Whole Genome Sequencing (WGS) a Powerful Tool …...Whole Genome Sequencing (WGS)–a Powerful Tool...
Transcript of Whole Genome Sequencing (WGS) a Powerful Tool …...Whole Genome Sequencing (WGS)–a Powerful Tool...
Whole Genome Sequencing (WGS)– a Powerful Tool for Traceback Analysis of Foodborne Pathogens
6th Annual American Food Sure Summit
Maria Hoffmann, Ph.D.Genomics Research Microbiologist
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The Complex Etiology of Foods
Shrimp – IndiaCilantro – MexicoRomaine – Salinas, CACheddar – WisconsinCarrots – IdahoGruyere – SwitzerlandPecans – GeorgiaSprouts – ChicagoRed Cabbage - NY
Shrimp – IndonesiaImitation Crab – AlaskaTuna Scrape – IndiaFish Roe – SeychellesSalmon – Puget SoundSoy Sauce – China Rice – ThailandSeaweed Wrap – CAAvocado – MexicoCucumber – MarylandWasabi – JapanPepper – Vietnam
Watermelon – DelawareBlackberries – GuatemalaBlueberries – New JerseyPineapple – GuamGrapes – CaliforniaKiwi – New ZealandApples – New YorkPears – OregonCantaloupe – Costa RicaHoneydew – ArizonaPapaya – MexicoBanana – Costa Rica
Salad Sushi Fruit platter
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Some perspective on the food supply
•Tracking and Tracing of food pathogens • Almost 200,000 registered food facilities (2/14)
• 81,574 Domestic and 115,753 Foreign• More than 300 ports of entry• More than 130,000 importers and more than 11 million
import lines/year• In the US there are more than 2 million farms
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Gold standard method for pathogen identification
PFGE: banding patterns determine discrimination within serovar.
PulseNet, est. 1996http://www.cdc.gov/pulsenet/
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• 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)
PFGE v/s WGS
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Pedigree vs Phylogeny
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DNA based pathogen surviellance not new
• Flu: 1990s – flu vaccines predicted from phylogenetic trees
• HIV: 1990s – early tracking of HIV transmission using phylogenetics
http://evolution.berkeley.edu/evolibrary/news/081101_hivorigins
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• CDC investigated a multistate (29 states) outbreak
• 410 confirmed cases between January 1st and July 7th, 2012
• Among the 326 case patient, 55 (17%) had been hospitalized
• Yellowfin tuna was implicated as source of this outbreak
• This product had been imported from an Indian corporation and was used to make spicy tuna sushi for restaurants and grocery stores
• At this time no reference genome
was available at NCBI
Salmonella enterica serovar Bareilly
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PFGE identical in red
NGS distinguishes geographical structure among closely related Salmonella Bareilly strains
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Different PFGE than the outbreak pattern
Same PFGE but not part of the
outbreak
Outbreak IsolatesMD isolates – in greenNY isolates – in purple
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Same PFGE cluster together (120 SNPs) outbreak isolates cluster together with 100% bootstrap Closest neighbor differ by 20 SNPs
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11720
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GenomeTrakr
Network of State, Federal, Hospital, University, Contract, and International laboratories sequencing and sharing data on pathogens.
1. Increased resolution of whole genome sequencing (WGS)
2. Both genomic data and metadata from foodborne pathogens
3. Data and filtered metadata housed in public databases at the National Center for Biotechnology Information (NCBI)
4. Publicly accessible
5. Real time comparison and analysis
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FDA’s GenomeTrakr
• Distributed network of labs to use whole genome sequencing
• Contributing members:
• 13 FDA labs
• 11 PulseNet labs (state public health labs)
• 5 Dept. of Agriculture labs
• 7 University labs
• 1 U.S. hospital lab
• 2 international labs (Argentina, Mexico)
• 3 private contracting labs
• Data curation and bioinformatic support/analyses provided by National Center for Biotechnology Information (NCBI) and FDA-CFSAN.
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GenomeTrakr verses PulseNet?
clinicals -> PNfood/env -> GT
clinicals -> PNfood/env -> GT
NCBI
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GenomeTrakr verses PulseNet?
clinicals -> PNfood/env -> GT
clinicals -> PNfood/env -> GT
NCBI
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Publicizing data
NCBI:Sequences and metadata
– fastq files in SRA DB, annotated assemblies in GenBank– metadata in BioSample DB (taxonomy, collected by, country and state, year,
isolation source)– Private: city, county, zipcode, firm names, product names, patient data (age,
sex, etc)
Analyses– Phylogenetic trees for each pathogen published daily at NCBI:
http://www.ncbi.nlm.nih.gov/projects/pathogens
GitHub:– CFSAN SNP Pipeline: http://snp-pipeline.readthedocs.org/en/latest/index.html
20http://www.ncbi.nlm.nih.gov/projects/pathogens
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New isolate check - Salmonella
SNPs distance to same category
SNPs distance to different category
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Look at close matches within SNP cluster
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Biosample: Isolate metadata
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1. Are there any new outbreaks or new clinical-food/environmental links?
2. Which clinical isolates belong to the same outbreak?3. Does this clinical isolate match any food/environmental
isolate?4. Does this food/environmental isolate match any clinical
isolate?5. Is this a resident strain? How long has it been in the
facility?
Routine Questions in Regulatory Investigations
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Salmonella Braenderup 2014 pre-outbreak
• In 2014, FDA conducted baseline environmental sampling in nut butter processing facilities
• A few of the samples tested positive for S. Braenderup and a PFGE pattern matched several cases of recent salmonellosis without a common link
• WGS was performed on both environmental and clinical isolates and found to be extremely close (2 SNP differences)
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Salmonella Braenderup
env. swab
clinical
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Comparing Traditional and Retrospective Outbreaks in Nut Butters
❖ Salmonella Tennessee (Company A, Brand A Peanut Butter, 2006/2007): 715 cases, 129 hospitalizations, no deaths
❖ Salmonella Typhimurium (Company B, Brand B Peanut butter, 2008/2009): 714 cases, 166 hospitalizations, 9 deaths
❖ Salmonella Bredeney (Company C/Brand C Peanut butter, 2012): 42 cases, 10 hospitalizations, 0 deaths
Retrospective Outbreak Investigation
❖ Salmonella Braenderup (Company D/Brand D nut butter, 2014): 6 cases, 1 hospitalization, no deaths
Traditional Outbreak Investigations
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Timeline for Traditional Approach to Foodborne Illness Investigation
Contaminated foodenters commerce
Identify contaminated foodand confirm that product or
environmental samplePFGE pattern matches the clinical
sample pattern
Identify illnesses and get PFGE pattern from
clinical samples
Source of contaminationidentified too late to prevent most illnesses
CDC FDA/FSIS
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Timeline for Foodborne Illness InvestigationUsing Whole Genome Sequencing
Contaminated food enterscommerce
FDA, CDC, FSIS, and States use WGS in real-time and in parallel on clinical, food,
and environmental samples
Source of contaminationidentified early through WGS combined database queries
Averted Illnesses
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• Earlier intervention means:
1) Reduced amount of recalled product;
2) fewer sick patients
3) less impact overall and minimal damage to brand recognition.
Immediate benefits of WGS to industry, growers, and distributers
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The Fresh-cut Tomato Supply Chain is complex
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WGS-based monitoring can pinpoint root causes
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Benefits 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?
One Data Record - Many Possibilities
…..AAGCTTGGAGATCTACGTGTACCTAGTCGAAGACTGAGGCTCTA….
AMR
SNP
Serotype
wgMLST
Markers
Virulence
Resistance (Disinfectant, Heat,Heavy metal…)
Ecological FitnessBiofilm persistence
UnknownAdaptation
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AMR genotype prediction
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Resistome Tracker
• What is Resistome Tracker?An epidemiological tool that allows users to track the appearance and spread of resistance genes in bacteria from different sources around the world.
• What can Resistome Tracker help you do?Identify potential reservoirs for the dissemination of resistant bacteriaMay help speed the investigation of resistant outbreaks
• Who operates ResistomeTracker?The National Antimicrobial Resistance Monitoring System (NARMS)
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NARMS Resistomics
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EXPLORE
Geographic distribution of Salmonella with selected resistance gene, by year
Running sum of isolate number
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DISCOVER
Lists genes that arenew to NCBI; updated weekly
Search for the first instance of a gene in Salmonella basedon collection date
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Improving Food Safety
1. Identify source of foodborne outbreaks more quickly ~ WGS provides an integrated food safety surveillance system ~ permits international capacity building through integration of foreign
food safety entities into the GT network
2. Transparency of open data gives industry full access ~ Genome data made public in real-time~ Public software and analysis tools readily available to industry for viewing of results
3. Food Safety Modernization Act (2011) – preventive Controls, Improve Industry Practices
~ WGS compliments rapid testing methods with environmental
monitoring for repeat positives and problems w/ resident pathogens.
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Acknowledgements
• FDA• Center for Food Safety and Applied
Nutrition• Office of Regulatory Affairs• Center for Veterinary Medicine
• National Institutes of Health• National Center for
Biotechnology Information
• USDA/FSIS• Eastern Laboratory
• CDC• Enteric Diseases Laboratory
CFSAN contributors:Ruth TimmeMaria Sanchez-LeonEric BrownErrol StrainJames PettengillYan LuoMarc Allard
CVM contributors:Patrick McDermottHeather TateShaohua Zhao