Whole genome sequencing: New methods for traceback investigations

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Presentation transcript:

Whole genome sequencing: New methods for traceback investigations

Salmonella enterica subsp. enterica serovar Enteritidis

Wright County Egg, Galt Iowa

Size standard JEGX01.0004 (“pattern 4”) https://openi.nlm.nih.gov/detailedresult.php?img=PMC3220660_1756-0500-4-369-2&req=4

Wright County Egg, Galt Iowa Hillandale Farms, West Union

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

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

http://www.nature.com/news/technology-the-1-000-genome-1.14901

http://www.cdc.gov/pulsenet/pathogens/protocol-images.html#wgs

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

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

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…

Impact of WGS http://www.cdc.gov/listeria/pdf/whole-genome-sequencing-and-listeria-508c.pdf

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

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

Aug & Oct 2014: FDA inspections find positives http://www.cdc.gov/listeria/outbreaks/bean-sprouts-11-14/index.html Aug & Oct 2014: FDA inspections find positives 5 patients ill June-August 2014 2 deaths 2 recall eating sprouts

http://www.cdc.gov/salmonella/bareilly-04-12/index.html Jan-May 2012 Aroor, India 410 cases Bareilly 15 cases Nchanga

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.