1. Whole genome sequencing: New methods for traceback investigations

2. Salmonella enterica subsp. enterica serovar Enteritidis

3. Wright County Egg, Galt Iowa

4. Size standard JEGX01.0004 (“pattern 4”)

5. Wright County Egg, Galt Iowa
Hillandale Farms, West Union

6. Issues with PFGE Sometimes isn’t discriminatory enough;
Difficulty comparing patterns between labs;
Patterns can change slightly during outbreak;
Highly technical procedure to perform

7. Size of DNA (in bp): Campylobacter; 1,800,000 S. aureus; 2,700,000
Listeria; 2,900,000
Shigella: 4,500,000
Salmonella; 5,000,000
E. coli O157:H7; 5,500,000
Human; 2,900,000,000
Should provide:
Greater discrimination
More “discrete” data
Improved speed

10. “Shotgun” sequencing TGACATACCATTATGATCCATATCCGAGAC
GAGCACGTGACGGGGATAACCACGTAGA
CACTAAGACAGGAGTCAATATGACAT
CACGTAGATCACTAAGACAGGAGTCAATATGAC
CACGTAGATCACTAAGACAGGAGTCAATATGAC
GTAGATCACTAAGACAGGAGTCAATATGACAT
CATTATGATCCATATCCGAGACATTATG
TGACATACCATTATGATCCATATCCGAGAC
CCCATTTTGAGCACGTGACGGGGATA
AGTCAATATGACATACCATTATGATC
TCACTAAGACAGGAGTCAATATGACA

11. Sequence assembly Reads Consensus sequences Contig 1 Contig 2
ATCCGAGACATTATGACAAAAAGATGT
ACGGGGATAACCACGTAGATCACTAA
ACATACCATTATGATCCATATCCGAGACA
GGGGATAACCACGTAGATCACTAAGAC
ACATACCATTATGATCCATATCCGAGACATTATGA
AGCCCATTTTGAGCACGTGACGGGG
CGAGACATTATGACAAAAAGATGTATGC
CCATTTTGAGCACGTGACGGGGATAACCAC
ACCACGTAGATCACTAAGACAGGAGTCA
CATATCCGAGACATTATGACAAAAAGAT
GTGACGGGGATAACCACGTAGATCACT
TGACATACCATTATGATCCATATCCGA
CCCATTTTGAGCACGTGACGGGGA
TGATCCATATCCGAGACATTATGAC
TTTTGAGCACGTGACGGGGATAACCACGTAGA
Consensus sequences
AGCCCATTTTGAGCACGTGACGGGGATAACCACGTAGATCACTAAGACAGGAGTCA
TGACATACCATTATGATCCATATCCGAGACATTATGACAAAAAGATGTATGC
Contig 1
Contig 2

12. Analysis Single nucleotide polymorphisms (SNPs)
Isolate #1…AGCGTGCATAGCATTGACAGATACACCACACTTTAAGATTTTAGCC…
Isolate #2…AGCGTGCATAGCATTGACAGATACACCGCACTTTAAGATTTTAGCC…
Deletions (can be few to hundreds of thousands of nucleotides)
Isolate #2…AGCGTGCATAGCATTGA CACTTTAAGATTTTAGCC…
Insertions (can be few to hundreds of thousands of nucleotides)
Isolate #1…AGCGTGCATAGCATTGAAACAGATACACCACACTTTAAGATTTTAGCC…
Isolate #2…AGCGTGCATAGCATTGA CAGATACACCGCACTTTAAGATTTTAGCC…

13. Impact of WGS

14. Drawbacks of WGS Can be too discriminatory;
Analysis still not routine for most laboratories;
Initial equipment purchase expensive;
Infrastructure changes.

15. Creating a new world-wide database
GenomeTrakr (US-FDA, College Park, MD)
Sequencing foodborne pathogens
Collecting metadata

16. Aug & Oct 2014: FDA inspections find positives
Aug & Oct 2014: FDA inspections find positives
5 patients ill June-August 2014
2 deaths
2 recall eating sprouts

17.
Jan-May 2012
Aroor, India
410 cases Bareilly
15 cases Nchanga

18. Summary
Whole genome sequencing is rapidly replacing PFGE as the preferred molecular subtyping method
Improves our ability to identify outbreaks, and link isolates from patients and foods to outbreak
Many challenges ahead of us.