Starting from the bench – Prevention and control of

foodborne and zoonotic diseases

Martin Wiedmann

Department of Food Science

Cornell University, Ithaca, NY

E-mail: mw16@cornell.edu

Yrjo …

Overview

• Foodborne diseases – the scope • Laboratory-based subtyping techniques and their

application to detect disease outbreaks and characterize transmission routes

• Definition of pathogen subtypes within distinct molecular and epidemiological characteristics

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.

Overview

• Foodborne diseases – the scope • Laboratory-based subtyping techniques and

their application to detect disease outbreaks and characterize transmission routes

• Definition of pathogen subtypes within distinct molecular and epidemiological characteritics

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

12

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

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

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

PulseNet International

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

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

Use of DNA fingerprinting methods to understand pathogen transmission

22

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

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

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

Rapid Whole Genome Sequencing based subtyping of bacterial pathogens

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)

MLVA type frequency BGBQFJWIDAIBNACEAGVABAFBD

98 MLVA types

Some Salmonella (e.g. serovar Enteritidis) are poorly resolved by current subtyping

technologies

PFGE type frequency 4342215819692562332788231899879199

52 PFGE types

MLVA-PFGE type frequency B4B34G4B21BQ8I5W4J4D4BN692AI19AC2F2V4AG56J21

163 combined MLVA-PFGE types

Xbal SpeI

Den Bakker

et al. 2011.

AEM.

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

Overview

• Foodborne diseases – the scope • Laboratory-based subtyping techniques and their

application to detect disease outbreaks and characterize transmission routes

• Definition of pathogen subtypes within distinct molecular and epidemiological characteristics

L. monocytogenes full genome phylogeny

Molecular characterization of human, animal, and food isolates

Lineage I predominantly represents serotypes 1/2b and 4b, lineage

II predominantly represents serotypes 1/2a and 1/2c

Lineage

Human

isolates

(n=507)

Food isolates

(n=502)

Animal

isolates

(n=126)

Lineage I 54.4% 37.3% 40%

Lineage II 42.6% 62.4% 52%

Lineages III &

IV 2.4% 0.4% 8%

Number of isolates Ribotype

Food Human

P-value 1)

Comments

DUP-1030A 8 8 NS

DUP-1030B 0 10 ** not found in food

DUP-1038B 15 63 ****

DUP-1039A 12 31 **

DUP-1039B 18 43 **

DUP-1039C 35 25 NS

DUP-1042A 11 16 NS

DUP-1042B 18 72 ****

DUP-1042C 14 0 *** multiple food types, not in humans

DUP-1043A 30 16 *

DUP-1044A 11 28 **

DUP-1044B 1 19 *** rarely found in food

DUP-1044E 10 0 ** blue cheese only

DUP-1045B 14 11 NS

DUP-1052A 58 39 *

DUP-1053A 24 41 *

DUP-1062A 151 9 **** rarely found in humans

DUP-1062D 28 1 **** rarely found in humans

rare 22 42 * Ribotypes with 1-4 isolates

uncommon 22 33 NS Ribotypes with 5-8 isolates

Total 502 507 **** Overall analysis of ribotype vs. origin 1)

P-values refer to comparison of origin between ribotype specified in that row vs. all other ribotypes 1

where NS = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 2

Human virulence attenuation of ribotype DUP-1062A

• Isolates with ribotype DUP-1062A carry a premature stop codon in inlA, which leads to reduced invasion of human epithelial cells Wildtype

inlA (745 aa)

LM

Human intestinal epithelial cell

DUP-1062A

inlA (631 aa)

LM

Human intestinal epithelial cell

MA

Mutation type 3; DUP-1046B

& DUP-1062A

Food isolate from France (NV4;

Rousseaux et al., 2002)

Human fecal carriage strains from France (Olier

et al., 2002) N

C S 492

575 E

N

N S

606 F

S N 656

G

S N 700

J

S S

LRR B Repeats IR

LPXTG

MA

29 357 462 650 711

N C

800

A

Human fecal carriage strain L028

from France (Jonquieres et al., 1998)

Mutation type 1; DUP-1052A

& DUP-16635A

Mutation type 2; DUP-1025A

& DUP-1031A

N B S

460 Food isolate from France (NV8; Rousseaux et al.,

2002)

519 N D S Food isolate from France (NV7; Rousseaux

et al., 2002)

S N 677

H

S N 685

I Food isolate from France (NV5;

Rousseaux et al., 2002)

EGD-e (Glasser et al., 2001)

inlA premature stop codons in other strains

Van Stelten and Nightingale et al. AEM. 2008

Invasion into human intestinal epithelial cells of L. monocytogenes with and without premature stop

codons in inlA

Nightingale et al. AEM. 2008

L. monocytogenes with premature inlA stop codon: summary

• Found more commonly in food isolates than human isolates – France: inlA premature stop codon strains represent

35% of food isolates and 4% of human clinical isolates (Jacquet et al. 2004. JID 189)

– Also found in China and Portugal

• Attenuated virulence in guinea pigs

• Not all L. monocytogenes are equally likely to cause human disease and many L. monocytogenes in foods have reduced ability to cause human disease

Background Salmonella

den Bakker et al.

BMC Genomics.

2011

den Bakker et al.

BMC Genomics.

2011

Summary and conclusions

• It takes a village (of lab scientists) to “raise” an epidemiologists

• Some lab data can be useful for epidemiologists.. • Some epidemiologists can be useful for data

interpretation • The increasing granularity of lab data provides tremendous

new opportunities to (i) better understand disease transmission and (ii) better define mechanisms that may explain population-based and epidemiological observations

• Continued bridging of lab-based and epidemiological approaches is essential to a One-Health that delivers societal benefits

Acknowledgments

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

Cornell: Y. Groehn, K. J. Boor, Q. Sun NYSDOH: W. Wolfgang, N. Dumas, T. Root, D. Morse, D. Schoonmaker-Bopp, K. Musser, R. Limberger 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