Ecology of L. monocytogenes and Listeria spp. in natural and food associated environments Martin...

Ecology of L. monocytogenes and Listeria spp. in natural and food associated

environments

Martin Wiedmann

Department of Food Science

Cornell University

Ithaca, NY

E-mail: [email protected]

Phone: 607-254-2838

mailto:[email protected]

• The genus Listeria – members and their characteristics• Ecology of the genus Listeria – are Listeria spp. and L.

monocytogenes really ubiquitous?• “Ecology” of L. monocytogenes in food associated

environments – from farms to consumer kitchens• The genus Listeria – a diversity perspective on virulence

associated characteristics

The genus The genus ListeriaListeriaThe genus The genus ListeriaListeria

• Generally thought to be widely distributed in the environment.• soil, water, vegetation, sewage, animal feeds, farms, food processing

facilities, and urban and natural environments• Pathogenic members of the genus Listeria

• L. monocytogenes: causes disease in human and a variety of animals • L. ivanovii: causes disease in ruminants

• Non-pathogenic members of the genus Listeria• L. seeligeri (typically hemolytic, contains homologue of the L.

monocytogenes and L. ivanovii prfA cluster)• L. innocua• L. welshimeri• L. grayi

• Recently discovered species• L. marthii, L. rocourtii

Maximum likelihood reconstruction based Maximum likelihood reconstruction based on 9 locion 9 loci

addB, ldh, lmo1555, lmo2763, pbpA, polC, prs, rarA, and sigB

Listeria and Listeria and L. monocytogenes L. monocytogenes – are they really – are they really ubiquitous?ubiquitous?

• Over 2 years, 1805 samples were collected from four distinct natural environments and four distinct urban environments and tested for Listeria spp. and L. monocytogenes• Samples tested included water, soil, vegetation, and

“human contact surfaces” (e.g., floors, side walks etc.)• Isolates were characterized by molecular subtyping, including

sigB sequencing

ListeriaListeria spp. spp. prevalence prevalence

by site, by sample by site, by sample typetype

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

ADIRONDACKS CATSKILLS CT HILL WMA FLNF

SOIL

VEGETATION

WATER

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

ALBANY NYC ROCHESTER SYRACUSE

SOIL

VEGETATION

WATER

SW/FLOOR

HUMAN CONTACT

Listeria Listeria spp. prevalence was spp. prevalence was 23.4% and 22.3% in natural 23.4% and 22.3% in natural and urban environmentsand urban environments

Prevalence of different Prevalence of different Listeria spp. Listeria spp. by by environmentenvironment

Natural(907 samples)

Urban(898 samples)

L. monocytogenes 13* (6.1%) 67* (29.4%)

L. innocua 1* (0.5%) 49* (21.5%)

L. seeligeri 144* (67.3%) 90* (39.5%)

L. welshimeri 52* (24.3%) 22* (9.6%)

L. marthii 4 (1.9%) 0

TOTAL 214 228

• L. innocua and L. monocytogenes were overrepresented in urban environments

• L. seeligeri and L. welshimeri were overrepresented in natural environments

Prevalence of different Prevalence of different Listeria spp. Listeria spp. subtypessubtypes by by environment and locationsenvironment and locations

Natural Urban

ADK CATSK CTHILL FLNF ALB NYC ROCH SYR

All L. monocytogenes 4 5 2 2 27* 15 7 8

L. monocytogenes AT58 0 0 1 0 11* 1 3 3

All L. innocua 0 0 0 1 14 19 10 6

All L. seeligeri 15 28 50* 51* 24 18 19 29

L. seeligeri AT1 8 6 35* 15 5 1 4 4

L. seeligeri AT2 0 1 4 11* 0 0 0 3*

L. seeligeri AT12 3 4 1 10* 8* 2 2 3

All L. welshimeri 9 32* 7 4 9 2 5 6

L. welshimeri AT14 3 8* 1 0 0 0 0 0

L. welshimeri AT21 0 8* 0 1 0 0 0 2

Spatial clustering Spatial clustering ListeriaListeria species and species and sigBsigB allelic types using nearest neighbors analysisallelic types using nearest neighbors analysis

Environment Site Species or allelic types (AT)

No. of

positive

samples

Actual

MNNDa (km)

Random

MNND (km) p-valueb

Natural Catskills L. welshimeri 32 1.97 2.85 0.001

AT14 (L. welshimeri) 8 5.79 8.89 0.007

AT21 (L. welshimeri) 8 3.19 7.45 0.003

CT Hill L. seeligeri 50 0.14 0.18 0.001

AT1 (L. seeligeri) 35 0.18 0.21 0.013

Urban FLNF L. seeligeri 51 0.07 0.12 <0.0001

Albany AT58 (L. monocytogenes) 11 0.4863 0.749 <0.0001

NYC L. innocua 19 0.4868 1.013 0.003 aMNND=mean nearest neighbor distance bp-value for 1-sample t-test comparing actual and random mean nearest neighbor distances (MNND) values

6 isolates

ListeriaListeria Prevalence on Produce Farms Prevalence on Produce Farms

Sample typeL. monocytogenes L. species (including L.

mono)

Percent Positive (fraction) Percent Positive (fraction)

Fecal 42.9% (9/21) 66.7% (14/21)

Pooled Soil 13.6% (8/59) 23.7% (14/59)

Water 28.0% (14/50) 46.0% (23/50)

Drag swab 8.9% (5/56) 26.8% (15/56)

Overall 19.4% (36/186) 35.5% (66/186)

Samples collected Summer 2009, Fall 2009 and Winter 2010

SummarySummary• Listeria spp. are common in natural, urban, and agricultural

environments• Listeria spp. may differ in their association with different types

of environments• L. seeligeri and L. welshimeri were found to be more common among

natural environments

• L. innocua and L. monocytogenes were found to be more common among urban environments

• Most studies on foods and food processing environments seem to find L. monocytogenes and L. innocua as the most prevalent Listeria spp. (e.g., MacGowan et al., 1994)

• Specific Listeria subtypes may be able to establish persistent populations in specific environments, resulting in spatial clustering

L. monocytogenes L. monocytogenes prevalenceprevalence

• Pristine environments: 1.3% (n=900)• Urban environments: 7.3% (n=900)• Ruminant farms

• Bovine farms with listeriosis cases: 24.3% (n=616)

• Bovine farms without listeriosis cases: 20.1% (n=643)

• Small ruminant farms with listeriosis: 32.9% (n=322)

• Small ruminant farms without listeriosis: 5.9% (n=475)

• Food processing environments: from <0.1% to 30% or more• Ready-To-Eat foods: 0.17 – 4.7 % (e.g., Gombas et al., 2004)

0

5

10

15

20

25

30

35

40

45

50

Fecal Soil Feed Water

Sample Category

% L

M P

osit

ive S

am

ple

s

BOVINE CONTROL BOVINE CASE

a b a a a a a a

n=163

n=160

n=138

n=158

n=162

n=162n=156

n=160

0

5

10

15

20

25

30

35

40

45

50

Fecal Soil Feed WaterSample Category

% L

M P

osit

ive S

am

ple

s

SMALL RUMINANT CONTROL SMALL RUMINANT CASE

n=120

n=85

n=120

n=86

n=120

n=76

n=115

n=75

a b a b a b a b

Cattle

Small ruminants

Nightingale et al. 2004. Appl. Environ. Microbiol. 70: 4458-4467

Seasonal Prevalence in Small Ruminant Seasonal Prevalence in Small Ruminant FarmsFarms

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Fecal Samples Feed Samples Soil Samples Water Samples

Sample Category

% L

M P

os

itiv

e S

am

ple

s

Spring Summer Fall Winter

P = 0.0129

P < 0.0001

P < 0.0001

P = 0.0006

Nightingale et al. 2005. J. Am. Vet. Med. Assoc. 227:1808-1814.

L. monocytogenesL. monocytogenes ecology and contamination ecology and contamination patterns in food processing plantspatterns in food processing plants

• Environmental Listeria contamination as significant problem in the food industry and a key food safety concern

• Better understanding of the ecology of L. monocytogenes in food plants is key to better control

Sample Source

*

VISIT 2

VISIT 3

VISIT 1

****

**

*****

*

***

Sample Ribotype Sample Source RiboPrint® Pattern

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 DSubtyping Results - Plant D

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

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 D (cont.)Subtyping Results - Plant D (cont.)

L. monocytogenesL. monocytogenes persistence in food persistence in food processing plantsprocessing plants

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

Martin Wiedmann / Cornell 10-2008

Overall, 61 isolates with ribotype 1043A (representing this PFGE type) were found over 9 years

PFGE pattern of a persistent strain in a PFGE pattern of a persistent strain in a smoked fish processing plantsmoked fish processing plant

PFGE – ApaI Pattern PFGE – AscI Pattern Site Date

2000 US outbreak - Environmental 2000 US outbreak - Environmental persistence of persistence of L. monocytogenes? 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

• Sequenced full genome of human isolate from 1998 (food isolate sequence was already available)

• Sequenced full genome of human and food isolate from 2000

• Isolates were virtually identical except of a major differences between both 1998 and both 2000 isolates in one prophage (inserted into comK)

Summary - Persistent Summary - Persistent L. monocytogenesL. monocytogenes in in food processing plantsfood processing plants

• Persistent environmental contamination has been reported in almost all types of food processing plants, including RTE seafood plants (e.g., Norton et al., 2001, Appl. Environ. Micro. 67: 198-205;

Rorvik et al., 2000, Appl. Environ. Micro. 66: 4779-4784), dairy plants (e.g.,

Kabuki et al., 2004. J. Dairy Sci. 87:2803–2812); meat plants (>4 years in at least one plant; Nesbakken et al., 1996, Int. J. Food Micro. 31:161-171); poultry processing plants (e.g., Ojeniyi et al., 1996, J. Appl. Bacteriol. 80: 395-401)

• A number of listeriosis outbreaks have been linked to persistent L. monocytogenes contamination in source plants

L. monocytogenesL. monocytogenes in supermarket (retail) in supermarket (retail) environmentsenvironments

• Retrospective study; subtyped 98 food and 40 environmental L. monocytogenes isolated from 50 supermarkets in New York State between 1997 and 2002

• 16 supermarkets showed evidence for persistence of one or more specific L. monocytogenes strains as indicated by isolation of the same EcoRI ribotype from food and/or environmental samples collected in a given establishment on different days

Sauders et al. 2004. J. Food. Prot. 67: 1417–1428

Subtype data from environmental sampling Subtype data from environmental sampling of 121 retail establishmentsof 121 retail establishments

• Cross-sectional study indentified 27 establishments that had two or more L. monocytogenes with the same ribotype (Between 7 and 12 samples/establishment were collected)• In 19 establishments two samples had LM with the same subtype • In 6 establishments three samples had LM with the same subtype• In 1 establishment four samples had LM with the same subtype• In 2 establishments five samples had LM with the same subtype

• Follow-up on 7 establishments identified evidence of L. monocytogenes persistence in 5 establishments (8 to19 months)

Sauders et al. 2009. J. Food Prot. 72: 2337–2349

Date Sample Description Ribotype (lineage) PFGE type (AscI/ApaI)

2/21/06 (C) Deli sink DUP-1052A (I) I (052/090)

(D1) Deli area floor drain DUP-1062A (II) II (085/098)

(E1) Raw meat area floor drain DUP-1052A (I) I (052/090)

(G) Dry aisle DUP-1052A (I) I (052/090)

(I) Grocery cart wheels DUP-1052A (I) I (052/090)

(J1) Produce area floor drain DUP-1052A (I) I (052/090)

3/13/07 (C) Deli sink DUP-1052A (I) Ia (084/091)

(D1) Deli area floor drain DUP-1038C (I) III (082/058)

(E1) Raw meat area floor drain DUP-1062A (II) IIa (025/097)

(F1) Seafood area floor drain DUP-1062A (II) IIa (025/097)

11/28/05 (B) Deli case DUP-1062A (II) IIa (025/097)

(C) Deli sink DUP-1042B (I) IV (065/040)

(I) Grocery cart wheels DUP-1042B (I) IVa (065/040)

(J1) Produce area floor drain DUP-1042B (I) IVa (065/040)

3/6/07 (J1) Produce area floor drain DUP-1062A (II) IIb (026/097)

Overall, 47/1779 samples (2.6%) were positive for LM.

New York City

0 - 50,000

50,001 - 100,000

100,001 - 250,000

250,001 - 500,000

500,001 - 1,000,000

1,000,001 - 2,500,000

Population

A, BBB, I16 (0.59)

B2 (0.25)

E, I5 (0.14) 2 (0.67)

2 (0.39) 1 (0.32)4 (0.54)

B

23 (0.47)A, CCC, E

15 (0.29)CC, F 2 (0.19)

H

9 (0.83)BBB

2 (0.27)D

5 (0.89)BBBB2 (0.37)

E

1 (0.43)B

6 (0.36)BB, E

1 (0.56)

2 (0.90)

2 (1.13)

2 (0.84)

3 (0.35)

3 (0.24)

1 (0.86)

1 (0.82)

1 (0.28)

16 (0.47)DD, FFF, GGG

1 (0.15)

1 (0.45)

H

Geographic Distribution of Human Listeriosis CasesGeographic Distribution of Human Listeriosis Cases

Number cases (rate/100,000)

Single localized geographic cluster

Example: Cluster G

Temporal Clusters: A, B, C, D, E, F, G, H, I

Spatial Clusters: B*, D, G*, H, I

*=Epidemiologically-linked cluster (outbreak)

Molecular subtyping data to identify Molecular subtyping data to identify ListeriaListeria persistence – a few words of persistence – a few words of

cautioncaution• Actual spatial location(s) of persistence may be difficult to

determine• Specific subtypes may persist or be associated with a

given larger geographical area• Some subtypes may be common, widely distributed, and

genetically stable• Subtype databases are needed to aid in interpretation of

repeat isolation of a given subtype

• Not only was PFGE type 7 found across different sources, it was also widely geographically distributed across New York State, and linked to previous outbreaks in the US and Switzerland over 20 years ago!

FSL ID Region Year

Source (general)

Source (specific)

E1-128 Central 2001 Farm Fecal E1-131 Central 2001 Farm Fecal N3-032 Western 2001 Farm Fecal N3-080 Central 2001 Farm Fecal N3-068 Western 2001 Farm Feedstuff F3-520 Metro NY 2003 Food Potato salad* F3-020 Western 2002 Human F2-633 Metro NY 2001 Human F2-644 Western 2001 Human F2-674 Metro NY 2001 Human S4-941 Central 2002 Environment Natural S4-049 Central 2001 Environment Syracuse S4-780 Central 2002 Environment Syracuse H4-246 Western 2003 Farm Water N3-796 Western 2002 Farm Water

2H 4Fm

3E 3Fm

2H 1Fd

H = Human

E = Environment

Fm = Farm

Fd = Food

PFGE type 7 distributions in New

York State

Fugett et al., 2007. J. Clin. Micro




associated genes and phenotypic characteristics

Maximum parsimony tree based on 10 loci

addB, ldh, lmo1555, lmo2763, pbpA, polC, prs, lmo0490, rarA, and sigB

inlA inlA premature stop premature stop codons in codons in L. L.

monocytogenesmonocytogenes

LO28, Jonquieres et al., 1998

Wildtype

inlA (745 aa)

LM

Human intestinal epithelial cell

DUP-1062A

inlA (631 aa)

LM

Human intestinal epithelial cell

MA

?

L. monocytogenes L. monocytogenes with premature with premature inlAinlA stop stop codon: a brief summarycodon: a brief summary

• Found more commonly in food isolates than human isolates• inlA premature stop codon strains represented 35% of food

isolates and 4% of human clinical isolates in France (Jacquet et al. 2004)

• inlA premature stop codon strains represented 45% of 502 L. monocytogenes isolates from Ready-To-Eat foods and 5% of 500 human clinical isolates in the US (Van Stelten et al., 2010).

• Attenuated virulence in guinea pigs, similar to trends previously observed in chick embryo model (Olier et al., 2002, 2003, 2005)

Summary and conclusionsSummary and conclusions• The genus Listeria is commonly found in various environments

• L. monocytogenes can be found in many locations and environments, including food processing plants, farms, cities, natural environments

• Persistence of L. monocytogenes in various environments, including food associated environments, is common and contributes importantly to human listeriosis cases and outbreaks

• Control of L. monocytogenes in the food system is challenging

• A considerable number of Listeria isolates, including many L. monocytogenes, appear to be avirulent or virulence attenuated, including isolates that contain some complements of the Listeria virulence genes • Role of virulence genes in ecology of Listeria spp. largely remains to

be elucidated

AcknowledgmentsAcknowledgments Current and past lab members, in particular: E. Fortes, A. Ho, E. Fugett, B.

Sauders, A. Hoffman, J. Thimothe, A. Roberts, D. Sue, K. Nightingale, M. Chung, K. Evans, R. Ivy, K. Windham

Previous laboratories: C. Batt, K. J. Boor, D. Norton, C. Keating, A. Johnson, M. Bodis

Collaborators: R. Huffman, Y. Grohn, Y. Schukken, J. Scarlett, P. McDonough, M. Smith, B. Njaa, R. Peters (CU College of Veterinary Medicine), Q. Sun (Cornell), J. Bruce (fromerly at Qualicon), J. Kiehlbauch, J. Hibbs, N. Dumas, and D. Morse (NYSDOH), J. Schermerhorn, J. Corby, D. Rice (NYS Ag & Markets), J. Scott, D. Gombas (NFPA, now GMA), P. Mead, B. Swaminathan, L. Graves (CDC), L. Kornstein (NYCDOH), T. Bannerman (Ohio DOH) and the Listeria Working Group

Other financial support: New York Sea Grant, USDA-NRI, USDA Special Research Grants, USDA – Food safety Initiative, ILSI N.A., NIH, and Qualicon

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