# Presentation: 141453

## 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:  607-254-2838**
**Fax:  607-254-4868**
**E-mail:  mw16@cornell.edu**

## Transmission and pathogenesis of L. mono-cytogenes and other foodborne diseases

**Manure**

**Food products**

**Humans**

**Animal feed/environment /protozoans**

**Food animals**

**Animal derived food products**

**Food Processing Plants**

## 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. 2500 human cases/year in the U.S., resulting in ****about 500 deaths/year**

## 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***

## 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**

## 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**

## Typing methods used

**Automated ribotyping **
**Uses the Qualicon automated RiboPrinter**
**Standard method uses the enzyme *****Eco*****RI, also utilize the enzyme *****Pvu*****II 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 different L. monocytogenes ribotypes

##  Subtyping results - Plant I

- Sample Source

- *

- VISIT 1

- VISIT 2

- VISIT 3

- VISIT 4

- VISIT 5

- *

- *

- *

- *

- *

- *

- *

- A
- A
- C
- C
- B
- D
- C
- C
- C
- C
- C
- 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 room

- 1045             (E)   Floor, finished product cold room

- 1045             (E)   Floor drain, raw materials area

- 1045             (E)   Floor drain, finished product area

- 1043             (E)   Floor, cold smoker

- 1043             (E)   Floor drain, raw materials area

- 1042C           (E)   Floor, finished product cold room

- 1042C           (E)   Floor, brining cold room

- 1045             (E)   Floor drain, brining cold room

- 1045             (E)   Slicer, finished product area

- 1045             (E)   Floor, finished product storage cooler

- 1042C           (R)   Raw Atlantic Salmon

- 1042C           (E)   Floor drain, finished product area

- 1042C           (E)   Floor drain, raw materials area

- 1042D           (E)   Cutting table, raw materials

- 1062             (R)   Raw Whitefish

- 1042C           (IP)  Brine solution, trout

- 1042C           (E)   Floor drain, raw materials area

- 1039A           (F)   Cold-Smoked Chilean Salmon

- 1039A           (R)   Raw Chilean Salmon

** ****Subtyping results - Plant I**

##  Subtyping results - Plant II

- 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

** ****Subtyping results - Plant II**

##  Subtyping results - Plant II (cont.)

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

## Ecology of L. monocytogenes in RTE processing plants

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

## Ribotypes: Distribution analysis

## Pilot study #2:Tracking to determine sources of persistent L. monocytogenes contamination in RTE processing plants

## In-plant Listeria Contamination Patterns

## Distribution of L. monocytogenes by sampling sites

- FCS’s

- Drains   	       Other environments    FCS

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

## Persistent L. monocytogenes environmental 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:Relationships between environmental L. monocytogenes and Listeria testing results

## Listeria spp. and L. monocytogenes detection

## 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**

## 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**

## 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**

## 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**

## 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**

## 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. 1990. Eur. J. Clin. Microbiol. Infect. Dis. 9:210‐213; Schwartz, B., et al. J. Infect. Dis. 159:680‐685)**

## Clonal Structure of L. monocytogenes

## 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**

## 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**