GenomeTrakr: A Pathogen Database

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GenomeTrakr: A Pathogen Database

Marc W. Allard, PhD

Senior Biomedical Research Services Officer

Division of Microbiology

FAO Expert workshop to develop case studies on the use of Whole Genome Sequencing (WGS) on food safety managment. Nov. 12, 2015

Eric W. Brown, PhD

Director

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Outline

• Technology shift

• GenomeTrakr: Reference database and

pathogen detection pipeline

• Benefits to industry, growers, and distributers.

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Outline

• Technology shift

• GenomeTrakr: Reference database and

pathogen detection pipeline

• Benefits to industry, growers, and distributers.

(4)

Current method for pathogen

identification

1. antigens are screened to identify serovar

2. PFGE: genome is cut into pieces. Sizes of these pieces and

the banding patterns they determine discrimination within

serovar.

4

PulseNet

(5)

• WGS is high resolution



3-5 million data points are collected for each isolate

• WGS analyses are statistically robust



Unlike PFGE patterns, WGS data can be analyzed in its

evolutionary context. Accurate and stable genetic changes within

pathogen genomes enable us to pin point specific common

sources of outbreak strains (farms, processing plants, food types,

and geographic regions).



Source Tracking is Key Application

(6)

PFGE identical

in red

NGS distinguishes geographical structure among

closely related

Salmonella

Bareilly strains

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Same PFGE but not part of the outbreak

Outbreak Isolates 2-5 SNPs

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Outline

• Technology shift

• GenomeTrakr: reference database and

pathogen detection pipeline

• Benefits to industry, growers, and distributers.

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• Bases of DNA (ATGC) are sequentially identified from a DNA template strand

• Next Generation Sequencing (NGS) extends this process across millions of reactions in a massive parallel fashion

• NGS involves rapid sequencing of large DNA stretches spanning entire genomes

– Technology shift

– 3-5 million data points for each isolate

• Increasing availability and affordability of NGS is rapidly changing the face of microbiology

DNA Sequencing

$0 $500 $1,000 $1,500 $2,000 $2,500 $3,000 $3,500 2007 2008 2009 2010 2011 2012 2013

Cost per bacterial genome

Desk-top FDA 1st $70/genome in 2014 $40/genome in 2015 w/ Higher througput Technology

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GenomeTrakr Fast Facts

• First distributed network of labs to utilize WGS

for pathogen identification

• GenomeTrakr network has sequenced more than

40,000 isolates, and closed more than 100

genomes through November 12, 2015.

• Currently sequencing more than 1,000 isolates a

month

• The need for increased number of well

characterized environmental (food, water,

facility, etc.) sequences may outweigh the need

for extensive clinical samples

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GenomeTrakr Labs

• 14 federal labs

• 14 state and university labs

• 1 U.S. hospital lab

• 5 labs outside of the U.S.

• Collaborations with independent academic researchers

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15 N u m b er o f S eq u en ces (as of t he l as t day of t he quar ter )

Total Number of Sequences in the GenomeTrakr Database

2013 2014 2015

Average Number of Sequences

Added Per Month in 2013 = 184 Added Per Month in 2014 = 1,049Average Number of Sequences

First sequences uploaded in Feb 2013

Public Health England uploads more than 8,000

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0 5 10 15 20 25 30 35 40 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68

Timeline for Foodborne Illness Investigation

Using Whole Genome Sequencing

Contaminated food enters commerce

FDA, CDC, FSIS, and States use WGS in real-time and in parallel on clinical, food,

and environmental samples

Source of contamination

identified early through WGS combined database queries

Averted Illnesses N um ber of C ases Days

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MINIMAL PATHOGEN METADATA

(FOODBORNE OUTBREAKS)

sample_name organism strain/isolate Category (attribute_package) 1a) Clinical/Host-associated 1a1) specific_host 1a2) isolation_source 1a3) host-disease OR 1b) Environmental/Food/Other 1b1) isolation_source

Food Industry can hold confidential metadata linked to public records

collection_date Geographic location 6a) geo_loc_name OR 6b) lat_lon collected by

Where

When

Who

What

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Immediate impacts of WGS to industry, growers,

and distributers, countries, states.

• Earlier intervention means:

1) Reduced amount of recalled product;

2) fewer sick patients which means fewer lawsuits;

3) less impact overall and minimal damage to

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Impacts to industry, growers, and distributers (continued).

• Regular testing throughout network:

1) identifies specific suppliers that are introducing contaminants;

2) identifies whether contaminant is resident to a facility or

transient;

3) knowledge of where contaminant is coming from allows industry

to fix the problem based on scientific evidence.

–Shift costs to the supplier who has introduced the contaminant. –How often is the root cause of the problem left unresolved

to occur again at a later date?

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Background: CFSAN SNP Pipeline

Documentation: http://snp-pipeline.rtfd.org Source Code: https://github.com/CFSAN-Biostatistics/snp-pipeline PyPI Distribution: https://pypi.python.org/pypi/snp-pipeline

Pettengill JB, Luo Y, Davis S, Chen Y, Gonzalez-Escalona N, Ottesen A, Rand H, Allard MW, Strain E. (2014) An evaluation of alternative

methods for constructing phylogenies from whole genome sequence data: a case study with

Salmonella. PeerJ 2:e620

http://dx.doi.org/10.7717/peerj.620

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Molecular Epidemiology and Ecology of

Multi-drug Resistance (MDR) Salmonella

in Tanzania

•Julius Medardus

•Sokoine University of agriculture

•Wondwossen A. Gebreyes

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Salmonella

lose or gain resistance

depending on the ecosystem.

Environment- SSu

(Gebreyes and Altier, 2002

Gebreyes et al., 2004, 2009)

GIT- ACSSuT

(Briggs and Fratamico, 1999)

25

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What triggers the recombination?

Interaction between bacterial factors and and

chemical intervention in pig production

Important

Element

In MDR

Common

In the

Environment

QAC

Quaternary ammonium compounds

qacE

on integrons and Quats are commonly used as

disinfectants.

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Co-selection: Heavy metal v. MDR

• Heavy metals in the ecosystem- Cu and Zn;

• _{Assoc. b/n AMR-type and HM- MIC;}

• Co-selection with MDR;

• Association with Invasive NTS strains?;

• Efflux pump genes-

pcoA

and

czcD

;

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Association

-

Heavy Metal tolerance and MDR

Salmonella

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Odds ratio between copper tolerance (<20mM) and MDR AmStTeKm was 4.6 (Chi-square=17.9; P<0.05)

The odds of having a high Zn MIC (>8mM) were 14.66 times higher in

isolates with R-type AmClStSuTe than in those with R-type AmStTeKm(P<0.05).

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FDA GenomeTrakr partnership

924 isolates submitted

to FDA-CFSAN

• Brazil (4)

• Ethiopia (401)

• Kenya (86)

• Mexico (63)

• Tanzania (64)

• Thailand (60)

• U.S. –OSU (247)

29

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Tanzania

• WGS- 45 food animal isolates completed

• All Unknown STs

• Plasmid types- ColRNAI, IncI1, IncI2, IncFII, ColpV2

(total 10)- Others?

• Kentucky (16/ 45) and Not conforming with any

known type (n=8)

• Pending- HM and biocide tolerance genes/ efflux

system…

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COMPARE is a large EU project with the intention to speed up the detection of and response to disease outbreaks among humans and animals worldwide through the use of new genome technology.

The above figure represents genomic information as the pathogen-independent language across locations, sectors and time.

http://www.compare-europe.eu/ Coordinator

Frank M. Aarestrup

Technical University of Denmark National Food Institute

[email protected] Tel: +45 35 88 62 81

Co-Coordinator

Marion Koopmans

Erasmus Medical Centre Department of Viroscience [email protected] Tel: +31 10 70 44 066

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Whole Genome Sequencing Program (WGS)

http://www.fda.gov/Food/FoodScienceResearch/WholeGenomeSequencingProgramWGS/default.htm#trakr

GenomeTrakr

• State and Federal laboratory network

collecting and sharing genomic data from foodborne pathogens

• Distributed sequencing based network

• Partner with NIH

• Open-access genomic reference database

• http://www.ncbi.nlm.nih.gov/bioproject/183844 • Can be used to find the contamination

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For more information:

• For information about joining the

GenomeTrakr network as a sequencing

lab, providing isolates to a current

member lab for sequencing, or using

the GenomeTrakr database as a

research tool, please contact FDA at

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FDA

Steven Musser Patrick McDermott Ruth Timme Marc Allard Peter Evans Eric Brown Justin Payne Charlie Wang Rebecca Bell Christine Keys Errol Strain Yan Luo

James Pettengill Hugh Rand Darcy Hanes Gopal Gopinathrao Chis Grim Palmer Orlandi David Melka Cary Pirone Davies Justin Payne Maria Hoffman Eric Stevens Andrea Ottesen Tim McGrath Don Burr Jie Zheng Cong Li George Kastanis Tim Muravunda Shaohua Zhao

National Institutes of health (NCBI)

David Lipman Jim Ostell William Klimke Martin Shumway Richa Agarwala

State Health Labs

Bill Wolfgang (NY) Dave Boxrud (MN) Anita Wright (FL) Elizabeth Driebe (AZ) Angela Fritzinger (VA)

Ailyn Perez-Osorio (WA) More to come…….

USDA

David Goldman Kristin Holt

Illumina

Susan Knowles Omayma Al-Awar Kelly Hoon

With Additional Thanks….

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ORA OCC OFS OC OAO OFVM/SRSC CFSAN CDER

CBER CDRH CVM NCTR FDA CHIEF SCIENTIST OIP OARSA

SCIENCE BOARD IAS FFC FERN JIFSAN ADVISORY COMMITTEE IFSH

MOFFETT CENTER CIO DAUPHIN ISLAND CFSAN-OCD CORE WESTERN CENTER

INTERNAL FDA STAKEHOLDERS

FDLI GMA VaFSTF CDC FBI PULSENET-LATIN AM. AM. ACAD MICROBIOL ASM FSIS ARS UNIV VERMONT MINN DOH AZ DOH UNIV FL VA DOH WA DOH TX DOH NY AG LAB IRISH FSA NOVA SE UNIV IGS BALTIMORE INFORM MEETING HONGKONG POLYT U NIST ITALIAN FSA EFSA

WHO-FOOD SAFETT DIR. WHO-GFN

CDC-EU

EMERGING INFECTIOUS DIS CONF DANISH TECH UNIV

NM STATE UNIV/ NM DOH CARLOS MALBRAN INST/ARG ST COULD UNIV/FOOD MICRO SENASICA GMI NY DOH/WADSWORTH CENT UNIV HAMBURG CHINA CDC NESTLE FERA-UK MD DOH IAFP APHL AFDO BELGIUM VaTech US ARMY US NAVY

MELBOURNE FSA (AUS) UNIV NEBRASKA

PUBLIC HEALTH ENGLAND DHS

DELMARVA TASKFORCE PENN STATE FOOD SCIENCE PROD MAN ASSOC

ILLUMINA

UNIV IRELAND/DUBLIN COLLEGE

NCBI/NIH

GSRS GLOBAL SUMMIT FAO/OIE

PUBLIC HEALTH CANADA CFIA

HEALTH CANADA INTL VTEC MEETING CPS-GA AOAC UNITED FRESH COLUMBIA HAWAII DOH CA DOH ALASKA DOH SOUTH DAK UNIV UNIV GA UNIV IOWA/DOH UNIV CHILE BRAZIL OSU VETNET TURKEY MEXICO IEH SILLAKER

NEW ENG BIOLAB PACIFIC BIO CLC-BIO/QIAGEN CON-AGRA DUPONT AGILENT UC-DAVIS HARVARD MED INFORM MEETING THAILAND