molecular subtyping studies to characterize listeria monocytogenes diversity and ecology in the...

Molecular subtyping studies to characterize Listeria monocytogenes

diversity and ecology in the retail environment

Martin Wiedmann

Department of Food Science

Cornell University

Ithaca, NY

E-mail: [email protected]

Phone: 607-254-2838

mailto:[email protected]

OverviewOverview

• Listeria monocytogenes subtyping• Listeria monocytogenes diversity• Listeria monocytogenes distribution and ecology in

urban, rural (farm), and food associated environments• Summary and implication for control of Listeria

monocytogenes

L. monocytogenes L. monocytogenes strain differentiationstrain differentiation(subtyping/fingerprinting)(subtyping/fingerprinting)

• Tools which allow sensitive differentiation of bacterial subtypes• Facilitates detection of outbreaks• Facilitates detection of contamination sources

• Strain differentiation methods commonly applied to L. monocytogenes include serotyping, ribotyping, Pulsed Field Gel Electrophoresis (PFGE)

RibotypingRibotypingExamples of different Examples of different L. monocytogenesL. monocytogenes

ribotypesribotypes

Pulsed Field Gel ElectrophoresisPulsed Field Gel Electrophoresis

MMMM MMMM MMMM

OverviewOverview


urban, rural (farm), and food associated environments• Summary and implication for control of Listeria

monocytogenes

DNA sequence data support that DNA sequence data support that L. L. monocytogenes monocytogenes represents three lineagesrepresents three lineages

0.01

B. subtilis concatenatedN429039CANE104339CAN

F236045BHSE104445AANE104639CANE112339CAN

F2632UNQHSF242146AHCF203245BFDE105245DANF255339CHC

F223762DFDN429230BAN

F214153AHCF263939BHSE105730BANE1047UNQAN

F272339CFDF251562AFDF266354CHSF237339CFDF202962DHS

F205530AFDF266930AHS

F2655UNQHCF266742BFD

F203044BHSF236642BHSF209142BHCF266142BHS

C140652AFDE105828AAN

F224338BHCE111942BANF229331AFDM202325AHC

E103942BANE104842BAN

F242744AHCF2140UNQHS

F264242BHSF249324AFD

E104142CANF252140AFDF260242AHC

F236942CFDF260142BHCF202242BHCF269342BHC

E104042BANF269561AHC

F252561AHC

//

72

65

8066

64

75

69

5867

53

6397

59

79

0.01

B. subtilis concatenatedN429039CANE104339CAN

F236045BHSE104445AANE104639CANE112339CAN

F2632UNQHSF242146AHCF203245BFDE105245DANF255339CHC

F223762DFDN429230BAN

F214153AHCF263939BHSE105730BANE1047UNQAN

F272339CFDF251562AFDF266354CHSF237339CFDF202962DHS

F205530AFDF266930AHS

F2655UNQHCF266742BFD

F203044BHSF236642BHSF209142BHCF266142BHS

C140652AFDE105828AAN

F224338BHCE111942BANF229331AFDM202325AHC

E103942BANE104842BAN

F242744AHCF2140UNQHS

F264242BHSF249324AFD

E104142CANF252140AFDF260242AHC

F236942CFDF260142BHCF202242BHCF269342BHC

E104042BANF269561AHC

F252561AHC

//

72

65

8066

64

75

69

5867

53

6397

59

79

0.1

F252561AHCE104842BAN

F203044BHSF224338BHCF236942CFDF202242BHCF266742BFDF269342BHC

E103942BANC140652AFDE105828AANF266142BHSE111942BANF229331AFDF236642BHSM202325AHC

E104042BANE104142CAN

F209142BHCF260142BHC

F2140UNQHSF242744AHC

F264242BHSF249324AFDF252140AFDF260242AHC

F202962DHSE104339CANE104445AAN

F205530AFDF223762DFD

F2632UNQHSF266354CHS

F266930AHSE105245DANF203245BFD

F214153AHCE104639CANE1047UNQANE105730BANE112339CAN

F236045BHSF237339CFDF242146AHC

F251562AFDF255339CHCF263939BHSF272339CFDN429039CANN429230BAN

F2655UNQHCF269561AHC

99

71

62

100

90

68

0.1

F252561AHCE104842BAN

F203044BHSF224338BHCF236942CFDF202242BHCF266742BFDF269342BHC

E103942BANC140652AFDE105828AANF266142BHSE111942BANF229331AFDF236642BHSM202325AHC

E104042BANE104142CAN

F209142BHCF260142BHC

F2140UNQHSF242744AHC

F264242BHSF249324AFDF252140AFDF260242AHC

F202962DHSE104339CANE104445AAN

F205530AFDF223762DFD

F2632UNQHSF266354CHS

F266930AHSE105245DANF203245BFD

F214153AHCE104639CANE1047UNQANE105730BANE112339CAN

F236045BHSF237339CFDF242146AHC

F251562AFDF255339CHCF263939BHSF272339CFDN429039CANN429230BAN

F2655UNQHCF269561AHC

99

71

62

100

90

68

Concatenated prs, sigB, gap actA

Lineage I, lineage II, lineage III Nightingale et al. 2005, J. Bact.

Cluster analysis of Cluster analysis of genome wide gene genome wide gene presence/absence presence/absence patterns further patterns further

supports supports L. L. monocytogenes monocytogenes

lineages lineages • Analysis were performed using

both a randomer serotype 1/2a and a randomer 4b chip

• 16 regions of difference (RD) were absent in lineage I strains,

III

I

II

Zhang et al., 2003. J. Bacteriol. 185:5573-5584.

Molecular characterization of human, Molecular characterization of human, animal, and food isolatesanimal, and food isolates

Lineage Human isolates (n=507)

Food isolates (n=502)

Animal isolates (n=126)

Lineage I 54.4%** 37.3%** 40%

Lineage II 42.6%** 62.4%** 52%

Lineage III 2.4%* 0.4%* 8% ** P<0.0001; * P<0.01

Each lineage contains between 30 and 50 EcoRI ribotypes

Number of isolates Ribotype

Food Human

P-value 1) Comments

DUP-1030A 8 8 NS DUP-1030B 0 10 ** not found in food DUP-1038B 15 63 **** DUP-1039A 12 31 ** DUP-1039B 18 43 ** DUP-1039C 35 25 NS DUP-1042A 11 16 NS DUP-1042B 18 72 **** DUP-1042C 14 0 *** multiple food types, not in humans DUP-1043A 30 16 * DUP-1044A 11 28 ** DUP-1044B 1 19 *** rarely found in food DUP-1044E 10 0 ** blue cheese only DUP-1045B 14 11 NS DUP-1052A 58 39 * DUP-1053A 24 41 * DUP-1062A 151 9 **** rarely found in humans DUP-1062D 28 1 **** rarely found in humans

rare 22 42 * Ribotypes with 1-4 isolates uncommon 22 33 NS Ribotypes with 5-8 isolates

Total 502 507 **** Overall analysis of ribotype vs. origin 1) P-values refer to comparison of origin between ribotype specified in that row vs. all other ribotypes 1

where NS = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 2

Human virulence attenuation of Human virulence attenuation of ribotype DUP-1062Aribotype DUP-1062A

• Isolates with ribotype DUP-1062A carry a premature stop codon in inlA, which leads to reduced invasion of human epithelial cells

Wildtype

inlA (800 aa)

LM

Human intestinal epithelial cell

DUP-1062A

inlA (700 aa)

LM

Human intestinal epithelial cell

MA

Mutation type 3; DUP-1046B& DUP-1062A

Food isolate from France (NV4; Rousseaux et al., 2002)

Human fecal carriage strains from France (Olier et al., 2002)

NC S 492

575E

N

NS

606F

SN 656

G

SN 700

J

SS

LRR B RepeatsIRLPXTG

MA

29 357 462 650 711

N C

800

A

Human fecal carriage strain L028 from France (Jonquieres et al., 1998)

Mutation type 1; DUP-1052A & DUP-16635A

Mutation type 2; DUP-1025A & DUP-1031A

NB S

460 Food isolate from France (NV8; Rousseaux et al., 2002)

519ND S Food isolate from France (NV7; Rousseaux et al.,

2002)

SN 677

H

SN 685

I Food isolate from France (NV5; Rousseaux et al., 2002)

EGD-e (Glasser et al., 2001)

inlA inlA premature stop codons in other strainspremature stop codons in other strains

In vitro evaluation of invasion into human intestinal In vitro evaluation of invasion into human intestinal epithelial cells of epithelial cells of L. monocytogenes L. monocytogenes with and without with and without

premature stop codons in premature stop codons in inlAinlA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

L. monocytogenes Strain

% L

M Inva

sion o

f C

aco

-2 C

ells

10403S ²

inlA

DU

P-1052A

DU

P-1052A

muta

tion 1

DU

P-1025A

DU

P-1025A

muta

tion 2

10403S

EG

D-E

DU

P-1062A

muta

tion 3

10403S ²

inlA

B 10403S ²

inlB

10403S m

uta

tion 3

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

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

food isolates and 4% of human clinical isolates (Jacquet et al. 2004. JID 189:2094-2100)

• Also found in China and Portugal• Attenuated virulence in guinea pigs

• Not all L. monocytogenes are equally likely to cause human disease and many L. monocytogenes in foods have reduced ability to cause human disease

L. monocytogenes L. monocytogenes subtype data for 121 subtype data for 121 retail operations retail operations

• 156 isolates represented lineage I (55.8%) and II (44.2%)• Molecular serotyping classified isolates into profiles B

(serotypes 1/2b, 3b, and 7; 44 isolates), D (serotypes 4b, 4d, and 4e; 43 isolates), and A (serotypes 1/2a and 3a; 59 isolates)

• Isolates include ribotypes linked to human outbreaks, including DUP-1042B (13.5%), DUP-1038B (3.2%), and DUP-1044A (3.2%)

• Some ribotypes identified at retail include only isolates with inlA mutations (one ribotype, 8.3%) and some include isolates with and without inlA mutations (5 ribotypes, 38.4%)• Isolates will be specifically characterized for inlA mutations

OverviewOverview


urban, rural (farm), and food associated environments• Summary and implications for control of Listeria

monocytogenes

L. monocytogenes L. monocytogenes prevalenceprevalence

• Pristine environments: 1.3% (n=900) [23.4% for Listeria spp.]• Urban environments: 7.3% (n=900) [22.3% for Listeria spp.]• 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)

• Raw foods

• Ready-To-Eat foods: 0.17 – 4.7 % (Gombas et al., 2004)

L. monocytogenes L. monocytogenes in urban environmentsin urban environments

• Albany, NY• 214 samples

tested• 27 positive for

LM• 10 isolates

were ribotype DUP-1038B (over three samplings and >1 year

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

L. monocytogenesL. monocytogenes ecology and contamination ecology and contamination patterns in processing plants and retail patterns in processing plants and retail

operationsoperations

Martin Wiedmann / Cornell 10-2008

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

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

• Persistent environmental contamination in RTE seafood and dairy plants (Norton et al., 2001, Appl. Environ. Micro. 67: 198-205, Kabuki et al., 2004. J. Dairy Sci. 87:2803–2812)

• 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)

L. monocytogenesL. monocytogenes in retail environments in retail environments

• 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

• 17 ribotypes were found among human clinical isolates as well as among food and environmental isolates

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

L. monocytogenesL. monocytogenes in retail environments - in retail environments - exampleexample

Est. Code Source Sample Description

Date Collected (No. isolates) Ribotype

Persistent Ribotype (Lineage)

V Food Oven roasted turkey 08/17/00 DUP-1062A DUP-1062A (II)Food Smoked turkey breast 08/25/00 DUP-1062A Environment Swab; display cooler 08/29/00 DUP-1062AEnvironment Swab 08/29/00 DUP-1062AEnvironment Swab 08/29/00 DUP-1062AEnvironment Swab; deli case 09/21/00 DUP-1062A

W Environment Swab 10/27/00 DUP-1053A Not applicableEnvironment Swab 10/27/00 DUP-1053A

X Food Ham 11/07/00 DUP-1042C DUP-1042C(I)Environment Swab 11/20/00 DUP-1042CEnvironment Swab 11/20/00 DUP-1042CEnvironment Swab 12/04/00 DUP-1042CEnvironment Swab 12/04/00 DUP-1042CEnvironment

Y EnvironmentEnvironment

Z EnvironmentEnvironmentEnvironmentEnvironment

AA FoodEnvironment

BB FoodFoodEnvironment

EnvironmentCC Food

FoodFoodFoodFoodFoodEnvironment

Environment

DD FoodFoodFoodEnvironmentFoodFood 07/19/02

EE FoodFoodFoodEnvironment

EnvironmentEnvironmentEnvironment

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

• 27 establishments that had two or more L. monocytogenes with the same ribotype• in 19 est. two samples had LM with the same subtype, in 6

three samples had LM with the same subtype, in 1 four samples had LM with the same subtype, and in two five samples had LM with the same subtype

• In 11 establishments that had isolates with identical ribotypes in multiple environmental samples, at least one isolate was from a food contact surface

• In 7 establishments isolates with the same ribotype were found in multiple drain or floor samples

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)


7/10/06 (Food) Intact, deli ham DUP-1042B (I) V (064/032)

(C) Deli sink DUP-1042B (I) V (064/032)

(D1) Deli area floor drain DUP-1042B (I) V (064/032)

(E1) Raw meat area floor drain DUP-1042B (I) V (064/032)

(G) Dry aisle DUP-1042B (I) V (064/032)

(H) Dairy case DUP-1052A (I) VI (061/083)

3/12/07 (D1) Deli area floor drain DUP-1042B (I) V (064/032)

8/2/05 (F1) Seafood area floor drain DUP-1051D (I) VII (068/037)

(J1) Produce area floor drain DUP-1051D (I) VII (068/037)

(J4) Bakery floor drain DUP-1051D (I) VIIa (069/036)

3/12/07 (J1) Produce area floor drain DUP-1052A (I) VIII (040/070)

(F1) Seafood area floor drain DUP-1051D (I) VII (068/037)


12/13/05 (C) Deli sink DUP-1052A (I) VIII (040/070)

(D1) Deli area floor drain DUP-1052A (I) VIIIa (044/070)

(J1) Produce area floor drain DUP-1052A (I) IX (044/085)

3/5/07 (C) Deli sink DUP-1052A (I) VIIIb (040/079)

(D1) Deli area floor drain DUP-1052A (I) IX (044/085)

(J1) Produce area floor drain DUP-1052A (I) IXa (040/088)

(G) Dry aisle DUP-1052A (I) IX (044/085)

Patterns of human listeriosisPatterns of human listeriosis

O-9

2N

-92

D-9

2J-

93F

-93

M-9

3A

-93

M-9

3J-

93J-

93A

-93

S-9

3O

-93

N-9

3D

-93

J-94

F-9

4M

-94

A-9

4M

-94

J-94

J-94

A-9

4S

-94

O-9

4N

-94

D-9

4J-

95F

-95

M-9

5A

-95

M-9

5J-

95J-

95A

-95

S-9

5O

-95

N-9

5D

-95

J-96

F-9

6M

-96

A-9

6M

-96

0

1

2

3

4

5

6

7

8

9

Cluster HCluster ACluster ECluster BCluster ICluster DCluster JCluster CCluster KCluster LCluster FCluster MNonclusters

A

B

E D

H

I J L M

C

F

K

Sauders et al. EID vol. 9:672 – 680 (2003)

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)

OverviewOverview


urban, rural (farm), and food associated environments• Trends in human listeriosis• Summary and implications for control of Listeria

monocytogenes

SummarySummary• L. monocytogenes contamination in retail environments is not

uncommon• Not surprising as L. monocytogenes is not uncommon in

surrounding environments• Size and inspection scores seem to have little predictive value

for L. monocytogenes presence• L. monocytogenes can persist in retail environments • At least some L. monocytogenes found in retail environment

appear to have potential to cause human listeriosis • Patterns of cases and small outbreaks suggest that “local events”

may contribute to human listeriosis cases • Risk assessment suggests that contamination at retail is one

contributor to human listeriosis cases

Establishment size Establishment size and and L. mono-L. mono-

cytogenes cytogenes prevalenceprevalence

No significant association between the prevalence of L. monocytogenes (positive samples/number of samples) and the number of employees (P = 0.229) and area (in square feet) of the food facility (P = 0.834).

ConclusionsConclusions• Control of L. monocytogenes at retail is challenging• Contamination of RTE products from environmental sources at retail is a

major concern• Identification of “high risk” retail operations may be best achieved by

environmental testing/monitoring• Environmental testing not only identifies the problem but also provides

initial insights into potential control strategies• Environmental testing should focus on L. monocytogenes

• Persistence of L. monocytogenes in environmental niches is an important issue• Identification and elimination of niches is important• Niches can and maybe outside of retail environment

• Need for innovative strategies to reduce L. monocytogenes introduction into retail

• Need to comprehensively quantify contributions, to human listeriosis, of L. monocytogenes contamination at processing, retail, restaurant, and home and institutional kitchens

Support for this work was provided by the United States Department of Agriculture, Cooperative State Research, Education and Extension

Service, National Integrated Food Safety Initiative Special Emphasis Grant Project Number 2005-51110-03278

AcknowledgmentsAcknowledgments

More acknowledgmentsMore acknowledgments E. Fortes, B. Sauders, J. Thimothe, V. Lappi, K. Nightingale, S. Roof Collaborators: J. Kiehlbauch, J. Hibbs, N. Dumas, and D. Morse

(NYSDOH), J. Schermerhorn, J. Corby, D. Rice, S. Stitch, and others (NYS Ag & Markets)

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

molecular subtyping studies to characterize listeria monocytogenes diversity and ecology in the...

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