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Serovar Identification of

Foodborne Pathogens

Lenore Kelly, Ph.D.

Americas’ Food Research Scientist

Agilent Technologies, Inc.

Bart Weimer, Ph.D.

School of Veterinary Medicine

UC Davis

BGI@UCDavis

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Serovar Identification of

Foodborne Pathogens

Lenore Kelly, Ph.D.

Americas’ Food Research Scientist

Agilent Technologies, Inc.

Bart Weimer, Ph.D.

School of Veterinary Medicine

UC Davis

BGI@UCDavis

Contamination of food with pathogenic or toxin producing bacteria

or fungi are a major concern in food safety.

Major pathogenic bacteria: Salmonella enterica enterica,

pathogenic E. coli (EHEC),Listeria,Campylobacter, Yersinia enterocolitica

Example outbreaks: 2012 – Pet food and Salmonella

2011 – Re-emergence of E. coli O104

2010 – Eggs and S. Enteritidis

2008 – Peanut butter and S. Typhimurium

2006 – Tomatoes, peppers and S. Typhimurium

Estimated cost is $78,000,000 per year in the U.S.

Estimated economic impact in the EU >€5 billion caused by Campylobacter and

Salmonella

(Source: EFSA website)

Microorganisms as Threat to Food Safety and Quality

Food Safety News Jan 2012

Salmonella &

Human Illness

• 76 million cases annually

• 325,000 hospitalizations

• 3000-5000 deaths/year

• Economic burden due to Salmonella in US is ~$78 billion annually

• Aging population in industrialized countries leading to increased outbreaks

• Hypervirulence emerging (Heithoff et al. 2012 PLoS Pathogen)

Salmonella Serotyping Source:Wikipedia

Major food sources: fresh broiler, turkey and

pork meat, eggs and products thereof

Typing of Salmonella is dominated by traditional

methods (PFGE, phage typing, MLST, MLVA)

Molecular markers have been identified but are

not commonly used in subtyping

Starting point: Proof-of-concept design by

Greg Richmond

Source: EFSA/ECDC Report on Trends and Sources of

Zoonoses, Zoonotic Agents and Food-borne Outbreaks

in 2010 (2012)

Salmonella Diversity

Salmonella species Number of

serovars

S. enterica 2,557

S. enterica subsp. enterica 1,531

S. enterica subsp. salamae 505

S. enterica subsp. arizonae 99

S. enterica subsp. diarizonae 336

S. enterica subsp. houtenae 73

S. enterica subsp. indica 13

S. bongori 22

Total (genus Salmonella) 2,579

Project aims to

combine 2 of 3 steps

Faster response: < 30 h

More informative results

Salmonella Characterization

Detection Genus, species

qPCR, etc. 8-30 h

Traditional: 3-5 days

Serotyping Serogroup, Serovar

Serology: 4-5 days

Strain Typing Genovar, Genome

PFGE, MLST, etc.

5-10 days

Challenging ! > 2,500 serovars

Paucity of genome sequences – difficult molecular subtyping

Sensitivity Specificity

Surveillance

Outbreak response

Outbreak investigation

Complex Problems:

Where are the Solutions?

• Rapid detection strategies

• Genomics and systems biology

• Balance regulation with science

• Bacterial mitigation & reduction strategies

Rapid Detection

Technologies to enhance food safety & security

Eliminate enrichment

Fast

Sensitive

Reliable

Food, water & environment E. coli (B. Findley)

Challenges for Bacterial Detection • Large volumes

• Varying matrix types

• Solids & liquids

• Pre-enrichment

• Physiological state

• Breadth of pathogens

• Varying concentrations

• Zero tolerance

• Many organisms

• Salmonella

• E. coli O157:H7 (and others)

• Campylobacter

• Vibrio

• Yersinia

• Shigella

• Staphylococcus aureus

• Pasteurella multocida

• Mycobacterium avium

• Clostridium perfringens

• Clostridium difficile?

Rapid Detection Technologies

• ImmunoFlow™ GlycoBind®

• ImmunoDNA® TissueTag®

Target capture and concentration

platform

ELISA Presumptive ID

Genome-based Confirmation

Innovation in Methods

• Culture independent methods (CIM) to capture &

concentrate

• Coupling NGS & biomarkers with existing methods and

CIM

• Increase speed

• Increase information diversity

– Serotypes

– Pathogens

So here is where we are now

3 Protocols

• Capture

• “Static” or flow

• Ab, host receptor, liposome

• Wash

• Load other reagents

• Detection

• Chemiluminescent

• DNA

• Flow ELISA

• 30 minutes

• Flow capture with static detection

• 45 minutes

• “Static” ELISA

• 1.5 hours

• Flow capture with DNA detection

• ~3 hours, moving to 30 min

Variables for each

step Base Protocols

Molecular Serology in 2 hours

Report

serogroup &

serotype

Nano

electrophoresis

(30 min)

Heat 95C

for 5 min

Use 1uL as

PCR

template

PCR

(45 min)

Pick

colony

Action based on

serology in a single day

Multiplex PCR for

Salmonella serology Typ

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Band 2

positive

control

Band 1

positive

control

Sero-group

• Validation in

process

• Some primers

modified due

to SNPs

• 100% accurate

in 2 different

blinded

panels

Molecular serotype

Where can we go from here?

• How can we move microbiology testing to high-throughput,

and use modern analytical instrumentation?

Project Goal

Develop a novel tool that integrates PCR with mass code tagging

for multiplex detection and Mass Spectroscopic identification of

food-borne pathogens. This is a new method for serotyping &

characterization of food-associated organisms.

This project was developed at Agilent Labs, Santa Clara, CA

MassCode Liquid Arrays as a Tool for Multiplexed High-

Throughput Genetic Profiling, Gregory S. Richmond*, Htet Khine,

Tina T. Zhou, Daniel E. Ryan, Tony Brand, Mary T. McBride, Kevin

Killeen, PLoS ONE, April 2011, p 18967

This study is currently being validated by both the US FDA in

Bethesda MD and at the UC Davis sites.

What is the Principle of MassCode Detection?

September 24, 2012

target DNA

MassCode protocol

results in double-labeled

double-stranded amplicon

Clean up of PCR products

UV cleavage of tags

MS Detection and Data Analysis

MassCode

tags

MassCode Tag System Workflow

MassCode

tagged primers

w/ PCR

StrataPrep

cleanup

extract gDNA primary enrichment

10-40plex

96 samples

6-hour post-enrichment

detect

Reagents, consumables, instrumentation and software provided by Agilent

356

MassCode Tags A Novel Reporter for Biomolecules

– MassCode labels give biomolecules a digital code

370 374 366 356 352 378 382 T 390 386 394 398

422 434 426 470 466 478 482 T 450 462 458 454 446 442 438 430

T 518 506 514 486 494 498 502 510 534 538 530 526 522 543 547

T 589 601 593 617 613

418 414 402 406 410

474

542

609 605 597 573 569 577 581 585 565 561 557

621 625

633 629 645 649 641 637 T 657 653 661 665 685 669 677 673

T 693 705 701 697 689 733 713 717 721 709 725 729

Daltons

Dalto

ns

– 93 unique tags = high density multiplexing

– A true liquid array: Solution based; No Beads; No solid supports

A Stable Modular Design MassCode tagged primer

Mass post-

cleavage = 356

Stable positive

ion portion

+

Variable mass

portion

Nucleotides

of primer

Oligos are synthesized by Operon with a 6-amino-1-hexanol linker on the 5’-terminal

phosphate. The 6-amino- group is covalently coupled to a photocleavable MassCode tag.

UV light

A Stable Modular Design

Stable positive

ion portion

+

Variable mass

portion

Nucleotides

of primer

2

UV light

Mass post-cleavage = 729

Discrete Resolution of 93 tags

Selective Ion Monitoring of tags

No spectral overlap

15.5 in

26 in

29 in

Benchtop MSD

Simultaneous Monitoring of 44 tags Detection of 8 tags

MassCode PCR Software – Easy access to a MS

Software to facilitate setup and running of MS instrumentation

as well as analysis of resulting data

User interface has the look

and feel of QPCR software –

familiar to a biologist

New Analysis Features:

- Easy to spot, color-coded

results

- Full-blown analysis available,

all the way down to MS data

Making a new list of targets and controls is easy

Set up the MS run just like a PCR plate

Flow Injection looks like this from the run screen

It is easy to interpret results in Batch View

And to use the assay in a true multiplex fashion,

what we require are robust genetic sequences to

insure both specificity and exclusivity to type

bacteria

NGS Costs are Falling

(DeWitt et al., 2011)

Capacity Increasing

(Mardis et al., 2011)

HiSeq 2500

• 2 modes

• 11 days

• 1 day

• Data density

options

Genome Projects • Humans

• Genotype

• Exome

• Small RNA

• Individual microbes

• Pathogens

• Diagnostics

• Microbiome

• Communities

• Health & disease

• Expression

• RNAseq

• Multi-omics integration

(Relman et al., 2011)

Genomics Bottlenecks

(DeWitt et al., 2011)

Pathogen Evolution

• Vibrio evolution rapid

• Example for all enterics

• Also shown with environmental organisms

• Enterobacteria genome evolution

• HGT more common that appreciated

• Genome rearrangements influenced by local community

• Evidence for local pressure to induce population genome evolution

• Biogeography differences

• Likely to find footprints of geographical origin

• Requires large number of genomes to estimate

• Creates chimeric genomes

• Stress induces SNPs

• Mutations in DNA repair genes leads to SNPs

• Recombination events

• SNPs

• Large segments

• HGT

Shapiro et al., ‘12 Science; Denef & Banfield, ’12

Science

New Detection Paradigm

Specific gene

(PCR)

Genome

(multiplex)

Foundation for detection

Salmonella Phylogenomics

16s Alignment Whole Genome Alignment

Existing Limitations

• Molecular assays are limited by too few genomes to

develop robust biomarker genes quickly

• Genome evolution more complex that previously

appreciated

• Lack of genome sequence information to create robust

assays for improved detection

• Genome sequencing will enable technological advances

100K Genome Project

• Consortium

• Sequencing to be done at BGI@UCDavis

• Initial ~500 genomes will be closed

• Genomes will be placed in the public domain

• Metadata submission

• Additional partnerships

• Unique isolates

• World wide representation

• Food industry

• Government

• Academia

• Outcomes

• Population based assessment for new assay design

• Clinical vs. food isolates

• Outbreak and trace back

– Examine biogeography of genome to focus

– SNPs for local divergence

– Virulence and AR

• An entire collection of isolates that match genomes

Organisms of Interest

• Salmonella

• E. coli

• Listeria

• Campylobacter

• Vibrio

• Shigella

• Yersinia

• Clostridium

• Enterococcus

• Cronobacter

• Norovirus

• Hepatitis A

• Enteroviruses

Public/Private Partnership

• Founding Members

• UC Davis

• FDA

• Agilent Technologies

• Affiliate Members

• NCBI

• CDC

• Mars, Inc.

• Harvard hospital system

• RIVM

• DTU

• Walter Reed Hospital

SEEKING

ADDITIONAL

AFFILIATES

Acknowledgements

• Dr. Yi Xie

• Dr. Richard Jeannotte

• Dr. Holly Ganz

• Dr. Marie Forquin

• Dr. Prerak Desai

• Dr. Jigna Shah

• Ms. Nugget Dao

• Ms. Mai Lee Yang

Thanks to the sponsors:

FDA

USDA

DARPA

US Air Force

Cal. Dairy industry

Agilent Technologies

Thank You…

Bart Weimer Professor, UC Davis

Director, BGI@UC Davis

bcweimer@ucdavis.edu

530.754.0109

Lenore Kelly

Americas’ Food Research Biochemist

Lenore_kelly@agilent.com

408.345.8424

Questions?

European Reference Laboratory - FV