food testing
Unclassified / Non classifié
Canada’s food safety investigation and recall process
4
https://inspection.canada.ca/en/food-safety-consumers/how-we-decide-recall-food-product
Canada’s food safety investigation and recall process
4
https://inspection.canada.ca/en/food-safety-consumers/how-we-decide-recall-food-product
Unclassified / Non classifié
Health Canada - Health Risk Assessments (HRA)
•Follow Codex Alimentarius guidelines
✓CXG 63-2007 Principles and Guidelines for the Conduct of Microbiological Risk Management
✓CXG 30-1999 Principles and Guidelines for the Conduct of Microbiological Risk Assessment
✓4-steps: Hazard Identification, Hazard Characterization, Exposure Assessment, Risk Characterization
•Enabled through a Memorandum of Understanding between multiple departments and
agencies in Canada and driven via joint Standard Operating Procedure between the
Canadian Food Inspection Agency’s (CFIA) Office of Food Safety and Recall, and
Health Canada’s Food and Nutrition Directorate
•HRA Timeline: 8-48 hours for rapid decision making
5
Health Canada - Health Risk Assessments (HRA)
•Follow Codex Alimentarius guidelines
✓CXG 63-2007 Principles and Guidelines for the Conduct of Microbiological Risk Management
✓CXG 30-1999 Principles and Guidelines for the Conduct of Microbiological Risk Assessment
✓4-steps: Hazard Identification, Hazard Characterization, Exposure Assessment, Risk Characterization
•Enabled through a Memorandum of Understanding between multiple departments and
agencies in Canada and driven via joint Standard Operating Procedure between the
Canadian Food Inspection Agency’s (CFIA) Office of Food Safety and Recall, and
Health Canada’s Food and Nutrition Directorate
•HRA Timeline: 8-48 hours for rapid decision making
5
Unclassified / Non classifié
Microbiological Criteria* for B. cereus in Canada
6
Food Category n c m M
Instant Infant Cereal 10 1 10
2
10
4
Spices (Ready-to-eat only) 5 2 10
4
10
6
Raw Organ Derived Products and Herbal Products (in
tablets, capsules or powders, consumed at <10 g/day)
5 1 10
4
10
5
Powdered Protein, Meal Replacements, and Dietary
Supplements
5 2 10
1
10
2
*The microbiological guidelines above, along with other HC microbiological guidelines and standards, are being
reviewed as part of a Regulatory Modernization project currently under way.
Microbiological Criteria* for B. cereus in Canada
6
Food Category n c m M
Instant Infant Cereal 10 1 10
2
10
4
Spices (Ready-to-eat only) 5 2 10
4
10
6
Raw Organ Derived Products and Herbal Products (in
tablets, capsules or powders, consumed at <10 g/day)
5 1 10
4
10
5
Powdered Protein, Meal Replacements, and Dietary
Supplements
5 2 10
1
10
2
*The microbiological guidelines above, along with other HC microbiological guidelines and standards, are being
reviewed as part of a Regulatory Modernization project currently under way.
Unclassified / Non classifié
7
Bacillus group outbreaks in Canada reported in the
Publicly available international foodborne outbreak database
7
Bacillus group outbreaks in Canada reported in the
Publicly available international foodborne outbreak database
Unclassified / Non classifié
Outbreak Context - Canada
•A Canadian outbreak of note occurred in a long-term care institution where it was reported that
all clinical isolates were identified as B. thuringiensis, although B. thuringiensis was not
isolated from any of the food samples tested (Jackson, S.G. et al., 1995)
•A retrospective study to re-characterize Bacillus food-poisoning strains from 39 outbreaks in
British Columbia identified four outbreak-linked strains as B.thuringiensis; one of these had
initially been identified as B.cereus based on phenotype, but was later characterized by PCR
as B.thuringiensis (McIntyre, L.,et al., 2008)
8
Outbreak Context - Canada
•A Canadian outbreak of note occurred in a long-term care institution where it was reported that
all clinical isolates were identified as B. thuringiensis, although B. thuringiensis was not
isolated from any of the food samples tested (Jackson, S.G. et al., 1995)
•A retrospective study to re-characterize Bacillus food-poisoning strains from 39 outbreaks in
British Columbia identified four outbreak-linked strains as B.thuringiensis; one of these had
initially been identified as B.cereus based on phenotype, but was later characterized by PCR
as B.thuringiensis (McIntyre, L.,et al., 2008)
8
Unclassified / Non classifié
The B. cereus group in food – Take away messages
•Practically, food context testing is unable to distinguish between B. cereus and B. thurengiensis
•Outbreak data and literature indicate that B. cereus and some strains of B. thurengiensis can
cause foodborne illness
•Risk Assessment and Risk Management (i.e., the food safety investigation, HRA and recall
process in Canada) demand rapid timelines, accounting for uncertainty and the application of
precaution
•In October 2023, Health Canada clarified that MFLP-42 enumeration results shall be interpreted
as a confirmation of the levels reported for the B. cereus group in accordance with the
guidelines in the Interpretive Summary (i.e., the existing Health Canada microbiological criteria)
•Foods for which Health Canada has no microbiological guidelines shall be assessed on a case-
by-case basis, and all assessments are based on the specific situation being assessed
9
The B. cereus group in food – Take away messages
•Practically, food context testing is unable to distinguish between B. cereus and B. thurengiensis
•Outbreak data and literature indicate that B. cereus and some strains of B. thurengiensis can
cause foodborne illness
•Risk Assessment and Risk Management (i.e., the food safety investigation, HRA and recall
process in Canada) demand rapid timelines, accounting for uncertainty and the application of
precaution
•In October 2023, Health Canada clarified that MFLP-42 enumeration results shall be interpreted
as a confirmation of the levels reported for the B. cereus group in accordance with the
guidelines in the Interpretive Summary (i.e., the existing Health Canada microbiological criteria)
•Foods for which Health Canada has no microbiological guidelines shall be assessed on a case-
by-case basis, and all assessments are based on the specific situation being assessed
9
Unclassified / Non classifié
Thank you
Bureau of Microbial Hazards
Food and Nutrition Directorate
Health Products and Food Branch
Health Canada
[email protected]
10
Thank you
Bureau of Microbial Hazards
Food and Nutrition Directorate
Health Products and Food Branch
Health Canada
[email protected]
10
«Identification of Genetic Markers for the Detection of Bacillus
thuringiensis Strains of Interest for Food Safety »
13
th
Expert Group on BioPesticides Seminar, 25 February 2025
Mathilde BONIS* and Arnaud FELTEN
Anses, Maisons-Alfort, France,
*Laboratory for Food Safety,
«Staphylococcus, Bacillusand Clostridium Unit»
thuringiensis Strains of Interest for Food Safety »
13
th
Expert Group on BioPesticides Seminar, 25 February 2025
Mathilde BONIS* and Arnaud FELTEN
Anses, Maisons-Alfort, France,
*Laboratory for Food Safety,
«Staphylococcus, Bacillusand Clostridium Unit»
Bacterial foodborne outbeaks (FBOs) in France
About2000FBOsreportedannuallyinFrance,
~16000cases,and14deaths(SantéPublique
France,SPF)
Bacillus cereus (Bc): 1
st
cause of FBOs in France
since 2021
1
st
cause of FBOs due to bacterial toxins in Europe
(EFSA, 2022), with ~90% of Bc-FBO reported by Fr.
•In2022:462outbreaks,3961humancases,
86hospitalizations
•Sporadicdeaths(0à3/year)
•Probableunderestimation/under-reporting
?
Santé Publique France (SPF) data (https://www.santepubliquefrance.fr/)
About2000FBOsreportedannuallyinFrance,
~16000cases,and14deaths(SantéPublique
France,SPF)
Bacillus cereus (Bc): 1
st
cause of FBOs in France
since 2021
1
st
cause of FBOs due to bacterial toxins in Europe
(EFSA, 2022), with ~90% of Bc-FBO reported by Fr.
•In2022:462outbreaks,3961humancases,
86hospitalizations
•Sporadicdeaths(0à3/year)
•Probableunderestimation/under-reporting
?
Santé Publique France (SPF) data (https://www.santepubliquefrance.fr/)
Bacterial
strain
(virulence factors,
regulations, fitness
etc.)
Host immune status
(elderly people, NN, or more fragile
immunocompromised people)
Ingested
dose
(spores/vegetatives
cells)
FBO
FBO: the result of an equation with 3 variables
Sometimes difficult to
predict the pathogenicity
of a given strain
strain
(virulence factors,
regulations, fitness
etc.)
Host immune status
(elderly people, NN, or more fragile
immunocompromised people)
Ingested
dose
(spores/vegetatives
cells)
FBO
FBO: the result of an equation with 3 variables
Sometimes difficult to
predict the pathogenicity
of a given strain
Bc-associated FBOs
Cereulide +/-isoforms (ces)
Symptoms=nausea,
vomiting++(0.5-6h)
Untilorganfailure.
Severalcasesoffatal
outcomes
Vagus nerve stimulation
Symptoms=diarrhoea,
abdominalpain,nausea,
vomiting(6-24h)
In vivo production of enterotoxins
Hbl, Nhe, Cyt K1/2
+ Hémolysines, Phospholipases C, Sphingomyelinase
Protéases, Collagénases etc … +/-synergie
Enteropathogenic Bc
Cereulide +/-isoforms (ces)
Symptoms=nausea,
vomiting++(0.5-6h)
Untilorganfailure.
Severalcasesoffatal
outcomes
Vagus nerve stimulation
Symptoms=diarrhoea,
abdominalpain,nausea,
vomiting(6-24h)
In vivo production of enterotoxins
Hbl, Nhe, Cyt K1/2
+ Hémolysines, Phospholipases C, Sphingomyelinase
Protéases, Collagénases etc … +/-synergie
Enteropathogenic Bc
Bc group: a species complex
Molecular typing
e.g. panC-typing(Guinebretière et al., 2008)
Phenotypic traits
B. thuringiensisB. anthracisB. mycoides
Genomospecies:
2000 2005 2010 2015 2020
12ANI-based(92.5ANIthreshold)
genomospeciesassignmentproposedby
Carrolletal.in2020
+/-Subspecies+/-Biovars
Carroll L. et al., 2021
Growth temperature (°C)
Phylogenetic group
Molecular typing
e.g. panC-typing(Guinebretière et al., 2008)
Phenotypic traits
B. thuringiensisB. anthracisB. mycoides
Genomospecies:
2000 2005 2010 2015 2020
12ANI-based(92.5ANIthreshold)
genomospeciesassignmentproposedby
Carrolletal.in2020
+/-Subspecies+/-Biovars
Carroll L. et al., 2021
Growth temperature (°C)
Phylogenetic group
Diversity of Bc associated to FBOs in France
(Mozhaitseva K. et al., article under preparation)
PopulationstructureofB.cereuss.l.
(n=294)isolatedduringfoodborne
outbreaksinFrancebetween2004and
2023:
7 genomospecies (as defined by Carroll et al. 2020), 15 popCOGenT clusters, and subclusters etc.
Variety of virulence, AMR and heavy metal resistance genes
Enterotoxins
genesces
(Mozhaitseva K. et al., article under preparation)
PopulationstructureofB.cereuss.l.
(n=294)isolatedduringfoodborne
outbreaksinFrancebetween2004and
2023:
7 genomospecies (as defined by Carroll et al. 2020), 15 popCOGenT clusters, and subclusters etc.
Variety of virulence, AMR and heavy metal resistance genes
Enterotoxins
genesces
Europe(EuropeanComissionregulationNo1441/2007):
-NospecificfoodsafetycriterionrelativetoBc,exceptedfordriedpowderedpreparationsintendedfor
infantsbelow6monthsofage:m50ufc/gM500ufc/g
France:
-Recommendations:
AFSSA2007-SA-0174:alimitof100ufc/gisrecommandedforcookedmeats,delicatessenproducts(readymealsand
starters)
AFSSA2008-SA-0359:listoffoodstobetargeted,especiallyready-mademeals,productswithspices,herbsoraromatics,
reconstituteddehydratedfoods
-Alertthreshold:10
5
cfu/g(DGAL/MUS/N2009-8188,2009)
-Noreferencelaboratoy(LNR/EURL)forBc(USBCL«LNR-like»,DGAL/MUS/2024-81,15/02/24)
Bacillus cereus and food safety regulation
The issue of the acceptable dose of Bc in food is regularly tackled
In Glasset et al., 2016 : < 10
5
cfu/g of presumptive Bc for 84% of FBO
-NospecificfoodsafetycriterionrelativetoBc,exceptedfordriedpowderedpreparationsintendedfor
infantsbelow6monthsofage:m50ufc/gM500ufc/g
France:
-Recommendations:
AFSSA2007-SA-0174:alimitof100ufc/gisrecommandedforcookedmeats,delicatessenproducts(readymealsand
starters)
AFSSA2008-SA-0359:listoffoodstobetargeted,especiallyready-mademeals,productswithspices,herbsoraromatics,
reconstituteddehydratedfoods
-Alertthreshold:10
5
cfu/g(DGAL/MUS/N2009-8188,2009)
-Noreferencelaboratoy(LNR/EURL)forBc(USBCL«LNR-like»,DGAL/MUS/2024-81,15/02/24)
Bacillus cereus and food safety regulation
The issue of the acceptable dose of Bc in food is regularly tackled
In Glasset et al., 2016 : < 10
5
cfu/g of presumptive Bc for 84% of FBO
8
Search for 3 toxin producing bacteria:
Staphyloccocus aureus*
Clostridium perfringens
Bacillus cereus
+
Salmonella spp.*
Listeria monocytogenes*
STEC
Campylobacter
+
Hygiene indicators (Enterobacteriaceae…)
Certified laboratory, LCSV (NF EN ISO 7932)
FBO investigation in France
(reportable disease since 1987)
* Existence of a French reference laboratory (LNR)
+/-Search for enteric viruses*, marine toxins etc…
ARS/DDPP
Epidemiological and food investigation + food sampling
+
USBCL, Bacillusand Clostridiumteam
(Bc«LNR-like»)
Bcconfirmation + characterization
Phenotypic traits : hemolysis,
lecithinase, starch hydrolysis
Genotypic traits :
-M13-PCR (RAPD) typing
-Phylogenetic group (panCpartial sequencing)
-PCR for hlyII, cytK1/2, nheA/B/C, HblC/D/A, ces
Detection/quantification of toxins :
Nhe, Hbl Bt crystals
NF EN ISO 7932, Amd 1:2020
NGS
Cereulide
Search for 3 toxin producing bacteria:
Staphyloccocus aureus*
Clostridium perfringens
Bacillus cereus
+
Salmonella spp.*
Listeria monocytogenes*
STEC
Campylobacter
+
Hygiene indicators (Enterobacteriaceae…)
Certified laboratory, LCSV (NF EN ISO 7932)
FBO investigation in France
(reportable disease since 1987)
* Existence of a French reference laboratory (LNR)
+/-Search for enteric viruses*, marine toxins etc…
ARS/DDPP
Epidemiological and food investigation + food sampling
+
USBCL, Bacillusand Clostridiumteam
(Bc«LNR-like»)
Bcconfirmation + characterization
Phenotypic traits : hemolysis,
lecithinase, starch hydrolysis
Genotypic traits :
-M13-PCR (RAPD) typing
-Phylogenetic group (panCpartial sequencing)
-PCR for hlyII, cytK1/2, nheA/B/C, HblC/D/A, ces
Detection/quantification of toxins :
Nhe, Hbl Bt crystals
NF EN ISO 7932, Amd 1:2020
NGS
Cereulide
Starting point on Bacillus thuringiensis:
RisksforpublichealthrelatedtothepresenceofBacillus
cereusandotherBacillusspp.includingBacillus
thuringiensisinfoodstuffs.
Published:20july2016 ?
*
12 %Bt-associated FBO (with
anothermicro-organism)
8 %Bt-associated FBO
(onlyBtdetected)
20%→Bt
+
Inpresenceofatleast1Btisolatefor20%Bc-
associatedFBOs
Btisolatesmorefrequentlyassociatedwithraw
vegetables(e.g.tomatoes9x)thanBcnon-Bt
isolates
Wholegenomesanalysesdonotdiscriminate
FBO-Btfrom7commercialBtkurstakioraizawai
250B cereus-associated FBOs
(France, 2007-2017)
*This project was funded and supported by ANSES (PPV project “Bt-TIAC”, 2018) via the tax on sales of plant protection products
Need to develop genomic tools to specifically
detect Bt biopesticide strains
RisksforpublichealthrelatedtothepresenceofBacillus
cereusandotherBacillusspp.includingBacillus
thuringiensisinfoodstuffs.
Published:20july2016 ?
*
12 %Bt-associated FBO (with
anothermicro-organism)
8 %Bt-associated FBO
(onlyBtdetected)
20%→Bt
+
Inpresenceofatleast1Btisolatefor20%Bc-
associatedFBOs
Btisolatesmorefrequentlyassociatedwithraw
vegetables(e.g.tomatoes9x)thanBcnon-Bt
isolates
Wholegenomesanalysesdonotdiscriminate
FBO-Btfrom7commercialBtkurstakioraizawai
250B cereus-associated FBOs
(France, 2007-2017)
*This project was funded and supported by ANSES (PPV project “Bt-TIAC”, 2018) via the tax on sales of plant protection products
Need to develop genomic tools to specifically
detect Bt biopesticide strains
Bt associated with FBOs: a clonal population
NGSofn=172Bc/Btgenomes(isolatedfromFBOsorBtpesticides)+62
variousBcgenomes(NCBI)
K-mer-basedphylogeny(size=17,sketchsize=10000),Neighbour-Joining
method
1 major clade made of 152 genetically close Bt isolates (Bt gp IV) with > 95% of
FBO-Bt + 100% of commercial Bt isolates ssp. kurstakiand aizawai
Bt israelensis
Bt morrisoni
Bt kurstaki
Bt aizawai
Bt pesticides:
Variantcallinganalysis(iVARCall2),n=154BtfrompanCgroupIV
Phylogeneticreconstructionfrompseudogenomesandaftercorrectionof
recombinationevents(ClonalFrameML,IQtree,K3Pmodel)
MedianSNP
differences/cluster
2
0
1
1
0 to 10 SNP of distance between Bt isolates, within a same cluster
Inter-cluster SNP distances significantly higher than intra-cluster SNP
distances, for each cluster
But definition of
4 clusters (a to d):
Ssp. kurstaki
Ssp. aizawai
NGSofn=172Bc/Btgenomes(isolatedfromFBOsorBtpesticides)+62
variousBcgenomes(NCBI)
K-mer-basedphylogeny(size=17,sketchsize=10000),Neighbour-Joining
method
1 major clade made of 152 genetically close Bt isolates (Bt gp IV) with > 95% of
FBO-Bt + 100% of commercial Bt isolates ssp. kurstakiand aizawai
Bt israelensis
Bt morrisoni
Bt kurstaki
Bt aizawai
Bt pesticides:
Variantcallinganalysis(iVARCall2),n=154BtfrompanCgroupIV
Phylogeneticreconstructionfrompseudogenomesandaftercorrectionof
recombinationevents(ClonalFrameML,IQtree,K3Pmodel)
MedianSNP
differences/cluster
2
0
1
1
0 to 10 SNP of distance between Bt isolates, within a same cluster
Inter-cluster SNP distances significantly higher than intra-cluster SNP
distances, for each cluster
But definition of
4 clusters (a to d):
Ssp. kurstaki
Ssp. aizawai
Figure 2. Phylogenetic tree of 230 Bc genomes, alongside the gene
presence-absence variation matrix
Core genome of 1854 genes (~5% of all genes), 37,167 accessory genes
1. Microbial genome-wide association studies (mGWAS) :
GWAS approach allowing the identification of genomic variants (genes, k-mers or SNPs)
that are statistically associated with a given trait
n=287 genomes (collection + NCBI)
3 levels of traits analysed
Study dataset (SD) → mGWAS analysis (n=230, 80%)
Validation dataset (VD) → in silico validation (n=57, 20%)
kurstaki
aizawai
https://www.wikiwand.com/en/Pan-genome
presence-absence variation matrix
Core genome of 1854 genes (~5% of all genes), 37,167 accessory genes
1. Microbial genome-wide association studies (mGWAS) :
GWAS approach allowing the identification of genomic variants (genes, k-mers or SNPs)
that are statistically associated with a given trait
n=287 genomes (collection + NCBI)
3 levels of traits analysed
Study dataset (SD) → mGWAS analysis (n=230, 80%)
Validation dataset (VD) → in silico validation (n=57, 20%)
kurstaki
aizawai
https://www.wikiwand.com/en/Pan-genome
Selectionof14specificBtmarkers
Bt_typing:Script(Python3)fortheautomatedsearch
ofthesemarkersfrompresumptiveBcgenome
(https://github.com/afelten-Anses/Bt_typing)
1. Microbial genome-wide association studies (mGWAS):GWAS
analysis
Gene name Replicon Annotation UniprotKB Trait
Marker
Sensitivity (%)
Cumulative
Sensitivity (%)
cwlA Chromosome
N-acetylmuramoyl-L-
alanine amidase CwlA
P24808 Bt 93.8 93.8
intQ Chromosome
Putative defective protein
IntQ
P76168 Bt 91.7 94.4
group_3916 ChromosomeHypothetical protein N/A Bt 88.9 95.1
group_20749ChromosomeHypothetical protein N/A Bt 86.8 97.2
group_20361Plasmid Hypothetical protein N/A Bt 84.7 98.6
sdpR Plasmid
Transcriptional
repressor SdpR
O32242 Bt 84.0 100.0
apr ChromosomeSubtilisin P04189 Bta 100.0 N/A
group_27293Plasmid Hypothetical protein N/A Btk 98.2 98.2
group_27336Plasmid Hypothetical protein N/A Btk 89.5 100.0
group_10114ChromosomeHypothetical protein N/A Cluster a 100.0 N/A
rapF Chromosome
Response regulator
aspartate phosphatase F
P71002 Cluster b 92.5 92.5
group_20667Plasmid Hypothetical protein N/A Cluster b 82.5 100.0
lexA ChromosomeLexA repressor P31080 Cluster c 100.0 N/A
clpP1 Chromosome
ATP-dependent Clp
subunit 1
B0B803 Cluster d 100.0 N/A
L1
L2
L3
Table 3.List of 14 genes with strong evidence and best combinations for Bacillus thuringiensis (Bt)
trait identification associated with three GWAS analysis.
Probably not covering the entire diversity of Bt species due to
the studied dataset (focus on Bt food safety)
Upgradable tool (for instance for putative new strains approved)
Does not allow identification of Bt pesticides at strain level
Bt-typing decisional tree:
EG2348 / SA-12 / PB-54 SA11 / ABTS-351
Cluster c Cluster d
Bt_typing:Script(Python3)fortheautomatedsearch
ofthesemarkersfrompresumptiveBcgenome
(https://github.com/afelten-Anses/Bt_typing)
1. Microbial genome-wide association studies (mGWAS):GWAS
analysis
Gene name Replicon Annotation UniprotKB Trait
Marker
Sensitivity (%)
Cumulative
Sensitivity (%)
cwlA Chromosome
N-acetylmuramoyl-L-
alanine amidase CwlA
P24808 Bt 93.8 93.8
intQ Chromosome
Putative defective protein
IntQ
P76168 Bt 91.7 94.4
group_3916 ChromosomeHypothetical protein N/A Bt 88.9 95.1
group_20749ChromosomeHypothetical protein N/A Bt 86.8 97.2
group_20361Plasmid Hypothetical protein N/A Bt 84.7 98.6
sdpR Plasmid
Transcriptional
repressor SdpR
O32242 Bt 84.0 100.0
apr ChromosomeSubtilisin P04189 Bta 100.0 N/A
group_27293Plasmid Hypothetical protein N/A Btk 98.2 98.2
group_27336Plasmid Hypothetical protein N/A Btk 89.5 100.0
group_10114ChromosomeHypothetical protein N/A Cluster a 100.0 N/A
rapF Chromosome
Response regulator
aspartate phosphatase F
P71002 Cluster b 92.5 92.5
group_20667Plasmid Hypothetical protein N/A Cluster b 82.5 100.0
lexA ChromosomeLexA repressor P31080 Cluster c 100.0 N/A
clpP1 Chromosome
ATP-dependent Clp
subunit 1
B0B803 Cluster d 100.0 N/A
L1
L2
L3
Table 3.List of 14 genes with strong evidence and best combinations for Bacillus thuringiensis (Bt)
trait identification associated with three GWAS analysis.
Probably not covering the entire diversity of Bt species due to
the studied dataset (focus on Bt food safety)
Upgradable tool (for instance for putative new strains approved)
Does not allow identification of Bt pesticides at strain level
Bt-typing decisional tree:
EG2348 / SA-12 / PB-54 SA11 / ABTS-351
Cluster c Cluster d
Genes
k-mers
cgSNPs
Pangenome
SNPs
→ No specific kmers (k=250)
detected
→ No specific SNP
detected with Snippy
What strategy ?
→ Resolution not sufficient
?
Search for strain-specific markers
Bt pesticide
strains to target:Bt strainSubspeciesAMM nb of genomes
ABTS-351kurstaki2010513 5
SA-11 kurstaki9200482 12
EG2348 kurstaki2160494 2
SA-12 kurstaki2170466 2
PB-54 kurstaki2200701 2
ABTS-1857aizawai2020241 3
GC-91 aizawai2170436 10
TOTAL 7 - - 36
Dataset n=66 Bt genomes, with reliable annotation:
Bt pesticide
strains
Bt non-pesticide
strains
k-mers
cgSNPs
Pangenome
SNPs
→ No specific kmers (k=250)
detected
→ No specific SNP
detected with Snippy
What strategy ?
→ Resolution not sufficient
?
Search for strain-specific markers
Bt pesticide
strains to target:Bt strainSubspeciesAMM nb of genomes
ABTS-351kurstaki2010513 5
SA-11 kurstaki9200482 12
EG2348 kurstaki2160494 2
SA-12 kurstaki2170466 2
PB-54 kurstaki2200701 2
ABTS-1857aizawai2020241 3
GC-91 aizawai2170436 10
TOTAL 7 - - 36
Dataset n=66 Bt genomes, with reliable annotation:
Bt pesticide
strains
Bt non-pesticide
strains
2. Pangenome SNP (/INDEL) analyses
Satisfactory results for GC-91, SA-11 and [SA-11/ABTS-351]
Promising for SA-12 and EG2348 → to validate (insufficient nb of genomes)
Unsatisfactory for ABTS-1857, ABTS-351 and PB-54 (genome diversity ?)
No marker to differentiate SA-11 and ABTS-351 (cluster d)
Global analysis
Identification of 2 SNPs distinguing these two strains
[SA-11 /
ABTS-351]
Search for
2 SNPs
SA-11
ABTS-351
Specific analysis for [SA-11/ABTS-351]
Search for [SA-11/ABTS-351]
markers
Satisfactory results for GC-91, SA-11 and [SA-11/ABTS-351]
Promising for SA-12 and EG2348 → to validate (insufficient nb of genomes)
Unsatisfactory for ABTS-1857, ABTS-351 and PB-54 (genome diversity ?)
No marker to differentiate SA-11 and ABTS-351 (cluster d)
Global analysis
Identification of 2 SNPs distinguing these two strains
[SA-11 /
ABTS-351]
Search for
2 SNPs
SA-11
ABTS-351
Specific analysis for [SA-11/ABTS-351]
Search for [SA-11/ABTS-351]
markers
Plant family Food product n
Samples tested positive
for Bc
n (%)
a
Samples tested
positive for Bt
n (%)
b
Bc CFU/g
(mean)
a
Bc CFU/g
(min-max)
a
Bt CFU/g
(mean)
c
Bt CFU/g
(min-max)
c
Tomato 32 20 (63%) 20 (63%) 4.8 x 10
3
10
1
-7.7 x 10
4
4.8 x 10
3
10
1
-7.7 x 10
4
Pepper 24 16 (67%) 16 (67%) 2.5 x 10
3
10
1
-3.0 x 10
4
2.5 x 10
3
10
1
-3.0 x 10
4
Cucumber 20 7 (35%) 6 (30%) 5.3 x 10
1
10
1
-2.1 x 10
2
6.8 x 10
1
10
1
-2.1 x 10
2
Zucchini 8 3 (38%) 0 (0%) 2.0 X 10
1
10
1
-3.0 x 10
1
< Ld < Ld
Brassicaceae Cabbages 24 14 (58%) 9 (38%) 1.1 x 10
4
10
1
-9.6 x 10
4
1.7 x 10
4
10
1
-9.6 x 10
4
Rosaceae Apple 23 15 (65%) 14 (61%) 6.6 x 10
1
10
1
-2.4 x 10
2
6,9 x 10
1
10
1
-2.4 x 10
2
Asteraceae Lettuce 18 17 (94%) 8 (44%) 3.0 x 10
2
3.0 x 10
1
-1.6 x 10
3
4,0 x 10
2
4.0 x 10
2
Lamiaceae Aromatic herbs 8 8 (100%) 6 (75%) 2.1 x 10
2
2.0 x 10
1
-7.5 x 10
2
1,4 x 10
2
3.0 x 10
1
-3.5 x 10
2
Vitaceae Grape 2 0 (0%) 0 (0%) < Ld < Ld < Ld < Ld
TOTAL TOTAL 159 100 (63%) 79 (50%) 3.0 x 10
3
10
1
-9.6 x 10
4
4,3 x 10
3
10
1
-9.6 x 10
4
Solanaceae (n=56)
Cucurbitaceae (n=28) Markers tested on Bt isolated from fresh fruits/vegetables
1. Collect of 161 organic fruits/vegetables
•Intended to be consumed unpeeled, raw or minimally processed,
•Randomly collected in supermarkets, organic shops, retail markets
•Origin France ~ 95%
2. Isolation of viable B cereus(NF ISO 7932)
IsolationofBcin64%offoodsamples
Mean=3.0 x 10
3
cfu/g
3. Search for Btcrystals (NF ISO 7932, Amd1)
Presence of Bt in 50% of tested food samples
Mean=4.3 x 10
3
cfu/g
Highest prevalence for
aromatic herbs, peppers and
tomatoes
Highest counts reported for
cabbage (9.6 x 10
4
cfu/g)
Samples tested positive
for Bc
n (%)
a
Samples tested
positive for Bt
n (%)
b
Bc CFU/g
(mean)
a
Bc CFU/g
(min-max)
a
Bt CFU/g
(mean)
c
Bt CFU/g
(min-max)
c
Tomato 32 20 (63%) 20 (63%) 4.8 x 10
3
10
1
-7.7 x 10
4
4.8 x 10
3
10
1
-7.7 x 10
4
Pepper 24 16 (67%) 16 (67%) 2.5 x 10
3
10
1
-3.0 x 10
4
2.5 x 10
3
10
1
-3.0 x 10
4
Cucumber 20 7 (35%) 6 (30%) 5.3 x 10
1
10
1
-2.1 x 10
2
6.8 x 10
1
10
1
-2.1 x 10
2
Zucchini 8 3 (38%) 0 (0%) 2.0 X 10
1
10
1
-3.0 x 10
1
< Ld < Ld
Brassicaceae Cabbages 24 14 (58%) 9 (38%) 1.1 x 10
4
10
1
-9.6 x 10
4
1.7 x 10
4
10
1
-9.6 x 10
4
Rosaceae Apple 23 15 (65%) 14 (61%) 6.6 x 10
1
10
1
-2.4 x 10
2
6,9 x 10
1
10
1
-2.4 x 10
2
Asteraceae Lettuce 18 17 (94%) 8 (44%) 3.0 x 10
2
3.0 x 10
1
-1.6 x 10
3
4,0 x 10
2
4.0 x 10
2
Lamiaceae Aromatic herbs 8 8 (100%) 6 (75%) 2.1 x 10
2
2.0 x 10
1
-7.5 x 10
2
1,4 x 10
2
3.0 x 10
1
-3.5 x 10
2
Vitaceae Grape 2 0 (0%) 0 (0%) < Ld < Ld < Ld < Ld
TOTAL TOTAL 159 100 (63%) 79 (50%) 3.0 x 10
3
10
1
-9.6 x 10
4
4,3 x 10
3
10
1
-9.6 x 10
4
Solanaceae (n=56)
Cucurbitaceae (n=28) Markers tested on Bt isolated from fresh fruits/vegetables
1. Collect of 161 organic fruits/vegetables
•Intended to be consumed unpeeled, raw or minimally processed,
•Randomly collected in supermarkets, organic shops, retail markets
•Origin France ~ 95%
2. Isolation of viable B cereus(NF ISO 7932)
IsolationofBcin64%offoodsamples
Mean=3.0 x 10
3
cfu/g
3. Search for Btcrystals (NF ISO 7932, Amd1)
Presence of Bt in 50% of tested food samples
Mean=4.3 x 10
3
cfu/g
Highest prevalence for
aromatic herbs, peppers and
tomatoes
Highest counts reported for
cabbage (9.6 x 10
4
cfu/g)
Bt strain prediction
4. Search for SNP markers in the genome of n=87 Bt isolates (from 78 food products)n % n % n %
Isolates assigned to ABTS-351 47 54% 20 51% 6 35%
Isolates assigned to SA-11 28 32% 9 23% 10 59%
Isolates assigned to SA-12 3 3% 3 8% 0 0%
Isolates assigned to EG2348 1 1% 1 3% 0 0%
TOTAL 79 91% 33 85% 16 94%
Unassigned isolates- 8 9% 6 15% 1 6%
TOTAL - 87 100% 39 100% 17 100%
All food productsSolanaceae Rosaceae
Isolates with Bt pesticide
strain prediction
(n=79/87, 91%)
91% of Bt assigned to a Bt pesticide strain (predictions in line with
clusters attribution and Cry family signatures)
ABTS-351 (54%) > SA-11 (32%), consistent with Bt sales data
Reversed ranking for Rosaceae
8 (9%) unassigned isolates, including 5 with profiles similar to ABTS-
1857 (typing results, Cry families)
6food products (8%) with a mix of Bt strains
Le Strat A. et al., Article under preparation
BNV-D© database
(https://ventes-produits-phytopharmaceutiques.eaufrance.fr/search,
accessed on February 07, 2025
Bt sales in France, 2022 (BNVD)
ABTS-351
SA-11
Other
4. Search for SNP markers in the genome of n=87 Bt isolates (from 78 food products)n % n % n %
Isolates assigned to ABTS-351 47 54% 20 51% 6 35%
Isolates assigned to SA-11 28 32% 9 23% 10 59%
Isolates assigned to SA-12 3 3% 3 8% 0 0%
Isolates assigned to EG2348 1 1% 1 3% 0 0%
TOTAL 79 91% 33 85% 16 94%
Unassigned isolates- 8 9% 6 15% 1 6%
TOTAL - 87 100% 39 100% 17 100%
All food productsSolanaceae Rosaceae
Isolates with Bt pesticide
strain prediction
(n=79/87, 91%)
91% of Bt assigned to a Bt pesticide strain (predictions in line with
clusters attribution and Cry family signatures)
ABTS-351 (54%) > SA-11 (32%), consistent with Bt sales data
Reversed ranking for Rosaceae
8 (9%) unassigned isolates, including 5 with profiles similar to ABTS-
1857 (typing results, Cry families)
6food products (8%) with a mix of Bt strains
Le Strat A. et al., Article under preparation
BNV-D© database
(https://ventes-produits-phytopharmaceutiques.eaufrance.fr/search,
accessed on February 07, 2025
Bt sales in France, 2022 (BNVD)
ABTS-351
SA-11
Other
Conclusions
1. Identification and in silico validation of molecular markers (genes) specific to “Bt” (as
defined by the dataset), its subspecies aizawai, kurstakiand four previously described
proximity clusters associated with these subspecies
Bt_typing: command line tool based on a 14-marker workflow, to carry out a
computational search for Bt-related markers from a putative Bc genome assembly
(https://github.com/afelten-Anses/Bt_typing)
2. Identification and the in silico validation of SNP/INDEL molecular markers for Bt
pesticide strains assignment
Satisfying for strains SA-11, ABTS-351, and GC-91 (>90% of Bt sales in Fr)
To confirm for EG2348 and SA-12 (insufficient nb of genomes)
Not conclusive for PB-54 and ABTS-1857
Only few SNPs to distinguish SA-11 and ABTS-351
Bt of interest for food
safety:
GC-91
EG2348 / SA-12 /
PB-54
Ssp. aizawai:
Ssp. kurstaki:
Cluster c
Cluster d
SA11 / ABTS-351
ABTS-1857
Cluster a
Cluster b
Article in preparation
Identification of several markers allowing to discrimination of Bt pesticide kurstakiand aizawaiat different
levels:
1. Identification and in silico validation of molecular markers (genes) specific to “Bt” (as
defined by the dataset), its subspecies aizawai, kurstakiand four previously described
proximity clusters associated with these subspecies
Bt_typing: command line tool based on a 14-marker workflow, to carry out a
computational search for Bt-related markers from a putative Bc genome assembly
(https://github.com/afelten-Anses/Bt_typing)
2. Identification and the in silico validation of SNP/INDEL molecular markers for Bt
pesticide strains assignment
Satisfying for strains SA-11, ABTS-351, and GC-91 (>90% of Bt sales in Fr)
To confirm for EG2348 and SA-12 (insufficient nb of genomes)
Not conclusive for PB-54 and ABTS-1857
Only few SNPs to distinguish SA-11 and ABTS-351
Bt of interest for food
safety:
GC-91
EG2348 / SA-12 /
PB-54
Ssp. aizawai:
Ssp. kurstaki:
Cluster c
Cluster d
SA11 / ABTS-351
ABTS-1857
Cluster a
Cluster b
Article in preparation
Identification of several markers allowing to discrimination of Bt pesticide kurstakiand aizawaiat different
levels:
Thank you for your attention
UGVB
Arnaud Felten
BES team
Armel Gallet
Regulatedproducts
assessmentdepartment, DEPR
Arnaud Duboisset(CVDS)
Xavier Sarda(URSA)
Gaelle Vial (URSA)
Chantal Arar (UCIV)
Unit Staphyloccocus, Bacillusand
Clostridium, SBCL
Arnaud Fichant
Armelle Le Strat
Véronique Maladen
Sylvie Pairaud
Angélie Dijoux
Olivier Firmesse
Jacques-Antoine HENNEKINNE
Projects“Bt-TIAC”(2018)etOSABt(2019-2020)werefundedandsupportedbyANSESviathetaxonsalesofplant
protectionproducts(https://www.anses.fr/fr/content/la-phytopharmacovigilance)
UGVB
Arnaud Felten
BES team
Armel Gallet
Regulatedproducts
assessmentdepartment, DEPR
Arnaud Duboisset(CVDS)
Xavier Sarda(URSA)
Gaelle Vial (URSA)
Chantal Arar (UCIV)
Unit Staphyloccocus, Bacillusand
Clostridium, SBCL
Arnaud Fichant
Armelle Le Strat
Véronique Maladen
Sylvie Pairaud
Angélie Dijoux
Olivier Firmesse
Jacques-Antoine HENNEKINNE
Projects“Bt-TIAC”(2018)etOSABt(2019-2020)werefundedandsupportedbyANSESviathetaxonsalesofplant
protectionproducts(https://www.anses.fr/fr/content/la-phytopharmacovigilance)
Restricted Use -À usage restreint
Bacillus thuringiensis:
Taxonomy, Agricultural Use,
and Implications for food
safety
OECD, 25
th
February 2025
Bacillus thuringiensis:
Taxonomy, Agricultural Use,
and Implications for food
safety
OECD, 25
th
February 2025
Restricted Use -À usage restreint
Taxonomy evolution
Hugenholtz et al. 2021: Prokaryotic taxonomy and nomenclature in the age of
big sequence data. The ISME Journal (2021) 15:1879–1892
https://doi.org/10.1038/s41396-021-00941-x
Key events in prokaryotic taxonomy and
nomenclature over the past 100 years
2
Taxonomy evolution
Hugenholtz et al. 2021: Prokaryotic taxonomy and nomenclature in the age of
big sequence data. The ISME Journal (2021) 15:1879–1892
https://doi.org/10.1038/s41396-021-00941-x
Key events in prokaryotic taxonomy and
nomenclature over the past 100 years
2
Restricted Use - À usage restreint
The Prokaryotic Species Concept
•Phenotype-practical identification of isolates rather than constructing an evolutionary framework
•Numerical taxonomy - quantitative comparisons of phenotypic properties
•Birth of phylogenetic analysis
•rRNA, DNA hybridization, … - evolution with new methods
•Polyphasic taxonomy (phenotype + genotype)
•Whole Genome based taxonomy (Work in Progress)
➢Increasing amounts of data and complexity of mathematical tools
➢Operational definitions for species concept
Past
Future
3
The Prokaryotic Species Concept
•Phenotype-practical identification of isolates rather than constructing an evolutionary framework
•Numerical taxonomy - quantitative comparisons of phenotypic properties
•Birth of phylogenetic analysis
•rRNA, DNA hybridization, … - evolution with new methods
•Polyphasic taxonomy (phenotype + genotype)
•Whole Genome based taxonomy (Work in Progress)
➢Increasing amounts of data and complexity of mathematical tools
➢Operational definitions for species concept
Past
Future
3
Restricted Use - À usage restreint
Phylogenetic Trees Only Describe Evolution, Not Risk of an Organism
•Several attempts in phylogenetic analysis to determine the clustering and species attribution of B. cereus s.l.
•From ANI to rps (genes that control other genes), From rps to molecular targets.
•Phylogenetic trees reconstructed from different genes in the same organism can differ.
•Determining what genes causes/activate a molecular effect will carry false information if you start from the wrong
organism.
•Evolutionary relatedness does not necessarily correspond to pathogenicity in Bacillus sp.
4
ANI: Average Nucleotide Identity
rps: ribosome protein subunits
Phylogenetic Trees Only Describe Evolution, Not Risk of an Organism
•Several attempts in phylogenetic analysis to determine the clustering and species attribution of B. cereus s.l.
•From ANI to rps (genes that control other genes), From rps to molecular targets.
•Phylogenetic trees reconstructed from different genes in the same organism can differ.
•Determining what genes causes/activate a molecular effect will carry false information if you start from the wrong
organism.
•Evolutionary relatedness does not necessarily correspond to pathogenicity in Bacillus sp.
4
ANI: Average Nucleotide Identity
rps: ribosome protein subunits
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Genomics & Phenotype
Genomics is not the
answer to all questions
Gene regulation, expressions, condition of
expressions, environmental niche of the
organism, etc.
5
Wong et al. 2005: Phenotypic differences in genetically identical organisms: the epigenetic perspective;
Human Molecular Genetics, Vol. 14. Review, Issue 1.
Genomics & Phenotype
Genomics is not the
answer to all questions
Gene regulation, expressions, condition of
expressions, environmental niche of the
organism, etc.
5
Wong et al. 2005: Phenotypic differences in genetically identical organisms: the epigenetic perspective;
Human Molecular Genetics, Vol. 14. Review, Issue 1.
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Taxonomic changes of Bacillus thuringiensis (Bt):
where do we want to get?
Taxonomy
Unique
identity
Identification
methods
Evolution
Markers
6
Taxonomic changes of Bacillus thuringiensis (Bt):
where do we want to get?
Taxonomy
Unique
identity
Identification
methods
Evolution
Markers
6
Restricted Use - À usage restreint
Mechanism of action to be understood
•Exceptionally high variability of toxin production between single strains (distribution, genetic
organization, gene expression and toxin secretion)
•Several virulence factors known, but their individual relevance for the adverse effect is not yet
understood:
•nhe, hbl, cytk, SMAse, etc.
•Lack of agreement in scientific community on valid models for enterotoxicity
•E.g. Pig, rat, nematode, CaCo cell models, HeLa cell models, rabbit intestinal loop assay
•No known virulence factors that allow distinguising enterotoxigenic strains from non-toxigenic strain
Virulence Factors Enterotoxicity
Bacillus cereus senso lato
i.e. no Validated Effect Biomarkers available for enterotoxicity (yet)
7
nhe: nonhemolytic enterotoxin; Hbl: hemolysin BL; Cytk: cytotoxin K; SMAse: Sphingomyelinase
Mechanism of action to be understood
•Exceptionally high variability of toxin production between single strains (distribution, genetic
organization, gene expression and toxin secretion)
•Several virulence factors known, but their individual relevance for the adverse effect is not yet
understood:
•nhe, hbl, cytk, SMAse, etc.
•Lack of agreement in scientific community on valid models for enterotoxicity
•E.g. Pig, rat, nematode, CaCo cell models, HeLa cell models, rabbit intestinal loop assay
•No known virulence factors that allow distinguising enterotoxigenic strains from non-toxigenic strain
Virulence Factors Enterotoxicity
Bacillus cereus senso lato
i.e. no Validated Effect Biomarkers available for enterotoxicity (yet)
7
nhe: nonhemolytic enterotoxin; Hbl: hemolysin BL; Cytk: cytotoxin K; SMAse: Sphingomyelinase
Restricted Use - À usage restreint
Enterotoxicity Effect Biomarker
•Proficient scientific work being developed in the recent years
•Critical analysis requires Bacillus cereus s.l. of proven enterotoxigenic
effect
•European Food Borne Outbreaks (FBO) reporting: FBOs are categorised
as ‘strong-evidence’ or ‘weak-evidence' based on the strength of
evidence implicating a suspected food vehicle as the cause of the
outbreak
•Standard proceedures for identifying causative agents in FBO can vary
significantly by country making these data challenging to use for
characterizing causative agents.
8
Enterotoxicity Effect Biomarker
•Proficient scientific work being developed in the recent years
•Critical analysis requires Bacillus cereus s.l. of proven enterotoxigenic
effect
•European Food Borne Outbreaks (FBO) reporting: FBOs are categorised
as ‘strong-evidence’ or ‘weak-evidence' based on the strength of
evidence implicating a suspected food vehicle as the cause of the
outbreak
•Standard proceedures for identifying causative agents in FBO can vary
significantly by country making these data challenging to use for
characterizing causative agents.
8
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Strong Evidence Requires Strict Adherence to:
Epidemiological
evidence
Strong evidence includes statistical associations in well-conducted
analytical epidemiological studies or convincing descriptive evidence.
Microbiological
evidence
Strong evidence includes the identification of anindistinguishable
causative agent in a human case and in a food, a food component, or its
environment, which is unlikely to have been contaminated
coincidentally or after the event, or the identification of a causative
agent, such as a toxin or bio-active amine, in the food vehicle, in
combination with clinical symptoms and an onset of illness in outbreak
cases strongly indicative/pathognomonic to the causative agent.
Product-tracing and distribution chain is identified for all or a large proportion of cases
which can provide strong evidence in case a common point along the
food-production o were exposed and for whom a place of exposure/
point of sale could be identified.
Descriptive
environmental
evidence
Alone is almost invariably weak.
Fig 1 – Extract of 2022 EFSA/ECDC report: Trends in the number of strong-evidence
and weak-evidence outbreaks (left axis) and outbreak reporting rate (per 100,000)
(right axis), 2013–2022
Dark red = strong evidence
Light red = weak evidence
Weak evidence gives a broad picture but cannot be used to determine causality.
Weak evidence may then be used to build a faulty pathogenic genotype.
9
Source: EFSA 2014. Update of the technical specifications for harmonised reporting of food-borne outbreaks through the
European Union reporting system in accordance with Directive 2003/99/EC. EFSA Journal 2014; 12(3):3598, 25 pp.
doi:10.2903/j.efsa.2014.3598
Strong Evidence Requires Strict Adherence to:
Epidemiological
evidence
Strong evidence includes statistical associations in well-conducted
analytical epidemiological studies or convincing descriptive evidence.
Microbiological
evidence
Strong evidence includes the identification of anindistinguishable
causative agent in a human case and in a food, a food component, or its
environment, which is unlikely to have been contaminated
coincidentally or after the event, or the identification of a causative
agent, such as a toxin or bio-active amine, in the food vehicle, in
combination with clinical symptoms and an onset of illness in outbreak
cases strongly indicative/pathognomonic to the causative agent.
Product-tracing and distribution chain is identified for all or a large proportion of cases
which can provide strong evidence in case a common point along the
food-production o were exposed and for whom a place of exposure/
point of sale could be identified.
Descriptive
environmental
evidence
Alone is almost invariably weak.
Fig 1 – Extract of 2022 EFSA/ECDC report: Trends in the number of strong-evidence
and weak-evidence outbreaks (left axis) and outbreak reporting rate (per 100,000)
(right axis), 2013–2022
Dark red = strong evidence
Light red = weak evidence
Weak evidence gives a broad picture but cannot be used to determine causality.
Weak evidence may then be used to build a faulty pathogenic genotype.
9
Source: EFSA 2014. Update of the technical specifications for harmonised reporting of food-borne outbreaks through the
European Union reporting system in accordance with Directive 2003/99/EC. EFSA Journal 2014; 12(3):3598, 25 pp.
doi:10.2903/j.efsa.2014.3598
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Potential sources of False Positives when Characterizing Bc strains
•Spore forming bacteria are easier to isolate and culture
•Similar illnesses to Bc, but more difficult to detect:
•Enteric viruses
•noroviruses most identified but also astrovirus, adenovirus, sapovirus, rotavirus, enterovirus, Hepatitis A and Aichi
virus
•persistence of these viruses within high-risk food commodities-leafy greens, soft fruits and vegetables, red fruits and
bivalve mollusks
•culture techniques need a week or more for completion and require specialized laboratory equipment and skills.
•Clostridium – requires enrichment step and/or toxin identification
10
Potential sources of False Positives when Characterizing Bc strains
•Spore forming bacteria are easier to isolate and culture
•Similar illnesses to Bc, but more difficult to detect:
•Enteric viruses
•noroviruses most identified but also astrovirus, adenovirus, sapovirus, rotavirus, enterovirus, Hepatitis A and Aichi
virus
•persistence of these viruses within high-risk food commodities-leafy greens, soft fruits and vegetables, red fruits and
bivalve mollusks
•culture techniques need a week or more for completion and require specialized laboratory equipment and skills.
•Clostridium – requires enrichment step and/or toxin identification
10
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Potential for Circular Rationalization (Self-Reinforcing)
for Virulence Factors
Self-fulfilling loop
•A Bacillus cereus strain is identified as likely causative agent
•The strain may contain virulence factors also found in commercial strains
•Without an adequate mammalian model for verification, characteristics of this strain can
be assumed to describe a pathogenic strain
•Identification of strains with similar characteristics from FBO results in a positive
feedback loop when those strains are used in future models
•These potential false positive models may then be applied to new fields, like genomics,
bioinformatics, etc. where researchers may not be able to validate the categorization
previously assigned
11
Potential for Circular Rationalization (Self-Reinforcing)
for Virulence Factors
Self-fulfilling loop
•A Bacillus cereus strain is identified as likely causative agent
•The strain may contain virulence factors also found in commercial strains
•Without an adequate mammalian model for verification, characteristics of this strain can
be assumed to describe a pathogenic strain
•Identification of strains with similar characteristics from FBO results in a positive
feedback loop when those strains are used in future models
•These potential false positive models may then be applied to new fields, like genomics,
bioinformatics, etc. where researchers may not be able to validate the categorization
previously assigned
11
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Risk Assessment based on Three Key Attributes – No single one is
enough
Genomic Characterization:
-Identification, phylogeny, evolution
But also need to consider…
-Genetic regulation
-Virulence factors
-Constitutive vs. induced gene expression
Ecology, Biovar:
-Optimal growth conditions
-Physiological profile/ phenotypic characteristics
-Association with specific niche (e.g. presence of
Cry-protein crystals)
Mammalian exposure data:
-Literature on direct exposure to humans and
other mammals
-Identified in FBO cases
-Commercial Bt strain history of use in
agriculture
12
Risk Assessment based on Three Key Attributes – No single one is
enough
Genomic Characterization:
-Identification, phylogeny, evolution
But also need to consider…
-Genetic regulation
-Virulence factors
-Constitutive vs. induced gene expression
Ecology, Biovar:
-Optimal growth conditions
-Physiological profile/ phenotypic characteristics
-Association with specific niche (e.g. presence of
Cry-protein crystals)
Mammalian exposure data:
-Literature on direct exposure to humans and
other mammals
-Identified in FBO cases
-Commercial Bt strain history of use in
agriculture
12
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IBMA
International Biocontrol Manufacturers Association AISBL
Rue de Trèves 61
1040 Brussels
Belgium
WWW.IBMA-GLOBAL.ORG
Thank you
REGULATORY AFFAIRS CONSULTANT
Ulf Heilig
[email protected]
Jennifer Lewis
[email protected]
EXECUTIVE DIRECTOR
Jérémy Belzunces
[email protected]
TECHNICAL PROJECT MANAGER
Isabelle Pinzauti Babrzyński
[email protected]
ADVOCACY AND COMMUNICATION
MANAGER
Soizick Menais
[email protected]
OFFICE MANAGER
PROJECT MANAGER
Britta Schnittger
[email protected]
IBMA CONSULTANT
Jeroen Meussen
[email protected]
13
José Carvalho
IBMA Microbials Professional Group, Chair
[email protected]
IBMA
International Biocontrol Manufacturers Association AISBL
Rue de Trèves 61
1040 Brussels
Belgium
WWW.IBMA-GLOBAL.ORG
Thank you
REGULATORY AFFAIRS CONSULTANT
Ulf Heilig
[email protected]
Jennifer Lewis
[email protected]
EXECUTIVE DIRECTOR
Jérémy Belzunces
[email protected]
TECHNICAL PROJECT MANAGER
Isabelle Pinzauti Babrzyński
[email protected]
ADVOCACY AND COMMUNICATION
MANAGER
Soizick Menais
[email protected]
OFFICE MANAGER
PROJECT MANAGER
Britta Schnittger
[email protected]
IBMA CONSULTANT
Jeroen Meussen
[email protected]
13
José Carvalho
IBMA Microbials Professional Group, Chair
[email protected]
1FMC CORPORATION
Restricted Use -À usage restreint
Industry perspective on
taxonomy evolution of Bacillus thuringiensis
Nina Joergensen and Rosa Criollo
OECD EGBP seminar February 25, 2025
Restricted Use -À usage restreint
Industry perspective on
taxonomy evolution of Bacillus thuringiensis
Nina Joergensen and Rosa Criollo
OECD EGBP seminar February 25, 2025
2FMC CORPORATION
Restricted Use - À usage restreint
Lesson learned – FMC Bt strain
2FMC CORPORATION
•Until recently, CRY proteins served as the genetic markers for all approved
Bt strains.
•A novel FMC Bacillus thuringiensis strain, which does not contain CRY
protein, has initiated the registration phase since 2022
➢EU: Process initiated
➢USA: The process is ongoing, with inquiries from authorities regarding identity –
all addressed.
➢Canada: The process is ongoing, with inquiries from authorities regarding
identity – all addressed.
➢Brazil: Registered since November 2024. Additional information on identity was
provided to authorities during the review process.
•The ongoing scientific debate about the Bacillus cereus (Bc) group, including
Bacillus thuringiensis (Bt), has required extra data during the authority review
to clarify the identity and safety of the FMC strain.
Restricted Use - À usage restreint
Lesson learned – FMC Bt strain
2FMC CORPORATION
•Until recently, CRY proteins served as the genetic markers for all approved
Bt strains.
•A novel FMC Bacillus thuringiensis strain, which does not contain CRY
protein, has initiated the registration phase since 2022
➢EU: Process initiated
➢USA: The process is ongoing, with inquiries from authorities regarding identity –
all addressed.
➢Canada: The process is ongoing, with inquiries from authorities regarding
identity – all addressed.
➢Brazil: Registered since November 2024. Additional information on identity was
provided to authorities during the review process.
•The ongoing scientific debate about the Bacillus cereus (Bc) group, including
Bacillus thuringiensis (Bt), has required extra data during the authority review
to clarify the identity and safety of the FMC strain.
3FMC CORPORATION
Restricted Use - À usage restreint
Scientific evidence on presence/non-presence of
CRY protein for taxonomic identification
➢Within the Bacillus cereus (Bc) group, Bacillus thuringiensis (Bt) has
traditionally been identified by the presence of crystalline (CRY) proteins.
➢However, the ability to synthesize these crystals is encoded by plasmid-
borne CRY genes, which can be acquired or lost by the organisms.
➢Additionally, CRY genes have been detected in other Bacillus species
closely related to Bt.
➢Some strains designated as Bt neither carry CRY genes nor produce other
visible crystal proteins.
➢Recent scientific literature indicates that the presence or absence of
virulence plasmids and CRY genes is largely uncorrelated with the
phylogenetic position of the host bacteria.
Conclusion: Presence of CRY proteins should not be used as an
identification method for Bt organisms in a regulatory context
Restricted Use - À usage restreint
Scientific evidence on presence/non-presence of
CRY protein for taxonomic identification
➢Within the Bacillus cereus (Bc) group, Bacillus thuringiensis (Bt) has
traditionally been identified by the presence of crystalline (CRY) proteins.
➢However, the ability to synthesize these crystals is encoded by plasmid-
borne CRY genes, which can be acquired or lost by the organisms.
➢Additionally, CRY genes have been detected in other Bacillus species
closely related to Bt.
➢Some strains designated as Bt neither carry CRY genes nor produce other
visible crystal proteins.
➢Recent scientific literature indicates that the presence or absence of
virulence plasmids and CRY genes is largely uncorrelated with the
phylogenetic position of the host bacteria.
Conclusion: Presence of CRY proteins should not be used as an
identification method for Bt organisms in a regulatory context
4FMC CORPORATION
Restricted Use - À usage restreint
Taxonomic classification completed for FMC Bt strain
4FMC CORPORATION
Phylum
Class
Order
Family
Genus
Species
Strain
Clone
16S rRNA
(1 locus)
MLST
(7 loci)
Ribosomal
MLST
(53 loci)
WGS
(all loci) ▪16S rRNA
✓Sanger sequencing and WGS sequence
✓Phylogenetic trees
▪MLST
✓Phylogenetic tree
✓panC group phylogenetic assignment
▪WGS analysis
✓ANI (average nucleotide identity)
✓dDDH (DNA-DNA hybridization)
✓Core genome-based phylogeny
✓Phylogenetic tree using GTDB-Tk marker
genes (Genome Taxonomy Database Tool
kit)
All analysis conclude the FMC strain is a Bt
Restricted Use - À usage restreint
Taxonomic classification completed for FMC Bt strain
4FMC CORPORATION
Phylum
Class
Order
Family
Genus
Species
Strain
Clone
16S rRNA
(1 locus)
MLST
(7 loci)
Ribosomal
MLST
(53 loci)
WGS
(all loci) ▪16S rRNA
✓Sanger sequencing and WGS sequence
✓Phylogenetic trees
▪MLST
✓Phylogenetic tree
✓panC group phylogenetic assignment
▪WGS analysis
✓ANI (average nucleotide identity)
✓dDDH (DNA-DNA hybridization)
✓Core genome-based phylogeny
✓Phylogenetic tree using GTDB-Tk marker
genes (Genome Taxonomy Database Tool
kit)
All analysis conclude the FMC strain is a Bt
5FMC CORPORATION
Restricted Use - À usage restreint
Lessons learnt from FMC for novel Bt strain
Regulatory framework that are not regularly adjusted to state of art scientific
developments can result in request for unsuitable data requirements
➢Request for additional phenotypic, physiological and biochemical characteristics
to support identity of strain
➢List of toxins to address varies widely between countries and is often
unpredictable, leading to post-submission deficiencies.
➢The need to perform a Mouse IP assay to rule out B. anthracis contamination.
➢Post-registration requirements for routine quality control in production include
increased contamination monitoring and the Mouse IP assay.
To avoid uncertainties related to identity during registration of Bt strain, we would like to
invite to a debate on the following regulatory topics related to taxonomy/Biological
properties on B. thuringiensis strains.
➢Higher acceptance of genomic methods for taxonomy and toxin screening for B.
thuringiensis species
➢To avoid unnecessary repeated animal sacrifice, replace Mouse IP assay with
genomic or in vitro methods
➢A harmonized approach to establish a list of toxins to be considered in the
registration dossier
Restricted Use - À usage restreint
Lessons learnt from FMC for novel Bt strain
Regulatory framework that are not regularly adjusted to state of art scientific
developments can result in request for unsuitable data requirements
➢Request for additional phenotypic, physiological and biochemical characteristics
to support identity of strain
➢List of toxins to address varies widely between countries and is often
unpredictable, leading to post-submission deficiencies.
➢The need to perform a Mouse IP assay to rule out B. anthracis contamination.
➢Post-registration requirements for routine quality control in production include
increased contamination monitoring and the Mouse IP assay.
To avoid uncertainties related to identity during registration of Bt strain, we would like to
invite to a debate on the following regulatory topics related to taxonomy/Biological
properties on B. thuringiensis strains.
➢Higher acceptance of genomic methods for taxonomy and toxin screening for B.
thuringiensis species
➢To avoid unnecessary repeated animal sacrifice, replace Mouse IP assay with
genomic or in vitro methods
➢A harmonized approach to establish a list of toxins to be considered in the
registration dossier
6FMC CORPORATION
Restricted Use - À usage restreint
Take home message
1.CRY protein is plasmid-borne and not species specific. Therefore, relying
on CRY protein presence for Bt identification should not be used for
species identification in a regulatory context.
2.Considering the state-of-the-art developments in genomics, the only
approach to explicitly recognize a specific strain of B. thuringiensis is
through whole genome sequence (WGS).
3.Regulatory authorities globally to consider moving away phenotypic,
physiological and biochemical characteristics as main route of identifying
B. thuringiensis and accept WGS tool as a reliable methodology for
identification in their regulatory framework
4.Harmonized approach needed for identification of toxins relevant for
registration purposes and replacement of Mouse IP assay as post
registration requirement in quality control settings.
6FMC CORPORATION
Restricted Use - À usage restreint
Take home message
1.CRY protein is plasmid-borne and not species specific. Therefore, relying
on CRY protein presence for Bt identification should not be used for
species identification in a regulatory context.
2.Considering the state-of-the-art developments in genomics, the only
approach to explicitly recognize a specific strain of B. thuringiensis is
through whole genome sequence (WGS).
3.Regulatory authorities globally to consider moving away phenotypic,
physiological and biochemical characteristics as main route of identifying
B. thuringiensis and accept WGS tool as a reliable methodology for
identification in their regulatory framework
4.Harmonized approach needed for identification of toxins relevant for
registration purposes and replacement of Mouse IP assay as post
registration requirement in quality control settings.
6FMC CORPORATION
7FMC CORPORATION
Restricted Use - À usage restreintAPPENDIX
Restricted Use - À usage restreintAPPENDIX
8FMC CORPORATION
Restricted Use - À usage restreint
Background literature
8FMC CORPORATION
▪BaekI, Lee K, GoodfellowM, Chun J (2019). ComparativeGenomicand PhylogenomicAnalyses ClarifyRelationships Withinand BetweenBacilluscereus
and Bacillusthuringiensis: Proposalfor the Recognitionof TwoBacillusthuringiensisGenomovars. Front Microbiol. 2019 Aug23;10:1978. doi:
10.3389/fmicb.2019.01978.
▪Carroll LM et al (2021). Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond. Critical Reviews in Food Science and
Nutrition. doi: 10.1080/10408398.2021.1916735.
▪Carroll, LM, Cheng RA, and Kovac J. (2020). No assembly required: Using BTyper3 to assess the congruency of a proposed taxonomic framework for the
Bacillus cereus group with historical typing methods. Front Microbiol. 11 (2285):580691. doi:10.3389/fmicb. 2020.580691.
▪Chen M, Wei X, Zhang J, Zhou H, Chen N, Wang J, Feng Y, Yu S, Zhang J, Wu S, Ye Q, Pang R, Ding Y, Wu Q (2022). Differentiation of Bacilluscereusand
BacillusthuringiensisUsing Genome-GuidedMALDI-TOF MS Basedon Variations in RibosomalProteins. Microorganisms. 2022 Apr27;10(5):918. doi:
10.3390/microorganisms10050918.
▪Chun J, OrenA, VentosaA, Christensen H, ArahalDH, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S, TrujilloME, (2018). Proposedminimal standards
for the useof genomedata for the taxonomyof prokaryotes. International Journal of Systematicand EvolutionaryMicrobiology, 68: 461–466.
doi:10.1099/ijsem.0.002516.
▪DunlapCA (2019): Taxonomyof registeredBacillusspp. strainsusedas plant pathogenantagonists. BiologicalControl July2019; 134, 82–86.
▪EFSA BIOHAZ Panel (EFSA Panel on BiologicalHazards) (2016): Scientific opinion on the risksfor public healthrelatedto the presenceof Bacilluscereus
and otherBacillusspp. includingBacillusthuringiensisin foodstuffs. EFSA Journal 2016;14(7):4524, 93 pp. doi: 10.2903/j.efsa.2016.4524.
▪Liu Y, Lai Q, GokerM, Meier-KolthoffJP, Wang M, Sun Y, Lei Wang L., ShaoZ (2015). Genomicinsightsintothe taxonomicstatus of the Bacilluscereus
group. Sci. Rep. 5:14082. doi: 10.1038/srep14082
▪Liu Y, Lai Q, GokerM, Meier-KolthoffJP, Wang M, Sun Y, Lei Wang L., ShaoZ (2015). Genomicinsightsintothe taxonomicstatus of the Bacilluscereus
group. Sci. Rep. 5:14082. doi: 10.1038/srep14082. Liu Y, Lai Q, Du J, ShaoZ. (2017). Geneticdiversityand population structureof the Bacilluscereus
groupbacteriafrom diverse marine environments. SciRep. Apr6;7(1):689. doi: 10.1038/s41598-017-00817-1. PMID: 28386130; PMCID: PMC5429728.
▪MéricG, MageirosL, PascoeB, WoodcockDJ, MourkasE, LambleS, BowdenR, JolleyKA, Raymond B, SheppardSK (2018). Lineage-specificplasmid
acquisitionand the evolution of specializedpathogensin Bacillusthuringiensisand the Bacilluscereusgroup. Mol. Ecol.;27:1524–1540. doi:
10.1111/mec.14546.
▪Torres Manno, M.A., Repizo, G.D., Magni, C. et al. (2020). The assessment of leading traits in the taxonomy of the Bacillus cereus group. Antonie van
Leeuwenhoek 113, 2223–2242. doi:10.1007/s10482-020-01494-3
▪Zheng J, Gao Q, Liu L, Liu H, Wang Y, Peng D, RuanL, Raymond B, Sun M. (2017). ComparativeGenomicsof BacillusthuringiensisRevealsa Path to
SpecializedExploitationof Multiple InvertebrateHosts. mBio. 8(4):e00822-17. doi: 10.1128/mBio.00822-17. PMID: 28790205; PMCID: PMC5550751.
Restricted Use - À usage restreint
Background literature
8FMC CORPORATION
▪BaekI, Lee K, GoodfellowM, Chun J (2019). ComparativeGenomicand PhylogenomicAnalyses ClarifyRelationships Withinand BetweenBacilluscereus
and Bacillusthuringiensis: Proposalfor the Recognitionof TwoBacillusthuringiensisGenomovars. Front Microbiol. 2019 Aug23;10:1978. doi:
10.3389/fmicb.2019.01978.
▪Carroll LM et al (2021). Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond. Critical Reviews in Food Science and
Nutrition. doi: 10.1080/10408398.2021.1916735.
▪Carroll, LM, Cheng RA, and Kovac J. (2020). No assembly required: Using BTyper3 to assess the congruency of a proposed taxonomic framework for the
Bacillus cereus group with historical typing methods. Front Microbiol. 11 (2285):580691. doi:10.3389/fmicb. 2020.580691.
▪Chen M, Wei X, Zhang J, Zhou H, Chen N, Wang J, Feng Y, Yu S, Zhang J, Wu S, Ye Q, Pang R, Ding Y, Wu Q (2022). Differentiation of Bacilluscereusand
BacillusthuringiensisUsing Genome-GuidedMALDI-TOF MS Basedon Variations in RibosomalProteins. Microorganisms. 2022 Apr27;10(5):918. doi:
10.3390/microorganisms10050918.
▪Chun J, OrenA, VentosaA, Christensen H, ArahalDH, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S, TrujilloME, (2018). Proposedminimal standards
for the useof genomedata for the taxonomyof prokaryotes. International Journal of Systematicand EvolutionaryMicrobiology, 68: 461–466.
doi:10.1099/ijsem.0.002516.
▪DunlapCA (2019): Taxonomyof registeredBacillusspp. strainsusedas plant pathogenantagonists. BiologicalControl July2019; 134, 82–86.
▪EFSA BIOHAZ Panel (EFSA Panel on BiologicalHazards) (2016): Scientific opinion on the risksfor public healthrelatedto the presenceof Bacilluscereus
and otherBacillusspp. includingBacillusthuringiensisin foodstuffs. EFSA Journal 2016;14(7):4524, 93 pp. doi: 10.2903/j.efsa.2016.4524.
▪Liu Y, Lai Q, GokerM, Meier-KolthoffJP, Wang M, Sun Y, Lei Wang L., ShaoZ (2015). Genomicinsightsintothe taxonomicstatus of the Bacilluscereus
group. Sci. Rep. 5:14082. doi: 10.1038/srep14082
▪Liu Y, Lai Q, GokerM, Meier-KolthoffJP, Wang M, Sun Y, Lei Wang L., ShaoZ (2015). Genomicinsightsintothe taxonomicstatus of the Bacilluscereus
group. Sci. Rep. 5:14082. doi: 10.1038/srep14082. Liu Y, Lai Q, Du J, ShaoZ. (2017). Geneticdiversityand population structureof the Bacilluscereus
groupbacteriafrom diverse marine environments. SciRep. Apr6;7(1):689. doi: 10.1038/s41598-017-00817-1. PMID: 28386130; PMCID: PMC5429728.
▪MéricG, MageirosL, PascoeB, WoodcockDJ, MourkasE, LambleS, BowdenR, JolleyKA, Raymond B, SheppardSK (2018). Lineage-specificplasmid
acquisitionand the evolution of specializedpathogensin Bacillusthuringiensisand the Bacilluscereusgroup. Mol. Ecol.;27:1524–1540. doi:
10.1111/mec.14546.
▪Torres Manno, M.A., Repizo, G.D., Magni, C. et al. (2020). The assessment of leading traits in the taxonomy of the Bacillus cereus group. Antonie van
Leeuwenhoek 113, 2223–2242. doi:10.1007/s10482-020-01494-3
▪Zheng J, Gao Q, Liu L, Liu H, Wang Y, Peng D, RuanL, Raymond B, Sun M. (2017). ComparativeGenomicsof BacillusthuringiensisRevealsa Path to
SpecializedExploitationof Multiple InvertebrateHosts. mBio. 8(4):e00822-17. doi: 10.1128/mBio.00822-17. PMID: 28790205; PMCID: PMC5550751.
Lieve Herman, BIOHAZ Panel
Institute of Agricultural, Fisheriesand Food Research
FlemishCommunity, Belgium
BACILLUS THURINGIENSISQPS
ASSESSMENT
THE 13TH EXPERT GROUP ON BIOPESTICIDESSEMINAR ON ‘EMERGING
RISK ASSESSMENT APPROACHES FORBIOPESTICIDES’
OECD, PARIS, FEBRUARY25
Institute of Agricultural, Fisheriesand Food Research
FlemishCommunity, Belgium
BACILLUS THURINGIENSISQPS
ASSESSMENT
THE 13TH EXPERT GROUP ON BIOPESTICIDESSEMINAR ON ‘EMERGING
RISK ASSESSMENT APPROACHES FORBIOPESTICIDES’
OECD, PARIS, FEBRUARY25
QUALIFIED PRESUMPTION OF SAFETY (QPS)
QPS
Safety assessment
approach for
microorganisms
intentionally added
at different stages
into the food and
feed chain
Simplified
safety
evaluation
of strains
belonging to
QPS list
microorganisms
species
bacteria,
yeast, protists,
microalgae
families
viruses
support to
FIP
NIF
FEEDCO
PREV
Food enzymes
Novel food
Feed/ food additives
Plant protection
products (PPPs)
Genetically modified
microorganisms
(GMM)
Assessment
Identity
Body of
knowledge
Safety
Genetic
Info
QPS
Safety assessment
approach for
microorganisms
intentionally added
at different stages
into the food and
feed chain
Simplified
safety
evaluation
of strains
belonging to
QPS list
microorganisms
species
bacteria,
yeast, protists,
microalgae
families
viruses
support to
FIP
NIF
FEEDCO
PREV
Food enzymes
Novel food
Feed/ food additives
Plant protection
products (PPPs)
Genetically modified
microorganisms
(GMM)
Assessment
Identity
Body of
knowledge
Safety
Genetic
Info
4 PILLARS OF QPS ASSESSMENT
•Taxonomic identification
•Only unambiguously defined microbiological TUs
•Body of knowledge
•Peer-reviewed papers published in journals and books (scientific literature databases)
•Safety concerns in relation to pathogenicity/virulence
•Case reports of human diseases, particularly infections or intoxications
•Reports of infection, intoxication or other diseases in livestock and wild animals
•Studies indicating the presence of virulence factors (e.g. toxins and enzymes
that might contribute to the pathogenicity of the microorganism)
•WGS analysis → genetic determinants encoding genes of potential concern
•Safety for the environment
3
•Taxonomic identification
•Only unambiguously defined microbiological TUs
•Body of knowledge
•Peer-reviewed papers published in journals and books (scientific literature databases)
•Safety concerns in relation to pathogenicity/virulence
•Case reports of human diseases, particularly infections or intoxications
•Reports of infection, intoxication or other diseases in livestock and wild animals
•Studies indicating the presence of virulence factors (e.g. toxins and enzymes
that might contribute to the pathogenicity of the microorganism)
•WGS analysis → genetic determinants encoding genes of potential concern
•Safety for the environment
3
Restricted Use - À usage restreint
•QPS assessment of B. thuringiensisuponinternalEFSA request
•Publishedin QPS Panel Statement 21
https://doi.org/10.2903/j.efsa.2025.9169
•Basedon Extended LiteratureSearch fromJanuary2015 tillJuly2024 (as follow up
of theEFSA opinion on “Risks for public health related to the presence of Bacillus
cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs” (EFSA,
2026)
•A totalof 5867 hits wereidentifiedand, fromthese, 64 referenceswereselectedas
relevant
•Additionalpapers wereincluded(alsopublishedbefore2015) tointerpretthedata in
a broadercontext thancoveredbytheELS search items
QPS ASSESSMENT BACILLUS THURINGIENSIS
•QPS assessment of B. thuringiensisuponinternalEFSA request
•Publishedin QPS Panel Statement 21
https://doi.org/10.2903/j.efsa.2025.9169
•Basedon Extended LiteratureSearch fromJanuary2015 tillJuly2024 (as follow up
of theEFSA opinion on “Risks for public health related to the presence of Bacillus
cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs” (EFSA,
2026)
•A totalof 5867 hits wereidentifiedand, fromthese, 64 referenceswereselectedas
relevant
•Additionalpapers wereincluded(alsopublishedbefore2015) tointerpretthedata in
a broadercontext thancoveredbytheELS search items
QPS ASSESSMENT BACILLUS THURINGIENSIS
Restricted Use - À usage restreint
•B. thuringiensis is a species with Standing in Nomenclature
•B. thuringiensis belongs to B. cereus sensu lato (s.l.), also known as the Bacillus
cereus group.
•The presence of crystal proteins, investigated by microscopical examination is
considered as the standard method for defining the strains as B. thuringiensis (used
in the QPS assessment as criterium to include the studies)
•Genome-based taxonomy using ANI values as threshold to separate different
genomospecies. The strains containing the genes for entomotoxins (indication for
the presence of the cytotoxins and the identification of B. thuringiensis) were
detected across various B. cereus s.l. lineages
•B. thuringiensis strains subspecies kurstaki, aizawai, israelensis, tenebrionis,
morrisoni only based on flagellin amino acid sequence and is not related to the
presence/absence of other genes e.g. virulence genes
IDENTITY
•B. thuringiensis is a species with Standing in Nomenclature
•B. thuringiensis belongs to B. cereus sensu lato (s.l.), also known as the Bacillus
cereus group.
•The presence of crystal proteins, investigated by microscopical examination is
considered as the standard method for defining the strains as B. thuringiensis (used
in the QPS assessment as criterium to include the studies)
•Genome-based taxonomy using ANI values as threshold to separate different
genomospecies. The strains containing the genes for entomotoxins (indication for
the presence of the cytotoxins and the identification of B. thuringiensis) were
detected across various B. cereus s.l. lineages
•B. thuringiensis strains subspecies kurstaki, aizawai, israelensis, tenebrionis,
morrisoni only based on flagellin amino acid sequence and is not related to the
presence/absence of other genes e.g. virulence genes
IDENTITY
Restricted Use - À usage restreint
•B. thuringiensisformulations(mixture of sporesandcrystals) commercialisedas
biopesticide
•B. thuringiensistransferredtoediblepartsof theplantsafterbiopesticideapplication
or fromthesoil, wheretheyare naturallypresent
•The sporescansurvivedehydrationandfood processing andend up in a varietyof
food productswheretheycanfurtherproliferateunderappropriateconditions
•European studies found B. thuringiensis on bell peppers, tomatoes and lettuce (prevalence
from 18% to 41%of tested samples) in concentrations ranging from 10
2
to 10
5
cfu/g
•Concentration from 1.95 x 10
4
cfu/g to 1.75 x 10
5
cfu/g on spinach treated with a B.
thuringiensis biopesticide and from 100 cfu/g till 850 cfu/g for the non-treated ones.
•Theoretically estimated concentration of B. thuringiensis spores on treated vegetables ranging
from 10
5
to 10
6
cfu/g just after application of the biopesticide; a decrease in concentration
during pre-harvest growth in open field conditions with about 1.5-2 log reduction in 5-15 days
and reaching a constant level of contamination at approximately 10
2
-10
3
cfu/g. It is not clear
if the reduction seen in open field grown vegetables would be as prominent in greenhouse
grown treated produce.
BODY OF KNOWLEDGE
•B. thuringiensisformulations(mixture of sporesandcrystals) commercialisedas
biopesticide
•B. thuringiensistransferredtoediblepartsof theplantsafterbiopesticideapplication
or fromthesoil, wheretheyare naturallypresent
•The sporescansurvivedehydrationandfood processing andend up in a varietyof
food productswheretheycanfurtherproliferateunderappropriateconditions
•European studies found B. thuringiensis on bell peppers, tomatoes and lettuce (prevalence
from 18% to 41%of tested samples) in concentrations ranging from 10
2
to 10
5
cfu/g
•Concentration from 1.95 x 10
4
cfu/g to 1.75 x 10
5
cfu/g on spinach treated with a B.
thuringiensis biopesticide and from 100 cfu/g till 850 cfu/g for the non-treated ones.
•Theoretically estimated concentration of B. thuringiensis spores on treated vegetables ranging
from 10
5
to 10
6
cfu/g just after application of the biopesticide; a decrease in concentration
during pre-harvest growth in open field conditions with about 1.5-2 log reduction in 5-15 days
and reaching a constant level of contamination at approximately 10
2
-10
3
cfu/g. It is not clear
if the reduction seen in open field grown vegetables would be as prominent in greenhouse
grown treated produce.
BODY OF KNOWLEDGE
Restricted Use - À usage restreint
•Diarrhealsyndrome: about90% of B. cereuss.l. foodborneoutbreaks
•This syndrome has been mainly associated with the production of heat-labile enterotoxins in
the small intestine.
•Genes coding for hemolysin BL (hbl), non-hemolytic enterotoxin (nhe), and cytotoxin K
(cytK).
•The emeticsyndrome
•This syndrome is caused by cereulide, an emetic toxin, preformed in the food due to B. cereus s.l.
growth.
•The cereulide is encoded by the ces gene cluster
SAFETY -BACILLUS CEREUSS.L. IN FOODBORNEDISEASE
•Diarrhealsyndrome: about90% of B. cereuss.l. foodborneoutbreaks
•This syndrome has been mainly associated with the production of heat-labile enterotoxins in
the small intestine.
•Genes coding for hemolysin BL (hbl), non-hemolytic enterotoxin (nhe), and cytotoxin K
(cytK).
•The emeticsyndrome
•This syndrome is caused by cereulide, an emetic toxin, preformed in the food due to B. cereus s.l.
growth.
•The cereulide is encoded by the ces gene cluster
SAFETY -BACILLUS CEREUSS.L. IN FOODBORNEDISEASE
Restricted Use - À usage restreint
•Most B. thuringiensis strains, also biopesticide strains (see table) contain the genes which
production is known related to the diarrheal syndrome: hbl and cytK2 genes and the genes of
the Nhe complex
•B. thuringiensis strains were not found positive for the presence of ces genes which
production is related to the emetic syndrome.
•B. thuringiensis and B. cereus s.s .also produce other virulence factors (e.g. phospholipase C,
sphingomyelinase, enterolysins). These may play a role in both insecticidal activity and
potential human impact.
•Expression of virulence factors has been demonstrated in food models for most of the tested
commercial B. thuringiensis strains; several assays such as cytotoxicity assays on cell lines and
Drosophila melanogaster model have been used to provide insights about some aspects of
virulence
BACILLUS THURINGIENSISAS POSSIBLESOURCE OF FOODBORNE
INFECTIONS–VIRULENCEPOTENTIAL
•Most B. thuringiensis strains, also biopesticide strains (see table) contain the genes which
production is known related to the diarrheal syndrome: hbl and cytK2 genes and the genes of
the Nhe complex
•B. thuringiensis strains were not found positive for the presence of ces genes which
production is related to the emetic syndrome.
•B. thuringiensis and B. cereus s.s .also produce other virulence factors (e.g. phospholipase C,
sphingomyelinase, enterolysins). These may play a role in both insecticidal activity and
potential human impact.
•Expression of virulence factors has been demonstrated in food models for most of the tested
commercial B. thuringiensis strains; several assays such as cytotoxicity assays on cell lines and
Drosophila melanogaster model have been used to provide insights about some aspects of
virulence
BACILLUS THURINGIENSISAS POSSIBLESOURCE OF FOODBORNE
INFECTIONS–VIRULENCEPOTENTIAL
Restricted Use - À usage restreint
•In routine foodborne outbreak investigations, the diagnosis at human clinical and
food level focuses on the identification to B. cereus s.l. with no further identification
down to the level of B. thuringiensis.
•Retrospective studies were carried out using strains collected during former
foodborne outbreak investigations where they were identified as B. cereus s.l.
•The outbreak investigations indicate that a substantial part of the foodborne
outbreaks with B. cereus s.l. are correlated with the presence of B. thuringiensis
biopesticide strains, mainly of the subspecies aizawai and kurstaki
•B. thuringiensis subsp. israelensis and tenebrionis/morrisoni biopesticide strains not
linked to foodborne outbreaks
•application target (not used in agriculture, or on non-edible plant parts)
•B. thuringiensissubsp. tenebrionis/morrisonidisruptednheApromoterandno cytK2 gene
B. THURINGIENSISBIOPESTICIDESTRAINSANDFOODBORNEDISEASE
•In routine foodborne outbreak investigations, the diagnosis at human clinical and
food level focuses on the identification to B. cereus s.l. with no further identification
down to the level of B. thuringiensis.
•Retrospective studies were carried out using strains collected during former
foodborne outbreak investigations where they were identified as B. cereus s.l.
•The outbreak investigations indicate that a substantial part of the foodborne
outbreaks with B. cereus s.l. are correlated with the presence of B. thuringiensis
biopesticide strains, mainly of the subspecies aizawai and kurstaki
•B. thuringiensis subsp. israelensis and tenebrionis/morrisoni biopesticide strains not
linked to foodborne outbreaks
•application target (not used in agriculture, or on non-edible plant parts)
•B. thuringiensissubsp. tenebrionis/morrisonidisruptednheApromoterandno cytK2 gene
B. THURINGIENSISBIOPESTICIDESTRAINSANDFOODBORNEDISEASE
Restricted Use - À usage restreint
•xx
BACILLUS THURINGIENSISAS POSSIBLESOURCE OF FOODBORNE
INFECTIONS–POSSIBLEINVOLVEMENTIN FOODBORNEOUTBREAKS
B. thuringiensis isolates
1)
Typing method
2)
Result Reference
8 biopesticide strains; 24 food strains, 7 outbreak
related strains linked to 3 outbreaks from which 2
were human faecal strains and 5 were food isolates
panC typing
and FTIR
spectroscopy
Intermixed clustering of food and human outbreak strains with
biopesticides B. thuringiensis aizawai and B. thuringiensis kurstaki
strains;
Johler et
al., 2018
13 biopesticide strains, 18 food strains, 3 of them
outbreak related and isolated from lettuce, 2 human
faecal strains outbreak related , the 5 outbreak related
strains (3 from food and 2 human faecal strains) are
the same strains as in Johler et al., 2018
wgSNPs and
cgSNP
5outbreakrelatedstrains(3fromfoodand2humanfecal
strains)differedby0-3wgSNPs(0-2cgSNPs)withtheB.
thuringiensisaizawaibiopesticidestrainABTS-1857.
Biggel et
al., 2022
19 biopesticide strains, 143 food strains, linked to 49
outbreaks and from which 21 strains were from
outbreaks where no other putative food pathogens
were detected during the outbreak investigation
cgSNPs
3)
Foodstrainslinkedto47outbreaksdifferedby0to10wgSNPs
fromB.thuringiensisaizawaiandB.thuringiensiskurstaki
biopesticidestrains.The47outbreaksrelatedto18,8%oftheB.
cereuss.l.associatedfoodborneoutbreaks.Levels in food
products for which B. thuringiensis was the only pathogen,
ranged from 10
2
to more than 10
7
cfu/g (median 9x10
2
), 2 food
strains linked to 2 outbreaks were not linked to any biopesticide
strain.
Bonis et
al., 2021
7 food strains, 5 strains from vomit sample of one
patient.
polyphasic
approach
No link found between strains isolated from vomit and from food. Pheepakp
raw et al.
2023
•xx
BACILLUS THURINGIENSISAS POSSIBLESOURCE OF FOODBORNE
INFECTIONS–POSSIBLEINVOLVEMENTIN FOODBORNEOUTBREAKS
B. thuringiensis isolates
1)
Typing method
2)
Result Reference
8 biopesticide strains; 24 food strains, 7 outbreak
related strains linked to 3 outbreaks from which 2
were human faecal strains and 5 were food isolates
panC typing
and FTIR
spectroscopy
Intermixed clustering of food and human outbreak strains with
biopesticides B. thuringiensis aizawai and B. thuringiensis kurstaki
strains;
Johler et
al., 2018
13 biopesticide strains, 18 food strains, 3 of them
outbreak related and isolated from lettuce, 2 human
faecal strains outbreak related , the 5 outbreak related
strains (3 from food and 2 human faecal strains) are
the same strains as in Johler et al., 2018
wgSNPs and
cgSNP
5outbreakrelatedstrains(3fromfoodand2humanfecal
strains)differedby0-3wgSNPs(0-2cgSNPs)withtheB.
thuringiensisaizawaibiopesticidestrainABTS-1857.
Biggel et
al., 2022
19 biopesticide strains, 143 food strains, linked to 49
outbreaks and from which 21 strains were from
outbreaks where no other putative food pathogens
were detected during the outbreak investigation
cgSNPs
3)
Foodstrainslinkedto47outbreaksdifferedby0to10wgSNPs
fromB.thuringiensisaizawaiandB.thuringiensiskurstaki
biopesticidestrains.The47outbreaksrelatedto18,8%oftheB.
cereuss.l.associatedfoodborneoutbreaks.Levels in food
products for which B. thuringiensis was the only pathogen,
ranged from 10
2
to more than 10
7
cfu/g (median 9x10
2
), 2 food
strains linked to 2 outbreaks were not linked to any biopesticide
strain.
Bonis et
al., 2021
7 food strains, 5 strains from vomit sample of one
patient.
polyphasic
approach
No link found between strains isolated from vomit and from food. Pheepakp
raw et al.
2023
Restricted Use - À usage restreint
1)Johleretal.,2018(PCR),Bonisetal.,2021(PCR),Biggeletal.,2022(PCR,WGS)
2)limited exposure is expected due to agricultural use when applied for mosquitoes (Brühl et al., 2020)
3)limited foodborne exposure is expected when used on potato foliage (Bonis et al., 2021)
4)of the nhe operon, the nheA gene has a disrupted promotor (Biggel et al., 2022)
BACILLUS THURINGIENSISBIOPESTICIDESTRAINS
Strain Application Presence of cytK2, nhe, hbl genes
1)
B. thuringiensis subsp. aizawai
GC-91 Fruit, vegetables, ornamentals cytK2, nhe, hbl
ABTS-1857 Fruit, vegetables, ornamentals cytK2, nhe, hbl
B. thuringiensis subsp. kurstaki
ABTS-351 Fruit, vegetables, ornamentals, forestry cytK2, nhe, hbl
SA-11 Fruit, vegetables, ornamentals, forestry cytK2, nhe, hbl
SA-12 Fruit, vegetables, ornamentals cytK2, nhe, hbl
PB-54 Fruit, vegetables, ornamentals cytK2, nhe, hbl
EG2348 Fruit, vegetables, ornamentals cytK2, nhe, hbl
B. thuringiensis subsp. israelensis
AM65-52 Mosquito control (not agricultural use)
2)
cytK2, nhe, hbl
BMP144 Mosquito control (not agricultural use) cytK2, nhe, hbl
B. thuringiensis subsp. tenebrionis/ morrisoni
NB-176 Potato foliage
3)
nhe
4)
, hbl
1)Johleretal.,2018(PCR),Bonisetal.,2021(PCR),Biggeletal.,2022(PCR,WGS)
2)limited exposure is expected due to agricultural use when applied for mosquitoes (Brühl et al., 2020)
3)limited foodborne exposure is expected when used on potato foliage (Bonis et al., 2021)
4)of the nhe operon, the nheA gene has a disrupted promotor (Biggel et al., 2022)
BACILLUS THURINGIENSISBIOPESTICIDESTRAINS
Strain Application Presence of cytK2, nhe, hbl genes
1)
B. thuringiensis subsp. aizawai
GC-91 Fruit, vegetables, ornamentals cytK2, nhe, hbl
ABTS-1857 Fruit, vegetables, ornamentals cytK2, nhe, hbl
B. thuringiensis subsp. kurstaki
ABTS-351 Fruit, vegetables, ornamentals, forestry cytK2, nhe, hbl
SA-11 Fruit, vegetables, ornamentals, forestry cytK2, nhe, hbl
SA-12 Fruit, vegetables, ornamentals cytK2, nhe, hbl
PB-54 Fruit, vegetables, ornamentals cytK2, nhe, hbl
EG2348 Fruit, vegetables, ornamentals cytK2, nhe, hbl
B. thuringiensis subsp. israelensis
AM65-52 Mosquito control (not agricultural use)
2)
cytK2, nhe, hbl
BMP144 Mosquito control (not agricultural use) cytK2, nhe, hbl
B. thuringiensis subsp. tenebrionis/ morrisoni
NB-176 Potato foliage
3)
nhe
4)
, hbl
Restricted Use - À usage restreint
•Bacillus cereus s.l.
•opportunistic , infections in vulnerable populations, such as premature neonates,
elderly and immunocompromised
•B. thuringiensis
•Several articles reporting on infections in immunocompromised humans e.g.
wound, eye, bloodstream infections
•All articles have methodological problems precluded an unambiguous taxonomic
assignment
SAFETY CONCERNS NOTRELATEDTOFOODBORNEDISEASE
•Bacillus cereus s.l.
•opportunistic , infections in vulnerable populations, such as premature neonates,
elderly and immunocompromised
•B. thuringiensis
•Several articles reporting on infections in immunocompromised humans e.g.
wound, eye, bloodstream infections
•All articles have methodological problems precluded an unambiguous taxonomic
assignment
SAFETY CONCERNS NOTRELATEDTOFOODBORNEDISEASE
Restricted Use - À usage restreint
•Bacillus thuringiensis is not recommended for the QPS
status due to safety concerns
CONCLUSION
•Bacillus thuringiensis is not recommended for the QPS
status due to safety concerns
CONCLUSION
THANKS: QPS WORKING GROUP AND BIOHAZ
PANEL
•Juan Evaristo Suarez
•Miguel Prieto Maradona
•Pier Sandro Cocconcelli
•Lolke Sijtsma
•Luisa Peixe - Panel member
•Pablo Salvador Fernández
Escámez
•Ingvar Sundh
•Marianne Chemaly
•Amparo Querol
•Lieve Herman – chair, Panel
member
14
PANEL
•Juan Evaristo Suarez
•Miguel Prieto Maradona
•Pier Sandro Cocconcelli
•Lolke Sijtsma
•Luisa Peixe - Panel member
•Pablo Salvador Fernández
Escámez
•Ingvar Sundh
•Marianne Chemaly
•Amparo Querol
•Lieve Herman – chair, Panel
member
14
Restricted Use - À usage restreint
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