The EU Reference Laboratory for Escherichia coli ... · coli, including Verotoxigenic E. coli...
Transcript of The EU Reference Laboratory for Escherichia coli ... · coli, including Verotoxigenic E. coli...
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
EU Reference Laboratory for Escherichia coliDip. Sanità Pubblica Veterinaria e Sicurezza Alimentare
Istituto Superiore di Sanità, Roma
www.iss.it/vtec
www.iss.it/seu
The EU Reference Laboratory for Escherichia coli, including Verotoxigenic E. coli (VTEC)
Activities and inter-relations with other EURLs
Stefano Morabito
http://www.iss.it/vtec
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
The “Zoonoses Directive” 2003/99/CE
Annex I A, First rank priority• Salmonella • Escherichia coli STEC • Campylobacter• Brucella • Listeria monocytogenes• Mycobacterium bovis• Echinococcus• Trichinella
Monitoring and surveillance of zoonotic agents in animals and food
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Intestinal E. coli - Kaper et al., 2004
The “E. coli problem”…
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
The “E. coli problem”…
Extraintestinal E. coli
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
(EFSA journal, 2015)
Is by far more complex than it seems
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Regulation EC No 882/2004 on official controls
The EU Reference Laboratory for Escherichia coli, including Verotoxigenic E. coli (VTEC)
Since 2006Unit for Foodborne Zoonoses and Veterinary EpidemiologyDepartment of Veterinary Public Health and Food Safety
Istituto Superiore di Sanità, Rome, Italy
www.iss.it/vtec
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
The network of RLs for Escherichia coli (VTEC)42 NRLs designated by
28 EU Member States+ 7 other European Countries
Additional Ref. Labs: Chile, Argentina, Iran, Nigeria, Egypt…
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Functions and duties of EU-RLs (882/2004, Art. 32)
To provide NRLs with analytical reference methods
To produce and distribute reference materials
To organise comparative testing (Proficiency Tests)
To conduct training for NRLs, (annual workshop,
courses, visits to the EU RL)
To provide scientific and technical assistance to the
Commission
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Methods development
Methods’ evaluationMethods developementPCR-based
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
First draft in 2008Published on 7 November 2012
Methods development
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
2011: Vegetables (spinach)
2008: Bacterial strains
Proficiency tests organized by the EU-RL VTEC
2009: Carcass swabs 2010: Milk
Detection of VTEC in food by ISO/TS 13136
2012: Water samples
2013/2014: Sprouts samples2015: Spent irrigation water from sprouting
2016: Ground beef2017: Spent irrigation water from sprouting
2017: Vegetables (rocket)2018: Sprouts samples
2012: Seeds for sprouting
Reg. 209/2013
Reg. 209/2013
Reg. 209/2013
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
2010: STEC more serogroups and virulence genes – Joint PT with ECDC labs
2007: STEC top five serogroups
Proficiency tests organized by the EU-RL VTEC
2008: STEC more serogroups2010: STEC more serogroups and virulence genes
STEC strains identification and typing
2012: STEC more serogroups and virulence genes – Joint PT with ECDC labs
2014: STEC + EPEC + EAEC2016: STEC + ETEC + EPEC + EAEC
2012/2013/2014/2015/2016: PFGE typing of STEC and other pathogenic E. coli
2017/2018: 1st interlaboratory study on WGS of STEC
2013: STEC + ETEC + EIEC + EPEC + EAEC
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Proficiency testing: Trend of participation
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Standard Training Programs are the tools for the capacity building
From 2015: STP on PT organisation & Bioinformatics courses
Visit our website (www.iss.it/vtec) for the latest STPs
New entry from 2017: STP on Bioinformatics
http://www.iss.it/vtec
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
A growing number of scientists receives training at the EU RL
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
NGS equipments adoption
on: Omicsmap.com June, 17, 2015
Friend or foe?
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
«the system has failed» paradox
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
ARIES: A Galaxy-based workspace for intensive data analyses
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Ask for an account: [email protected]
Collaborative projects: [email protected]; [email protected]
ARIES: CreditsThe Galaxy ARIES core group at the EU RL for E. coli:• Stefano Morabito (EU-RL VTEC): [email protected]
ARIES Scientific coordination, tools design, contact person• Arnold Knijn (SIDBAE): [email protected]
ARIES Administrator, tools integration, contact person• Valeria Michelacci (EU-RL VTEC): [email protected]
Tools design, contact person
• ARIES is a collaborative platform. It is open and based on the social science concept• We protect your data from hacking• We don’t backup your data (50GB workspace allocated to each user)• We don’t see your data (unless youre asking us to do so)• Read the Disclaimer at: https://w3.iss.it/site/aries/
ARIES: Users Policy
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
ARIES geographical spread – Workshop 2017
Total ~100 users
(and counting!)
Australia, Cina, India, Iran, USA
Including NRLs from 13 MSs:
BelgiumDenmarkFinlandGermanyIrelandItaly
LatviaThe NetherlandsPoland (2 NRLs)PortugalSloveniaUnited Kingdom
Users from outside Europe:
Other users from Europe:
France, Luxemburgh, Romania, Spain
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
Development of a pipeline for WGS typing of STEC
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
EURLS WORKING GROUP ON WGS
AIM:to promote the use of WGS across the EURLs' networks, build WGS capacity within the EU and ensure liaison with the work of the EURLs and the work of EFSA and ECDC on the WGS mandate sent by the Commission
MEMBERSHIP:EURL E. coli (coordinator)EURL Listeria monocytogenesEURL CPSEURL SalmonellaEURL CampylobacterEURL ParasitesEURL AREURL Foodborne virusesObservers: SANTE G4, EFSA, ECDC
Kick off:November 14th 2017
Second meeting:June 11th 2018
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
EURLs WORKING GROUP ON WGS
Tasks:- PTs- DNA extraction and sequencing- Bioinformatics tools- WGS data analysis- Bench marking- Training on WGS- Follow-up of ISO activities on WGS
EURL-VTEC nominated as an observer in the WG 25 “WGS for typing and genomic characterization” of ISO/TC 34/SC 9
“Food products-Microbiology”
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
EURLs WORKING GROUP ON WGS
First action:
Survey on the state of the art of the preparedness on the use of NGS across the NRLs networks
Basing on EFSA survey on NGS preparedness, with the aim of updating the results and targeting the working group actions on
the actual needs of the NRLs
Sent to the NRLs in March 2018
Deadline: April 16th 2018
Results to be discussed at the second meeting of the WG in June
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
E. coli network: Survey results
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WGS Metagenomics sequencing Bioinformatics analysis
NGS activities performed
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
E. coli network: Survey results
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Introduction WGS Wet-lab:sample and NGS
library preparation
WGS Wet-lab:quantification andquality control of
the preparedlibraries
WGS Dry-lab:quality control of
NGS data
WGS Dry-lab:genome assembly
WGS Dry-lab:SNP analysis
WGS Dry-lab:cgMLST
WGS Dry-lab:tools for in silicotyping; MLST,
MLVA,serotyping,
virulotyping,AMR
Interest in training on NGS organized by the EURL (N = 15)
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
1st PT on WGS of STEC
23 Laboratory (20 EU NRLs; 3 IT Ols) 6 STEC strains (STEC O26)Collected raw reads through an online system
Analyses:• QC• Serotyping• MLST (7 genes)• Identification of target genes (STEC virulence genes)• SNP-based phylogenesis with reference• SNP-based phylogenesis without reference• cgMLST
Purpose: to get info on the data produced by the network and on their usability to type STEC and for surveillance/monitoring
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
S Serotype errorM MLST errorV Virulence error
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1st PT on WGS of STEC –QC-
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
1st PT on WGS of STEC –SNP analysis without reference (kSNP3)
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
1st PT on WGS of STEC –cgMLST
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
1st PT on WGS of STEC –cgMLST
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Istituto Superiore di Sanità - Dip. Sanità Pubblica Veterinaria e Sicurezza Alimentare EU Reference Laboratory for E.coli
1st PT on WGS of STEC Take home messages
The Network is growing in size and skills on pathogenic E. coliMost of the NRLs in the E. coli Network do WGSThe data can be used as such to type STEC (a few errors but amendable)Surveillance and Monitoring of STEC may be largely affected by the lack of harmonizationThe inter EURL is committed to fill the gaps (as well as the inter EURL WG)The 1° PT on WGS of the EURL VTEC may serve as a quantitative basis for such an improvementOther networks?????
We are willing to help if our experience may be useful to build the capacity in other labs/networks
Diapositiva numero 1The “Zoonoses Directive” 2003/99/CE Diapositiva numero 3Diapositiva numero 4Diapositiva numero 5Diapositiva numero 6Diapositiva numero 7Diapositiva numero 8Diapositiva numero 9Diapositiva numero 10Diapositiva numero 11Diapositiva numero 12Diapositiva numero 13Diapositiva numero 14Diapositiva numero 15NGS equipments adoption«the system has failed» paradoxDiapositiva numero 18Diapositiva numero 19Diapositiva numero 20Diapositiva numero 21Diapositiva numero 22Diapositiva numero 23Diapositiva numero 24Diapositiva numero 25Diapositiva numero 26Diapositiva numero 27Diapositiva numero 28Diapositiva numero 29Diapositiva numero 30Diapositiva numero 31Diapositiva numero 32