24 June 2019: This OECD seminar presented and discussed the potential use of genome sequence, bioinformatic tools and databases in a regulatory decision process for microbial pesticides.

1. EFSA WGS activities for Food
Safety:
Potential value of bioinformatic
analysis in regulatory process
including pesticides
Beatriz Guerra and Jürgen Sturma
OECD Pesticides WG Bioinformatics Seminar:
Bioinformatics and regulation of microbial pesticides
24 June

2.  EFSA, sequencing and molecular studies
 WGS Foodborne pathogens and zoonotic bacteria
 Funded WGS projects
 EFSA WGS Umbrella Project
 WGS Regulated products:
 GMO
 FEED
 FIP
 Pesticides
 Conclussions
Outline
2

3. 3
EFSA, sequencing and
molecular studies

4. EFSA INTEREST on Using New sequencing
technologies
4
 Outbreak detection and investigation
 Detection, characterization and surveillance of emerging pathogens
 Monitoring of antimicrobial resistance
 ...
 For regulated products:
 GMOs
 Feed aditives
 Food enzymes
 Food aditives
 Novel foods
 Pesticides
...use the data generated by new Sequencing technologies (WGS, Metagenomics)
for Food Safety and Public Health Protection

5. Molecular studies in EFSA
5
Novel molecular
biology methods
Effects chemical
components on
living organisms
Genetic
polymorphisms
Novel computational
approaches to predict
drug metabolism and
toxicity
Enzyme
characterization
OMICs
Detection of
emerging
«high risk»
clones
WGS
Metagenomics
AMR
surveillance
(EUSR)
Outbreak
investigation
(ROAs)
Characterization/prediction
resistance/virulence
Transcriptomics
GMOs
Guidelines applications
regulatory products
Cloud
Novel food
Feed aditives
Pesticides Technical notes
Metabolomics
Big data
Biomarkers
…in EFSA
Proteomics
Source
attribution
Health claims

6. 6
Foodborne pathogens
and zoonotic bacteria

7. 7
Rapid outbreak assessment
Reference testing supporting EUSR-AMR

8. 8
“Technical support to collect and analyse whole genome sequencing (WGS)
data in the joint ECDC-EFSA molecular typing database”
at least L. monocytogenes, Salmonella, E.coli
Published May 2019
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/sp.efsa.2019.EN-1337
WP2 Joint ECDC-EFSA WGS Database
Request for scientific and technical assistance on harmonised monitoring of
antimicrobial resistance (AMR) in bacteria transmitted through food
Published June 2019
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2019.5709
BIOHAZ PANEL
“Self-tasking mandate for scientific opinion on the application and use of
next generation sequencing (including whole genome sequencing) for risk
assessment of foodborne microorganisms”
Deadline October 2019

9. 9
Funded WGS projects

10. Thematic grants, promote networking
ONE HEALTH
10
Molecular approaches for identifying and characterising microbial foodborne
pathogens, specifically using Whole Genome Sequence (WGS) analysis”
(GP/EFSA/AFSCO/2015/01)
www.engage-europe.euwww.innuendoweb.org
Co-funding EFSA: 1,000,000 €
Jan. 2016-July 2018
UH/UL/INSA/THL/EVIRA/VME/UPV-EMU DTU/PHE/APHA/BfR/NIPH-NIH/NVRI/IZSVe/IZSLT
Jan. 2016-Jan. 2018
https://www.efsa.europa.eu/it/supporting/pub/en-1498 https://www.efsa.europa.eu/it/supporting/pub/en-1431

11. 11
EFSA WGS Capacity building

12. Cloud infrastructure – Analysis and storage
12
GUIDs
Analysis
EFSA+
Scientist + Bioinformatician
S3 storage
~ 5TB - 5987 isolates
ER-DB
Linux AWS servers
Free license key

13. 13
Regulated products

14. GMO
14

15. GMO: Current use and infrastructure
15
Junction site analysis for detection of all insertion sites
Sequencing to demonstrate Genetic stability
GMO AZURE (microsoft) cloud for secure
storage and sharing Tools to visualise data
(FASTQ), view/perform alignments of SAM,
BAM or CRAM files (e.g. UGENE)
Quality check: Coverage, Bioinformatics,
Annotation (NCBI Sequin, EMBL GenBank),
Quality statistics (FASTQC)
Current infrastructure
and tools

16. Feedap Guidance
16
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2018.5206

17. FEEDAP Guidance
17
Characterisation of microbial strains which are the subject of applications for authorisation
of feed additives containing or produced with microorganisms.
Requirements:
 Taxonomic classification
 Antimicrobial susceptibility
 Potential toxigenicity and pathogenicity of the strains
 Antimicrobial production
 Production strains genetically modified - description of the genetic modification.
…Based on WGS data

18. CEP statement
18
https://efsa.onlinelibrary.wiley.com/do
i/full/10.2903/j.efsa.2019.5741

19. CEP Panel Statement
19
…. Whole genome sequence analysis (including chromosome(s) and
extrachromosomal genetic elements, e.g. plasmids) is required for the
characterisation of bacterial and yeast strains intended for use as
production strains. WGS analysis is also recommended for filamentous
fungi. WGS data provide information for the characterisation of the strains regarding
their potential functional traits of concern (e.g. virulence factors, production of or
resistance to antimicrobials of clinical relevance, production of known toxic
metabolites)...
Based on the FEEDAP Guidance, CEP statement requires…

20. FEEDAP and CEP
20

21. Pesticides
21
 Regulation (EC) No 1107/2009
 Provides a definition for ‘micro-organisms’
 “..any microbiological entity, including lower gungi and viruses, cellular
or non cellular, capable of replication or of transferring genetic
material”
 Regulations (EU) No 283 and 284, part B
 Data requirements for micro-organisms including viruses
 Data requirements for preparations of micro-organisms
including viruses
 Regulation (EC) No 546 (Annex part ii)
 ‘uniform principles’

22. Pesticides
22
 Requirements for e.g.
 Identity
 Biological properties
 Relationship to known pathogens
 Genetic stability
 Resistance or sensitivity to antibiotics
 Effects on human health
 Residues in/on treated products
 Fate and behaviour
 persistence and multiplication
 Mobility
 Ecotoxicology

23. Pesticides – areas of improvement
23
 Identity
 “best available technology should be used to identify and
characterise the MO at strain level”
 already the same wording in Directive 91/414, Annex IIB
 WGS data are not required
 Metabolites
 Data on metabolites should be submitted, if
 the metabolites contribute to the MoA (mode of action)
 Significant residues are expected
 Antimicrobial Resistance (AMR)

24. Pesticides
24
 WGS data should be submitted for Pesticides
 Potential uses of WGS analysis:
 Identification of the microorganism at strain level
 Identification of genes
 Related to the production of secondary metabolites / toxins
 Related to the possible antimicrobial resistance

25. Pesticides ongoing activities
25
 Commission ‘WG on biopesticides’
 Draft guidance document on secondary metabolites produced
by micro-organisms
 Draft guidance document on AMR
 Taking into consideration the FEED GD
 Discussion on the amendment for the data requirements for
micro-organisms
 EFSA internal discussions

26. 26
Conclusion

27. Conclusion…
27
EFSA is moving on in the area of WGS:
• Different needs, similar/different bioinformatic tools needed.
• Technology developments make it adequate to use WGS to characterize microorganisms,
also for those use as biopesticides.
• WGS-based information already required/recommended for other regulatory products.
• Very important harmonize the type of data required.
• Detailed information on analysis done/tools versions, etc needed.
and preparing to collect and deal with WGS data and WGS-based information.

28. 28
Beatriz Guerra
Senior Scientific Officer
Unit on Biological Hazards and Contaminants (BIOCONTAM)
Department of Risk Assessment and Scientific Assistance (RASA)
beatriz.guerra@efsa.europa.eu;
Jürgen Sturma
Scientific Officer
Pesticide Peer Review Unit
Department of Scientific Evaluation
of Regulated Products
Juergen.sturma@efsa.Europa.eu
Thanks for your attention!
And thank to all the colleagues from the different EFSA Units (BIOCONTAM, GMO,
FEED, FIP, Pesticides, TS) that providedshared information for this presentation