ecology of l. monocytogenes and listeria spp. in natural and food associated environments martin...
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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
• 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
• 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
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
• 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
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
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
• 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 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