Post on 16-Feb-2019
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
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.
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
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
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
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
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
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)
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.
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
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