martin wiedmann / cornell 5-01 environmental listeria testing and molecular subtyping to control...

Martin Wiedmann / Cornell 5-01

Environmental Environmental ListeriaListeria testing and testing and molecular subtyping to control molecular subtyping to control Listeria Listeria

monocytogenesmonocytogenes in RTE food in RTE food processing environmentsprocessing environments

Martin WiedmannDepartment of Food Science

Cornell UniversityIthaca, NY 14853

Phone: 607-254-2838Fax: 607-254-4868

E-mail: [email protected]

Transmission and pathogenesis of Transmission and pathogenesis of L. mono-L. mono-cytogenescytogenes and other foodborne diseases and other foodborne diseases

Manure

Food products

Humans

Animal feed/environment /protozoans

Food animals

Animal derived food products

Food Processing Plants


Listeria monocytogenesListeria 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. 2500 human cases/year in the U.S., resulting in

about 500 deaths/year


Human listeriosis cases - Three Human listeriosis cases - Three General ScenariosGeneral 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


Environmental Environmental ListeriaListeria testing 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


Pilot study #1:Pilot study #1: Application of molecular subtyping to Application of molecular subtyping to track track L. monocytogenes contamination L. monocytogenes contamination contamination RTE processing plantscontamination 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


Typing methods usedTyping 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


Examples of differentExamples of different L. monocytogenesL. monocytogenes ribotypes ribotypes


Sample Source

*VISIT 1

VISIT 2

VISIT 3

VISIT 4

VISIT 5

*

***

*

*

*

AACCBDCCC

CC

C

A

Sample Ribotype Sample Source RiboPrint® Pattern

1042C (E) Floor, brining cold room

1046A (E) Floor drain, brining cold room #2

1045 (E) Floor, brining cold room1045 (E) Floor, finished product cold room

1045 (E) Floor drain, raw materials area

1045 (E) Floor drain, finished product area1043 (E) Floor, cold smoker1043 (E) Floor drain, raw materials area

1042C (E) Floor, finished product cold room

1042C (E) Floor, brining cold room1045 (E) Floor drain, brining cold room

1045 (E) Slicer, finished product area1045 (E) Floor, finished product storage cooler

1042C (R) Raw Atlantic Salmon1042C (E) Floor drain, finished product area1042C (E) Floor drain, raw materials area

1042D (E) Cutting table, raw materials1062 (R) Raw Whitefish

1042C (IP) Brine solution, trout1042C (E) Floor drain, raw materials area

1039A (F) Cold-Smoked Chilean Salmon1039A (R) Raw Chilean Salmon

Subtyping results - Plant ISubtyping results - Plant I


Sample Source

*

VISIT 2

VISIT 3

VISIT 1

****

**

*****

*

***


1039C (E) Floor drain, raw materials area1039C (E) Floor drain, hallway to finished area1039C (IP) Troll Red King Salmon, in brine, head area1039C (IP) Troll Red King Salmon, in brine, belly area1039C (IP) Brine, Troll Red King Salmon1039C (IP) Faroe Island Salmon, in brine, head area1039C (F) Smoked Sable1039C (F) Cold-Smoked Norwegian Salmon1044A (E) Floor drain, brining cold room 11044A (R) Raw Troll Red King Salmon, head area1044A (IP) Brine, Faroe Island Salmon1045 (R) Raw Troll Red King Salmon, belly area1045 (IP) Faroe Island Salmon, in brine, head area1053 (IP) Norwegian Salmon, in brine1062 (E) Floor drain #1, raw materials preparation1039C (E) Floor drain #1, raw materials preparation1039C (E) Floor drain, brining cold room 11039C (E) Floor drain #2, raw materials preparation1039C (E) Floor drain #2, raw materials receiving1039C (E) Floor drain, finished product area1039C (E) Floor drain, hallway to finished area1039C (IP) Brine, Troll Red King Salmon1039C (F) Smoked Sable1044A (IP) Sable, in brine1044A (IP) Brine, Faroe Island Salmon1062 (IP) Brine, Norwegian Salmon

Subtyping results - Plant IISubtyping results - Plant II


VISIT 4

VISIT 5

*

*

*

*

**

*


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

1062 (E) Floor drain #2, raw materials receiving

1044A (IP) Troll Red King Salmon, in brine

1048 (E) Floor drain #2, raw materials preparation

1052 (F) Smoked Sable

1053 (R) Raw Atlantic Salmon, in spawn

1053 (IP) Atlantic Salmon, in brine, head area

1053 (IP) Atlantic Salmon, in brine, belly area

1062 (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

1062 (E) Floor drain #2, finished product area

1062 (E) Floor, finished product freezer

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

Subtyping results - Plant II (cont.)Subtyping results - Plant II (cont.)


Ecology of Ecology of L. monocytogenes L. monocytogenes in RTE in RTE processing plantsprocessing plants

• each processor had unique contamination pattern• specific strains persisted in facilities

processor ribotype frequency%

timevisits/(months)

B DUP-1042CDUP-1045

36.431.8

4 (5)4 (2.5)

C DUP-1044ADUP-1046B

25.025.0

3 (4)1 (na)

D DUP-1039C 48.9 5 (6)


Ribotypes: Distribution analysisRibotypes: Distribution analysis

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


Pilot study #2:Pilot study #2:

Tracking to determine sources of Tracking to determine sources of persistent persistent L. monocytogenes L. monocytogenes

contaminationcontamination in RTE processing in RTE processing plantsplants

In-plant Listeria Contamination Patterns

Positives for Listeria Genus / L. monocytogenes in environmental samples by week L= Listeria spp, 4 digit number indicates ribotype of L. monocytogenes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 # Positives

Drain 14 1043 1039c - - 1045b - 1042 1053 1045b 1052 1042 1042 1042 1052 1039c 1039c 13

Drain 17 1043 1052 1045b 1027a 1039c 1045b 1042 1052 1045b 1039c 1052 1043 1043 1042 1045b 1039c 16

Drain 18 1043 1039c 1043 1039c 1042 1044a 1062a 1062a 1045b L - 1039c 1062a 1039c 1052 1039c 14

Drain 24 (in cold smoke) - L 1027a 1027a 1039c L 1027a 1027a - - - - - - - - 5

Drain 46 - - - L 1039c 1039c 1062a 1039c 1039c 1052 1052 1039c 1039c 1039c 1039c 1039c 12

Under Tiromat (drain 49) 1039c 1039c 1062a 1039c 1062a L L - 1062a - - 1052 1039c L 1043 - 9

Drain 53 - - - - L 1039c L - L - - - 1039c L - - 2

Drain 54 - L 1062a - L L 1062c 1062c 1052 1039c 1062c L L 1039c 1043 1052 9

Nove box floor 1043 1045b 1043 L 1042 1044a 1043 1027a L - 1062a 1043 1043 L 1045b 1043 12

Floor near drains 12/14 - - - 1039c - 1044a - - 1044a L - - 1042 1043 L - 5

3 Crates 1039c 1039c 1052 1062a 1043 L L - L 1045b 1043 1039c - - L 1044a 9

Condensate line - - - 1052 - - - - - - - - - - - - 1

Overhead Waterline - 1039c - - - - - - - - L - - - - - 1

Door Handle - - - 1039c - - - - - - - L L - 1043 1043 3

Skinning machine, Finished - - - 1039c - - - - - - - - - - - - 1

Raw Scaling Machine - - - - - - - - - - - L - - - - 0

Plant Env. FCS’s

Incidence of Ribotype by Sample Location -- Plant A

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Inc

ide

nc

e

1062c

1062a

1053

1052

1045b

1044a

1043

1042

1039c

1027a

Drains Other environments FCS

Distribution of Distribution of L. monocytogenes L. monocytogenes by by sampling sitessampling sites


Persistent Persistent L. monocytogenesL. monocytogenes environmental contaminationenvironmental contamination

• Persistent environmental contamination in RTE seafood and dairy plants (Norton et al., 2001, Appl. Environ. Micro. 67: 198-205, 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. 31:161-171)

• Persistent environmental contamination in poultry processing plants (Ojeniyi et al., 1996, J. Appl. Bacteriol. 80: 395-401)

• Persistent environmental contamination in seafood plants (Rorvik et al., 2000, Appl. Environ. Micro. 66: 4779-4784)


Pilot study #3:Pilot study #3:Relationships between Relationships between

environmental environmental L. monocytogenes L. monocytogenes and Listeria and Listeria testing resultstesting results


ListeriaListeria spp. and spp. and L. monocytogenesL. monocytogenes detection detectionListeria

spp.L. mono-cytogenes

L.m. positive/Listeria spp.

positive samplesPlant A (Seafood)(n = 256)

138 112 81%

Plant B (Seafood)(n= 256)

61 3 5%

Plant C (Cheese)(n=97)

22 4 18%

Plant D (Cheese)(n=55)

11 8 73%

Plant E (Cheese)(n=94)

21 15 71%


SummarySummary

• 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


ConclusionsConclusions

• 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


Personal remarksPersonal 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


Molecular subtyping and the food Molecular subtyping and the food industry - a visionindustry - 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

Research hypothesesResearch 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


Host specificity and virulence differences Host specificity and virulence differences amongamong L. monocytogenes L. monocytogenes strainsstrains

• Three out of 13 L. monocytogenes serotypes account for 89-96% of human listeriosis cases (1/2a, 1/2b, and 4b) (McLauchlin, J. 1990. Eur. J. Clin. Microbiol. Infect. Dis. 9:210‑213; Schwartz, B., et al. J. Infect. Dis. 159:680‑685)

Clonal Structure ofClonal Structure of L. monocytogenes L. monocytogenes

Listeria monocytogenes

Lineage I Lineage II Lineage III

E 9.2 E 11.2 E 5.3, -D

E 5.2 E 5.3 G 6.2, temp H 9.0

G 8.1, G 5.8, E/G 5.8, H 7.1 H 7.1 H.7.1

11 RT 6 RT 2 RT 15 RT 3 RT 4 RT 3 RT 7 RT 1 RT 2 RT

Selected Preliminary ResultsSelected 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

Frequency amongGenetic SubtypeHuman cases

(n=275)Animal

cases (n=76)Smoked Fish

Processing (n=117)Lineage I 69% 42% 37% II 29% 47% 63% III 2% 11% 0%Ribotypes DUP-1038 15% 12% 3% DUP-1042 26% 12% 6% DUP-1046B 0% 0% 3%

AcknowledgmentsAcknowledgments 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

martin wiedmann / cornell 5-01 environmental listeria testing and molecular subtyping to control...

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