1. Department of Food Science E-mail: mw16@cornell.edu
Environmental Listeria testing and molecular subtyping to control Listeria monocytogenes in RTE food processing environments
Martin Wiedmann
Department of Food Science
Cornell University
Ithaca, NY 14853
Phone:
Fax:
Martin Wiedmann / Cornell 5-01

2. Transmission and pathogenesis of L
Transmission and pathogenesis of L. mono-cytogenes and other foodborne diseases
Animal feed/environment /protozoans
Food animals
Manure
Animal derived food products
Food products
Food Processing Plants
Humans

3. Listeria monocytogenes
Causes septicemia, abortion and encephalitis in humans and more than 40 animal species, but is also common in environment
Ubiquitous in the environment, can survive for prolonged periods in the environment (apparently outside a host)
Human listeriosis can occur as epidemic and sporadic cases
Affects predominantly elderly and immunocompromised people, pregnant women and newborns.
Approx human cases/year in the U.S., resulting in about 500 deaths/year
Martin Wiedmann / Cornell 5-01

4. Human listeriosis cases - Three General Scenarios
1. Isolated case
2. Cases due to a single event or lot of food
3. Clusters and isolated cases scattered by time and location.
- An unusually virulent strain of L. monocytogenes has become established in a food operation.
- Multiple lots of food are contaminated over time.
- The food supports the growth of L. monocytogenes
Martin Wiedmann / Cornell 5-01

5. Environmental Listeria testing
Environmental L. monocytogenes contamination appears to be the most common source of finished product contamination
Environmental Listeria testing often used as an indicator for conditions that may allow growth or persistence of L. monocytogenes
Better understanding of the ecology of Listeria spp. and L. monocytogenes in food plants is key to better control
Martin Wiedmann / Cornell 5-01

6. Pilot study #1: Application of molecular subtyping to track L
Pilot study #1: Application of molecular subtyping to track L. monocytogenes contamination contamination RTE processing plants
Followed environmental Listeria contamination patterns in 3 smoked fish processing plants for at least 6 months
Environmental isolates were characterized by molecular subtyping
Martin Wiedmann / Cornell 5-01

7. Typing methods used Automated ribotyping
Uses the Qualicon automated RiboPrinter
Standard method uses the enzyme EcoRI, also utilize the enzyme PvuII for further discrimination
Polymerase Chain Reaction (PCR)-restriction fragment length polymorphism (RFLP) for virulence genes actA and hly
Selected isolates are also characterized by serotyping, DNA sequencing of actA and the 16S rRNA gene and by tissue culture assays
Martin Wiedmann / Cornell 5-01

8. Examples of different L. monocytogenes ribotypes
Martin Wiedmann / Cornell 5-01

9. Subtyping results - Plant I
Sample Ribotype Sample Source RiboPrint® Pattern
Sample Source
1039A (R) Raw Chilean Salmon A C B D
1039A (F) Cold-Smoked Chilean Salmon
VISIT 1 *
1042C (E) Floor drain, raw materials area *
1042C (IP) Brine solution, trout
(R) Raw Whitefish
1042D (E) Cutting table, raw materials *
1042C (E) Floor drain, raw materials area *
1042C (E) Floor drain, finished product area
VISIT 2 *
1042C (R) Raw Atlantic Salmon
(E) Floor, finished product storage cooler
(E) Slicer, finished product area
(E) Floor drain, brining cold room *
1042C (E) Floor, brining cold room *
1042C (E) Floor, finished product cold room
VISIT 3
(E) Floor drain, raw materials area
(E) Floor, cold smoker
(E) Floor drain, finished product area *
1042C (E) Floor, brining cold room
VISIT 4
(E) Floor drain, raw materials area
(E) Floor, finished product cold room
(E) Floor, brining cold room
VISIT 5
1046A (E) Floor drain, brining cold room #2
Martin Wiedmann / Cornell 5-01

10. Subtyping results - Plant II
Sample Ribotype Sample Source RiboPrint® Pattern
Sample Source
VISIT 1
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
(R) Raw Troll Red King Salmon, belly area
(IP) Faroe Island Salmon, in brine, head area
(IP) Norwegian Salmon, in brine
(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
1044A (IP) Sable, in brine
(IP) Brine, Norwegian Salmon * * * * * * * *
VISIT 2 * * * * *
VISIT 3 * * *
Martin Wiedmann / Cornell 5-01

11. Subtyping results - Plant II (cont.)
VISIT 4
VISIT 5 *
Sample Ribotype Sample Source RiboPrint® Pattern
1039C (E) Floor drain #1, raw materials preparation
1039C (E) Floor drain #1, raw materials receiving
1039C (IP) Brine, Atlantic Salmon
1039C (F) Cold-smoked Salmon trimmings
(E) Floor drain #2, raw materials receiving
1044A (IP) Troll Red King Salmon, in brine
(E) Floor drain #2, raw materials preparation
(F) Smoked Sable
(R) Raw Atlantic Salmon, in spawn
(IP) Atlantic Salmon, in brine, head area
(IP) Atlantic Salmon, in brine, belly area
(E) Floor drain, brining cold room
1039C (E) Floor drain #2, raw materials preparation
1039C (E) Floor drain #2, raw materials receiving
1039C (F) Smoked Sea Bass
1042B (E) Floor drain #1, raw materials preparation
1042C (IP) Salmon-Trout, in brine
1044A (F) Smoked Sable
(E) Floor drain #2, finished product area
(E) Floor, finished product freezer
(E) Floor drain #1, raw materials preparation
Martin Wiedmann / Cornell 5-01

12. Ecology of L. monocytogenes in RTE processing plants
each processor had unique contamination pattern
specific strains persisted in facilities
Martin Wiedmann / Cornell 5-01

13. Ribotypes: Distribution analysis
Martin Wiedmann / Cornell 5-01

14. Pilot study #2: Tracking to determine sources of persistent L
Pilot study #2: Tracking to determine sources of persistent L. monocytogenes contamination in RTE processing plants
Martin Wiedmann / Cornell 5-01

15. In-plant Listeria Contamination Patterns

16. Distribution of L. monocytogenes by sampling sites
Plant Env.
FCS’s
Drains Other environments FCS

17. Persistent L. monocytogenes environmental contamination
Persistent environmental contamination in RTE seafood and dairy plants (Norton et al., 2001, Appl. Environ. Micro. 67: , Kabuki et al., in preparation)
Persistent environmental contamination in meat plants, >4 years in at least one plant (Nesbakken et al., 1996, Int. J. Food Micro. 31: )
Persistent environmental contamination in poultry processing plants (Ojeniyi et al., 1996, J. Appl. Bacteriol. 80: )
Persistent environmental contamination in seafood plants (Rorvik et al., 2000, Appl. Environ. Micro. 66: )
Martin Wiedmann / Cornell 5-01

18. Pilot study #3: Relationships between environmental L
Pilot study #3: Relationships between environmental L. monocytogenes and Listeria testing results
Martin Wiedmann / Cornell 5-01

19. Listeria spp. and L. monocytogenes detection
Martin Wiedmann / Cornell 5-01

20. Summary
Molecular subtyping of environmental L. monocytogenes isolates is required to differentiate persistent from transient contamination
Detection of Listeria spp. does not always correlate to presence of L. monocytogenes
Inclusion of drains and floors in environmental sampling plans increases the likelihood of detecting persistent contamination
Martin Wiedmann / Cornell 5-01

21. Conclusions
Control of L. monocytogenes in RTE processing plant environments can be achieved best by a combination of environmental testing for L. monocytogenes and molecular subtyping
Detection of persistent contamination and monitoring for persistent contamination is a crucial component of environmental testing
Combination of set sampling points and “variable” sampling locations may provide best approach to control and detect L. monocytogenes contamination
Martin Wiedmann / Cornell 5-01

22. Personal remarks
Efficient control of L. monocytogenes requires collaboration between industry, regulatory agencies, and academia
L. monocytogenes is likely to be present at least at low levels in almost all plants, rules should encourage industry to find L. monocytogenes and to take corrective action
Molecular subtyping can provide important data on sources and spread of environmental L. monocytogenes contamination
Regulatory environment needs to encourage industry to use these tools
Martin Wiedmann / Cornell 5-01

23. Molecular subtyping and the food industry - a vision
L. monocytogenes isolated from environmental or other samples collected in food plants are subtyped and assembled into a database
Benefit #1: Plants receive information on origin and spread of L. monocytogenes, allowing improved sanitation and control strategies
Benefit #2: Significant database of food isolates can be used to define subtypes which are present in food plants, but not associated with human disease
Martin Wiedmann / Cornell 5-01

24. Research hypotheses
L. monocytogenes subtypes (clonal groups) differ in ability to cause human and animal disease
Some subtypes may have adapted/specialized to cause disease in humans or animals (host specificity)
Some subtypes may have lost ability to cause disease in any mammalian species
Subtypes may differ in their ability to survive in processing plants and to survive under various stress conditions

25. Host specificity and virulence differences among L
Host specificity and virulence differences among L. monocytogenes strains
Three out of 13 L. monocytogenes serotypes account for 89-96% of human listeriosis cases (1/2a, 1/2b, and 4b) (McLauchlin, J Eur. J. Clin. Microbiol. Infect. Dis. 9:210‑213; Schwartz, B., et al. J. Infect. Dis. 159:680‑685)
Martin Wiedmann / Cornell 5-01

26. Clonal Structure of L. monocytogenes
Listeria monocytogenes
Lineage I Lineage II Lineage III
E E E 5.3, -D
E E G 6.2, temp
H 9.0
G 8.1, G 5.8, E/G 5.8,
H H H.7.1
11 RT 6 RT RT RT RT RT RT RT RT RT

27. Selected Preliminary Results
Preliminary statistical analyses indicate:
- Lineage I significantly more frequent among human cases as compared to animals cases
- Lineage III significantly more frequent among animal cases as compared to human cases
- Ribotype DUP-1038 significantly more common among human epidemic cases

28. Acknowledgments
K. Boor, A. Hoffman, C. Nadon, D. Norton, G. T. Jeffers, E. Fortes, C. Keating, A. Johnson, M. Bodis, and the Food Safety Laboratory
P. McDonough and M. Smith (CU College of Veterinary Medicine)
J. Bruce (Qualicon)
Financial support by New York Sea Grant, USDA-NRI, USDA Special Research Grant, ILSI N.A., and Qualicon