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WGS SERIES:Whole Genome Sequencing in the Industry
Case Studies
April 29, 2020
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Webinar Logistics
• Everyone is muted• Questions will be addressed during the Q&A session at the end of the
presentation• The presentation is being recorded• The recording/slides will be distributed following the presentation• Adjourn (60 minutes)• There will be 3 important survey questions at the conclusion of this
webinar. Your response is appreciated
Speaker
Dr. Maggie WeinrothDr. Weinroth is currently a postdoctoral researcher at the USDA ARS US
Meat Animal Research Center focusing on the integration of big data and sequencing technologies in meat safety. Her work encompasses the use of metagenomics to describe novel animal associated microbiomes
and the effect of management practices on microbiomes and resistomes of food animals.
Non-regulatory uses of Whole Genome
Sequencing (WGS) in the meat industry
Maggie Weinroth, Ph.D. USMARC ARS USDA
Outside of regulation, what can WGS be used for in the meat industry?
Characterization
Traceability
Integration with other NGS technologies
…monitoring programs rely on classical microbiological methods but may be complemented by molecular-based methods to obtain a more precise view of the hygienic state of the environment and a much more extended view of the ecological richness…
https://doi.org/10.1016/j.ijfoodmicro.2017.11.007
-Rantsiou et al. 2018
Characterization
• The nucleotide level of resolution afforded by WGS is unparalleled
• As more industries and stakeholders partake in WGS, more information on gene annotation and function can be gathered
• Once a genome is subjected to WGS, gene presence and absence can be assessed:
• Heat / Sanitizer resistance• Antibiotic Resistance • Virulence factors
Characterization: Heat Resistance1. Classified heat resistance vs. not
2. Find differences between groups
3. Put suspected heat resistance genes in susceptible E. coli
Traceability
• In facility traceability vs. traceability within a food system
• Persistence vs. new arrivals • Forward and backward
source determination
Traceability: looking to other industries
Integration with other NGS technologies
• WGS can be paired with other technologies to give an even more clear picture of what is happening
• 16S and metagenomics: microbial ecology
• Transcriptomics: what genes are being up or down regulated
Integration with other NGS
WGS looking forward
You can not manage what you don’t measure
Transition from characterization to intervention
Speaker
Dr. Scott HoodDr. Hood is the Director of Food Safety for the General Mills Global Scientific & Regulatory Affairs team. Dr. Hood has been an active leader in initiatives related to the microbiological safety of food,
including; the National Advisory Committee on the Microbiological Criteria for Food, the GMA Microbiological Safety Committee and
the University of Georgia Center for Food Safety.
15
Whole Genome Sequencing
Case Studies – Dry Products
April 2020
Scott Hood
16
Disclaimer
• General Mills does not use routinely whole genome sequencing (WGS).
• The case studies presented today includes investigations initiated to better understand the use of WGS and how it might be applied in the future.
• We operate with standards that require the area is cleaned and sanitized when environmental monitoring detects Salmonella or Listeria sp,. In addition, vector sampling is used to determine if Salmonella or Listeria can be found in adjacent areas. An escalation plan is in place if Salmonella or Listeria sp is found again.
17
Case Study Salmonella in environment of Dry, NRTE Product
Retrospective Analysis
Environmental Monitoring• Repeat
findings of Salmonella
Strain Characterization
• Serotyping• Ribotyping
Accelerate Remediation• Cleaning• Sanitizing• Capital
Improvements
WGS• Confirmed
persistent strain
Remediation
18
Case StudyListeria in environment of Dry, RTE product
Investigation
Environmental Monitoring• Repeat
findings of Listeria sp
Escalate • Test for Lm• Confirmed Lm
WGS• Confirm
persistent strain
Accelerate Remediation• Increased
sampling• Capital
Improvements
Remediation
19
1
2
3
Key Questions
Is the methodology and interpretation of the data sufficiently validated?
Methodology
Other Methods for Strain
CharacterizationDoes WGS data change the way we react?
Regulatory Risk
Does WGS data have to potential increase regulatory risk?
20
Thank You
Speaker
Dr. Martin WiedmannDr. Wiedmann is a Professor of Food Safety at Cornell University and
the Co-director of the New York State Integrated Food Safety Center of Excellence. He’s received 4 awards over the years for his work from
different well renowned organization such as the American Academy of Microbiology and International Life Science Institute.
Whole genome sequencing (WGS) applications to seafood, produce, and dairy
Martin Wiedmann, Dr. med. vet., Ph.D.Gellert Family Professor of Food Safety
Department of Food ScienceCornell University, Ithaca, NYE-mail: [email protected]
Phone: 607-254-2838
Take home messages
• Make sure someone in your organization knows how to use the NCBI Pathogen Detection database
• Have a serious discussion about use of subtyping as part of your environmental monitoring programs
• Can be WGS or another reliable approach • Consider WGS for root cause analysis of microbial pathogens
(beyond Listeria, Salmonella, and E. coli) and spoilage issues• Do a WGS pilot to test potential partners (labs etc.)
Outline
• NCBI Pathogen Detection database• WGS applications to seafood
• Listeria tracking• WGS applications to produce
• Listeria tracking• WGS applications to dairy
• Listeria tracking (just kidding)• B. cereus• Pseudomonas spoilage
https://www.ncbi.nlm.nih.gov/pathogens
Summary of L. monocytogenes data on NCBI (release 1382, August 16,2019)
Total number of isolates Number of clusters
Number of unclustered isolates
(> 50 SNPs to any other isolate in the
DB)Number of human
clinical isolates
Number of environmental/food
isolates
28,285 2,492 5,508 9,857 18,428
Isolates in the same SNP cluster differ from at least one other isolate in the cluster by at most 50 SNPs.
Outline
• NCBI Pathogen Detection database• WGS applications to seafood
• Listeria tracking• WGS applications to produce
• Listeria tracking• WGS applications to dairy
• Listeria tracking (just kidding)• B. cereus• Pseudomonas spoilage
20.0
1750 1760 1770 1780 1790 1800 1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020
H1-0258 D
T1-0029 A
N1-0110 L
M6-1133 I
H1-0081 A
M6-0810 A
T1-0261 I
M6-0150 A
R9-4438 P
M6-0755 E
H6-0175 F
M6-0306 A
H1-0328 AM6-0594 H
N1-0051* A
N1-0254 M
H1-0221 C
N1-0400 C
R6-0909 N
N1-0255 C
R9-4443 J
V1-0142 A
L4-0166 E
H1-0193 AT1-0077 A
T1-0938 I
M6-0296 A
M6-1145 J
M6-0958 A
V1-0034 A
N1-0256 C
T1-0027 Q
N1-0053 A
1987
1983
1832
2004
1987
1991
1998
2001
1991
1981
1976
2006
1981
1986
1983
1982
20001985
1996
1985
1990
1976
19991970
1974 1984
1983
1958
1980
1992
1980
2008
Company starts productionin the new facility
Company expandsto an adjacent building
The same prophage is found in FSL N1-0256, FSL N1-0400, FSL H1-0221 and FSL N1-0255
Loss of prophage
Replacement of original prophage by a new prophage
Acquision of prophage
Legend key
(II)
(II)
(I, II)
WGS-based characterization of isolates obtained from a single facility over 17 years
Reduced quat sensitivity in L. monocytogenes strains obtained from a seafood processing plant
Strains Cluster“Quatresistance” genes (based on WGS
MIC* for
BC BZT CPC Weiquat
FSL H1-0506 1 None 1 mg/L 2 mg/L 1mg/L 0.001%
FSL M6-0204 1 qacH 3 mg/L 4 mg/L 3mg/L 0.004%
FSL H1-0322 2 bcrABC 4 mg/L 7 mg/L 3mg/L 0.004%
FSL T1-0027 3a bcrABC 3 mg/L 5 mg/L 2mg/L 0.004%
FSL T1-0077 3b bcrABC 3 mg/L 6 mg/L 2mg/L 0.004%
BC= benzalkonium chloride, BZT= benzethonium chloride, CPC= cetylpyridinium chloride; MIC value indicates conditions for which no growth was detected (at the detection threshold OD600nm 0.15) after 24 h of incubation.
Outline
• NCBI Pathogen Detection database• WGS applications to seafood
• Listeria tracking• WGS applications to produce
• Listeria tracking• WGS applications to dairy
• Listeria tracking (just kidding)• B. cereus• Pseudomonas spoilage
Use of WGS to understand L. monocytogenes transmission in produce chain
• Longitudinal sampling of produce packing houses• Packing houses typically receive intact produce items (apples, pears,
peaches, tomatoes, cucumbers, etc.), perform minimal manipulations (e.g., washing, sorting etc.) and than pack them
• Products may be received directly from fields or orchards or from storage facilities
Other LMEmpty bins
Full bin in
Brush bed
Dra
in b
y flu
me
Sort
Drain by dryer
Dryer
Totes
Tray pack4 lane sizer
Break Room
Walk ways
Drain
Overhead door
Flume
West Dock
East Dock
Palletize
3 lane sizer
Drain
AT 61
AT 67
Drain w/ PVC
Visit 1
Other LMEmpty bins
Full bin in
Brush bed
Dra
in b
y flu
me
Sort
Drain by dryer
Dryer
Totes
Tray pack4 lane sizer
Break Room
Walk ways
Drain
Overhead door
Flume
West Dock
East Dock
Palletize
3 lane sizer
AT 61
AT 67
Drain w/ PVC
Drain
Visit 2
Other LMEmpty bins
Full bin in
Brush bed
Dra
in b
y flu
me
Sort
Drain by dryer
Dryer
Totes
Tray pack4 lane sizer
Break Room
Walk ways
Drain
Overhead door
Flume
West Dock
East Dock
Palletize
3 lane sizer
AT 61
AT 67
Drain w/ PVC
Visit 3
Drain
Other LMEmpty bins
Full bin in
Brush bed
Dra
in b
y flu
me
Sort
Drain by dryer
Dryer
Totes
Tray pack4 lane sizer
Break Room
Walk ways
Drain
Overhead door
Flume
West Dock
East Dock
Palletize
3 lane sizer
AT 61
AT 67
Drain w/ PVC
Visit 4
Drain
Other LMEmpty bins
Full bin in
Brush bed
Dra
in b
y flu
me
Sort
Drain by dryer
Dryer
Totes
Tray pack4 lane sizer
Break Room
Walk ways
Drain
Overhead door
Flume
West Dock
East Dock
Palletize
3 lane sizer
AT 61
AT 67
Drain w/ PVC
Visit 5-7
Drain
LM-1 Visit 1 Pan on south end of brush bed
LM-2 Visit 2 Outlet of PVC pipe NW corner
LM-3 Visit 2 Inside of end pan by dryer
LM-4 Visit 3 Trench leading to square drain by dryer
LM-5 Visit 1 Square drain by flume of bin dumper
LM-6 Visit 2 Square drain by flume of bin dumper
LM-7 Visit 2 Trench leading to square drain
LM-8 Visit 3 – Pre-production Square drain by flume of bin dumper
0.0005
LM-1
LM-2
LM-3
LM-4
LM-5
LM-6
LM-7
LM-8
100
100
Cluster 3(0-5 SNPs)
AT 67LM-1 5 Visit 7 DrainLM-2 20c Visit 3 Catch PanLM-3 20c Visit 4 Catch PanLM-4 20 Visit 1 PVC PipeLM-5 20c Visit 2 Catch Pan
LM-1 LM-2 LM-3 LM-4 LM-5
LM-1 0 18 17 17 17
LM-2 18 0 1 1 1
LM-3 17 1 0 0 0
LM-4 17 1 0 0 0
LM-5 17 1 0 0 0
From a different operation!
Other studies also found evidence for re-introduction events when isolates from multiple facilities were investigated using WGS (e.g., Hurley et al., 2019. mSphere 4 (4))
In one case, isolates with < 3 SNP differences were found in retail delis in 3 different states
A cautionary tale
Outline
• NCBI Pathogen Detection database• WGS applications to seafood
• Listeria tracking• WGS applications to produce
• Listeria tracking• WGS applications to dairy
• Listeria tracking (just kidding)• B. cereus• Pseudomonas spoilage
50 shades of gray: Pseudomonas causes gray discoloration in HTST milk
Gene name Annotation
trpD Anthranilate phosphoribosyltransferase
trpF N-(5'-phosphoribosyl)anthranilate isomerase
trpA Tryptophan synthase alpha chain
trpB Tryptophan synthase beta chain
iolG Inositol 2-dehydrogenase/D-chiro-inositol 3-dehydrogenase
mdh Malate dehydrogenase
Peptidase M
Xylose Isomerase Domain-Containing Protein
degT Pleiotropic regulatory protein
Oxidoreductase Domain Protein
trpC Indole-3-glycerol phosphate synthase
solR Transcriptional activator protein solR
rhtB Homoserine/homoserine lactone efflux protein
Key gene unique to Pseudomonas causing gray (and blue) color defects
This reaction requires 3 oxygens!!
Take home messages
• Make sure someone in your organization knows how to use the NCBI Pathogen Detection database
• Have a serious discussion about use of subtyping as part of your environmental monitoring programs
• Can be WGS or another reliable approach • Consider WGS for root cause analysis of microbial pathogens
(beyond Listeria, Salmonella, and E. coli) and spoilage issues• Do a WGS pilot to test potential partners (labs etc.)
Q & A
If your question wasn’t answered…
Please contact Scott Nichols at [email protected] or one of the trade organization representatives and we would be
happy to respond.
Thank you.
Brought To You By:
COVID-19 Resources
Cornell: https://instituteforfoodsafety.cornell.edu/coronavirus-covid-19/food-industry-resources/Food and Beverage Issue Alliance: https://www.feedingus.org/US Poultry: https://www.uspoultry.org/COVID-19/Western Growers: https://www.wga.com/covid-19-resources-page
Cornell Food Industry Virtual Office HoursThursday, April 30, 2020 | 4:00 to 5:00 pm (EDT)Join Zoom Meeting: https://cornell.zoom.us/j/98541351453?status=successMeeting ID: 985 4135 1453Dial by your location:
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Whole Genome
Sequencing in the
IndustryCase Studies
April 29, 2020
