Can molecular approaches reduce the 600 deaths per year caused by

Listeria monocytogenes and Salmonella?

Martin Wiedmann

Department of Food Science

Cornell University, Ithaca, NY

E-mail: mw16@cornell.edu

YES!

Take home messages

• Foodborne illnesses KILL real people every day! – Food scientists can prevent death or contribute to

them

• Molecular biology and genome sequencing is not just a lab toy anymore – Used routinely every day by FDA, CDC, etc. – Food microbiologists and food scientists that don’t

use these tool to assure food safety are foolish

• Molecular biology + “big data” = a new area for food safety

Take home messages

• Foodborne illnesses KILL real people every day! – Food scientists can prevent death or contribute to

them

• Molecular biology and genome sequencing is not just a lab toy anymore – Used routinely every day by FDA, CDC, etc. – Food microbiologists and food scientists that don’t

use these tool to assure food safety are foolish

• Molecular biology + “big data” = a new area for food safety

Microbial foodborne diseases - US

• Latest 2011 CDC study estimates 47.8 million cases of gastrointestinal illnesses ; 9.4 million due to known and 38.4 million due to unknown pathogens)

– 127,000 serious illnesses resulting in hospitalizations; 56,000 due to known and 71,000 due to unknown pathogens

– 3,037 deaths (range: 1,492–4,983); 1,351 due to known and 1,686 due to unknown pathogens

WHO statement on foodborne diseases

• Food and water-borne diarrhoeal illnesses present a “growing public health problem” that claim 2.2m lives annually – with 1.9m of these children.

• Many communicable diseases – including emerging zoonoses – are transmitted through food.

• 27 cases in June 1989, 1 deaths

Take home messages

• Foodborne illnesses KILL real people every day! – Food scientists can prevent death or contribute to

them

• Molecular biology and genome sequencing is not just a lab toy anymore – Used routinely every day by FDA, CDC, etc. – Food microbiologists and food scientists that don’t

use these tool to assure food safety are foolish

• Molecular biology + “big data” = a new area for food safety

Listeria monocytogenes

• Gram-positive animal and human food-borne pathogen • Facultative intracellular pathogen • Causes abortion, meningitis, and septicemia • Can grow at low temperatures • High infectious dose

• Causes an estimated 1,600 illness and 255 deaths/year in US

• As of May 2013, 47 L. monocytogenes genomes and 14 genomes for other Listeria spp. available in GenBank

Examples of L. monocytogenes ribotypes

18

DNA sequencing-based subtyping

Isolate 1 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTG

Isolate 2 AACATGCAGACTGACGATTCGTCGTAGGCTAGACGTTGACTG

Isolate 3 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTG

Isolate 4 AACATGCATACTGACGATTCGACGAAGGCTAGACGTTGACTG

2289

j2 -045

j1 -038

L99

j2 -068

j2 -003

10403S

j1 -047

c2-006

n1-064

c2-008

dd680

c2-011

n1-067

n1-079

9 2

9 2

9 2

8 8

7 5

9 2

9 2

9 2

7 5

9 1

8 9

Case study – human listeriosis outbreak

Human listeriosis cases - NYS 1/97-10/98

0

1

2

3

4

5

6

7

8

Jan

Mar

May Ju

lSep

Nov Ja

nM

arJu

nAug O

ct

Ribotyping results - Nov 8, 9 pm

Ribotyping results - Nov 8, 12 pm

Epidemic curve for 1/97 - 2/99 in NYS

0

1

2

3

4

5

6

7

8

Jan

Mar

May Ju

lSep Nov Ja

nMar

Jun

Aug Oct

Dec

Feb

1044A

Other Ribotypes

Similarity Search Results

Conclusions

• 101 human cases and 21 deaths in 22 US states linked to infection by the same sub-type of Listeria monocytogenes

• Outbreak traced back to a single specific plant in Michigan

Food isolate

Human case

Human case

0

10

20

30

40

50

60

70

1 8 15 22 29 36 43 50 57 64 71

Day of Outbreak

Num

ber o

f Cas

es

outbreak detected 1993

Meat recall

1993 Western States E. coli O157 Outbreak

726 cases

4 deaths

39 d

2002 Colorado E. coli O157 Outbreak

0

10

20

30

40

50

60

70

1 8 15 22 29 36 43 50 57 64 71

Day of Outbreak

Num

ber o

f Cas

es

outbreak detected 2002

18 d

If only 5 cases of E. coli O157:H7 infections were averted by the recall of ground beef

in the Colorado outbreak, the PulseNet system would have recovered all costs for

start up and operation for 5 years. (Elbasha et al. Emerg. Infect. Dis. 6:293-297, 2000)

Public Health Impact of Molecular Epidemiology

Use of DNA fingerprinting to track L. monocytogenes in processing plants

• Environmental Listeria contamination as significant problem in the food industry

• Controlling environmental L. monocytogenes contamination in food plants is key to better control (“Seek and destroy”)

Sample Source

*

VISIT 2

VISIT 3

VISIT 1

*

* *

*

* *

* *

* * *

*

*

* *

Sample Ribotype Sample Source RiboPrint® Pattern

1039C (E) Floor drain, raw materials area

1039C (E) Floor drain, hallway to finished area

1039C (IP) Troll Red King Salmon, in brine, head area

1039C (IP) Troll Red King Salmon, in brine, belly area

1039C (IP) Brine, Troll Red King Salmon

1039C (IP) Faroe Island Salmon, in brine, head area

1039C (F) Smoked Sable

1039C (F) Cold-Smoked Norwegian Salmon

1044A (E) Floor drain, brining cold room 1

1044A (R) Raw Troll Red King Salmon, head area

1044A (IP) Brine, Faroe Island Salmon

1045 (R) Raw Troll Red King Salmon, belly area

1045 (IP) Faroe Island Salmon, in brine, head area

1053 (IP) Norwegian Salmon, in brine

1062 (E) Floor drain #1, raw materials preparation

1039C (E) Floor drain #1, raw materials preparation

1039C (E) Floor drain, brining cold room 1

1039C (E) Floor drain #2, raw materials preparation

1039C (E) Floor drain #2, raw materials receiving

1039C (E) Floor drain, finished product area

1039C (E) Floor drain, hallway to finished area

1039C (IP) Brine, Troll Red King Salmon

1039C (F) Smoked Sable

1044A (IP) Sable, in brine

1044A (IP) Brine, Faroe Island Salmon

1062 (IP) Brine, Norwegian Salmon

DNA fingerprinting can identify persistence in plants

House bugs & pet Listeria

Samples

Plant B

n=129

Plant C

n=173

Plant D

n=229

P-value

Ribotype % Prevalence

1039C 0.0 0.0 10.0 0.0000

1042B 0.8 1.2 0.4 0.8221

1042C 6.2 0.6 0.4 0.0003

1044A 0.0 2.3 3.1 0.1494

1045 5.4 0.0 0.9 0.0006

1046B 0.0 2.3 0.0 0.0144

1053 0.0 0.6 1.7 0.2686

1062 0.8 0.6 2.6 0.1822

32

L. monocytogenes persisted in rubber floor mats despite sanitation

Listeria can be protected from sanitizer in “micro-cracks”, but can

be squeezed out by pressure if people stand on mats

2000 US outbreak - Environmental persistence of L. monocytogenes?

• 1988: one human listeriosis case linked to hot dogs produced by plant X

• 2000: 29 human listeriosis cases linked to sliced turkey meats from plant X

Whole Genome Sequencing

• It all started with the human genome project

• Sequencing of a bacterial genome is now feasible at costs of <$100/isolate • Costs will continue to drop

• Commonly used platforms include Illumina HiSeq/MiSeq; Life Technologies Ion Torrent; Roche 454; PacBio RS

• Public health applications of microbial whole genome sequencing are rapidly increasing, including investigation of nosocomial outbreaks

Salmonella • The genus Salmonella is divided into 2 species

• S. bongori and S. enterica, which is subdivided into 6 subspecies (enterica, salamae, arizonae, diarizonae, houtenae, indica)

• Over 2,500 recognized serotypes, e.g. S. enterica subsp. enterica serotype Typhimurium (Salmonella Typhimurium)

• Salmonellosis is one of the most common and widely distributed foodborne diseases

• Salmonella strains resistant to multiple antibiotics are a concern • A number of foodborne salmonellosis outbreaks have

been linked to multidrug resistant (MDR) strains

37

209 cases

28 cases

Xbal SpeI

Den Bakker

et al. 2011.

AEM.

Tip-dated maximum clade credibility tree based on SNP data for 47 Montevideo isolates

MLVA type frequency BGBQFJWIDAIBNACEAGVABAFBD

98 MLVA types

• Salmonella Enteritidis is most common cause of human salmonellosis – poorly resolved by current subtyping technologies.

PFGE type frequency 4

342215819692562332788231899879199

52 PFGE types

MLVA-PFGE type frequency B4B34G4B21BQ8I5W4J4D4BN692AI19AC2F2V4AG56J21

163 combined MLVA-PFGE types

S. Enteritidis Whole Genome Cluster Analysis

• Collaborative efforts between NYSDOH, FDA, and Cornell • Study Goals

– Determine utility of WGCA in detecting covert clusters of S. Enteritidis

– Determine feasibility for real-time WGCA in public health lab setting

• Approach – Retrospective study on an S. Enteritidis outbreak. – Prospective study of S. Enteritidis as they are acquired by the

lab. • WGS (Ion Torrent) performed at NYSDOH • Data analysis used SNV based phylogenetic trees

– SNV detection (either de novo or reference based) – Removal of SNVs introduced by recombination – Phylogenetic inference of population structure

Salmonella Enteritidis outbreak linked to long term care facility outbreak

• Sept. 2010: Connecticut Dept. of Health identifies a

Salmonella outbreak in a long term care facility.

– Outbreak was linked to cannoli from a Westchester bakery.

• Cases were linked to a another cluster in Westchester

NY.

• Both NY and CT cases consumed cannolis.

• Isolates had a common PFGE / MLVA DNA fingerprint.

NGS identifies additional outbreak cases

Contemporary isolates:

Two small clusters

Outbreak

Blue: Epidemiologically identified

Red: Additional cases identified with NGS

Rapid Whole Genome Sequencing based subtyping of L. monocytogenes

3 days • DNA extraction

• Library prep

24 h • Sequencing on Bench top sequencer (MiSeq, Ion Torrent)

12 h

• De novo assembly

• Rapid classification to subpopulation using pairwise distances based on average nucleotide identity values (BLAST)

• Inference of subpopulation structure based on SNP calling.

Collaboration with CDC (C. Tarr)

L. monocytogenes genome sequencing at CDC

Characterizing the mechanisms of L. monocytogenes growth inhibitors

• “96 of every 100 cases of listeriosis due to deli meats could be prevented if they all contained a Lm inhibitor”

• Lactate and di-acetate as commonly used growth inhibitors for selected foods where L. monocytogenes is a concern.

Experimental design

Stasiewicz et al. 2011 AEM

Treatment with lactate and acetate induces a shift toward

fermentative production of acetoin and away from aerobic

respiration and the production of lactate and acetate.

Summary

• Full genome sequencing has potential as both a primary and as a secondary subtyping method in outbreak investigations – Reduces scope of outbreaks – Identifies root causes, which prevents future outbreaks

• Other application of genomics and molecular biology can help prevent foodborne disease cases and outbreaks – Use of genomics and RNA-seq to develop control strategies

Take home messages

• Foodborne illnesses KILL real people every day! – Food scientists can prevent death or contribute to

them

• Molecular biology and genome sequencing is not just a lab toy anymore – Used routinely every day by FDA, CDC, etc. – Food microbiologists and food scientists that don’t

use these tool to assure food safety are foolish

• Molecular biology + “big data” = a new area for food safety

http://dashburst.com/infographic/big-data-volume-variety-velocity/

Classification Tree Predictions of Reservoirs

53

Strawn et

al. AEM.

2013

Overall summary and conclusions

• While genome sequencing is making “real world” contributions to food safety – Improved subtyping over PFGE – Identification of better target genes for detection – Translation of transcriptomics, metabolomics etc.

findings to improved prevention and treatment is in the early stages

• Genomics is only part of “big data” – Future generations of food scientists needs to be

able to play in the “big data” pond (what’s ROI anyway?)

Acknowledgments

Students and staff: H. den Bakker, T. Bergholz, M. Stasiewicz, H. Oliver, R. Orsi, K. Hoelzer, E. Fortes, R. Ivy, V. Ferreira Collaborators:

NYSDOH: W. Wolfgang, N. Dumas, T. Root, D. Morse, D. Schoonmaker-Bopp, K. Musser, R. Limberger CDPH: J. Fontana, A. Kinney Cornell: K. J. Boor, Q. Sun CDC: C. Tarr, P. Gerner-Smidt, B. Swaminathan, L. Graves, the Listeria Working Group FDA: M. Allard, E. Brown, E. Strain Life Technologies/ABI; Broad Institute

Financial support: New York Sea Grant, USDA-NRI, USDA Special Research Grants, USDA – Food safety Initiative, ILSI N.A., and NIH