The EU Reference Laboratory for Escherichia coli ... · coli, including Verotoxigenic E. coli...

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


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


Intestinal E. coli - Kaper et al., 2004

The “E. coli problem”…


The “E. coli problem”…

Extraintestinal E. coli


(EFSA journal, 2015)

Is by far more complex than it seems


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


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…


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


Methods development

Methods’ evaluationMethods developementPCR-based


First draft in 2008Published on 7 November 2012

Methods development


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


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


Proficiency testing: Trend of participation


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


A growing number of scientists receives training at the EU RL

2 2 2 3

811 11

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EU-NRL non EU-NRL/Third countries IT OLs


NGS equipments adoption

on: Omicsmap.com June, 17, 2015

Friend or foe?


«the system has failed» paradox


ARIES: A Galaxy-based workspace for intensive data analyses


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]


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


Development of a pipeline for WGS typing of STEC


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


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”


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


E. coli network: Survey results

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in house outsourced not performed in house outsourced not performed in house outsourced not performed

WGS Metagenomics sequencing Bioinformatics analysis

NGS activities performed

20/35 replies


E. coli network: Survey results

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in housepipeline

Commercialsoftware

Online server in housepipeline

Commercialsoftware

Online server in housepipeline

Commercialsoftware

Online server

SNP analysis wgMLST cgMLST

NGS analysis

02468

101214

NGS data production Analysis of NGS data Harmonising WGS workand assurance of quality

data

Interest in EURLProficiency Tests (N = 12)

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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)


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


S Serotype errorM MLST errorV Virulence error

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Single end

disclaimer

Depth

N50

Coverage

1st PT on WGS of STEC –QC-


1st PT on WGS of STEC –SNP analysis without reference (kSNP3)

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1st PT on WGS of STEC –cgMLST


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

The EU Reference Laboratory for Escherichia coli ... · coli, including Verotoxigenic E. coli...

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