Serovar Identification of Foodborne Pathogens - Agilent · Serovar Identification of Foodborne...
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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
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
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
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
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
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
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)
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
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
530.754.0109
Lenore Kelly
Americas’ Food Research Biochemist
408.345.8424
Questions?