Listeria monocytogenesListeria monocytogenes includes common includes common and widely distributed virulence and widely distributed virulence

attenuated attenuated clonalclonal groups and epidemic groups and epidemic clonesclones

Martin WiedmannDepartment of Food Science

Cornell UniversityIthaca, NY

E-mail: mw16@cornell.eduPhone: 607-254-2838

• L. monocytogenes in different hosts and environments –presence, persistence, and diversity

• L. monocytogenes population genetics and evolution: evidence for distinct lineages that differ in virulence

L. monocytogenes L. monocytogenes prevalenceprevalence

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

• Bovine farms with listeriosis cases: 24.35% (n=616)• Bovine farms without listeriosis cases: 20.06% (n=643)• Small ruminant farms with listeriosis: 32.92% (n=322) • Small ruminant farms without listeriosis: 5.89% (n=475)

• Food processing environments: from

E)

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 WaterSample Category

% L

M P

ositi

ve S

ampl

es

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

ositi

ve S

ampl

es

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

Fecal Fecal L. monocytogenesL. monocytogenes shedding in cattleshedding in cattleCows

Day 86 Day 87 Day 88 Day 89 Day 90 Day 91 Day 92 Day 93 Day 94 Day 95 Day 96 Day 97 Day 98 Day 994/12 4/13 4/14 4/15 4/16 4/17 4/18 4/19 4/20 4/21 4/22 4/23 4/24 4/25

1 (372) 1058A 1058A 1039C 1039E 1039C 18645/ 1045A 1039C 1042A2 (378) 1058A 1058A 1039E 1039E 1039C3 (324) 1058A 1058A4 (363) 1058A 1042B 1042B 1058A 18611/ 1044A 1045D 18645/ 1045A 18595/ 1042C 1042A 1058A5 (311)6 (380) 1058A 1058A 1058A 1042A 1045D 1042A7 (322)8 (309)9 (268) 1058A 1039E 1030A 1051D 1042A 1039C 1042A 1042B

10 (357) 1058A 1058A 1039C 1039E 1023A 1030A 18645/ 1045A 1039C 1042A 1039C 1058A11 (367) 1058A 1042B 1039E 1039E 1045D 18645/ 1045A 1042A 1039C12 (320) 1039C 1058A 1042B 1039C 1039C 1042B13 (338) 18611/ 1044A 1058A 1058A 1058A 1051D 1039E 1052A 1051D 1039C 1039C14 (334) 1058A 1058A 1039E 1042B 18645/ 1045A 1039C 1859515 (246)16 (327) 1058A 1058A 1039C 1042A 1042B17 (352) 1058A 1058A18 (348) 1058A 1058A 1058A 1042B 1039E 1039C 1042A 1039C 1039C 1042A19 (350)20 (290) 1058A 18611/ 1044A 1039C 1060A 18645/ 1045A 18645/ 1045A 1058A 1039C 1042B21 (358) 1058A 1045D 1058A 1039E 1039E 1030A 18645/ 1045A 18611/ 1044A 1042A 1058A22 (326) 1039C 1058A 1058A 1051D 1058A23 (336)24 (369) 1030A 1058A 1058A 1058A 1058A 1058A 18645/ 1045A 18595/ 1042C 1039C25 (355) 1045A 1058A 1058A 1060A 1039E 18595/ 1042C 1039E 18645/ 1045A 1039C 18627/ 1042A 1039C 1039E26 (362) 18595/ 1042C 1030A 1058A 1042B 1039E 1058A 18645/ 1045A 1039C27 (383) 1058A 1058A 1058A 1039E 1039E 18645/ 1045A 1042B 1058D 1051D 1042A 1058A28 (384)29 (308) 18595/ 1042C 1042B 1039E 1042B 1039E 1042A30 (266) 1058A 1052A 1039C 1058A 1051D 1042B31 (374) 18611/ 1044A 1039C 1042B 1039E 1039E 18645/ 1045A32 (346) 1039C 1058A 1039C 18645/ 1045A 1039E 1039E 1039E 1043 1039C 1030B 1045D 18595Silage

4/12 4/13 4/14 4/15 4/16 4/17 4/18 4/19 4/20 4/21 4/22 4/23 4/24 4/25spread1 1042B 1039C 18595/ 1042C 1042Bspread2 1058A 1042A 1039C 1042A 1042B

S1 1058A 18645/ 1045A 1042B 1039E 1039C 1039C 18611/ 1044AS2 1058A 1058A 1039C 1039C 18645/ 1045A 1038B 1039E

L. monocytogenesL. monocytogenes ecology and persistence in ecology and persistence in processing plantsprocessing plants

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 Subtyping Results –– seafood seafood processing plantprocessing plant

VISIT 4

VISIT 5

*

*

*

*

**

*

Sample Ribotype Sample Source RiboPrint® Pattern

1039C (E) Floor drain #1, raw materials preparation1039C (E) Floor drain #1, raw materials receiving1039C (IP) Brine, Atlantic Salmon1039C (F) Cold-smoked Salmon trimmings1062 (E) Floor drain #2, raw materials receiving1044A (IP) Troll Red King Salmon, in brine1048 (E) Floor drain #2, raw materials preparation1052 (F) Smoked Sable1053 (R) Raw Atlantic Salmon, in spawn1053 (IP) Atlantic Salmon, in brine, head area1053 (IP) Atlantic Salmon, in brine, belly area1062 (E) Floor drain, brining cold room1039C (E) Floor drain #2, raw materials preparation1039C (E) Floor drain #2, raw materials receiving1039C (F) Smoked Sea Bass1042B (E) Floor drain #1, raw materials preparation1042C (IP) Salmon-Trout, in brine1044A (F) Smoked Sable1062 (E) Floor drain #2, finished product area1062 (E) Floor, finished product freezer1062 (E) Floor drain #1, raw materials preparation

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

L. monocytogenesL. monocytogenes persistence in plantspersistence in 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

2000 US outbreak 2000 US outbreak -- Environmental 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. monocytogenesenvironmental contaminationenvironmental contamination

• 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 environmentsin retail environments• Subtyped 98 food and 40 environmental L. monocytogenes

isolates collected from retail establishments in New York State between 1997 and 2002 • Isolates collected from 50 different retail establishments

were characterized. • 16 retail establishments 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. monocytogenes L. monocytogenes diversity in different diversity in different environmentsenvironments

• 92 EcoRI ribotypes among 1,001 L. monocytogenes isolates collected over a period of 5 years in New York State from various sources (Sauders et al., 2006. JFP)• Isolates came from natural environments, n=13; urban environments,

n=67; human clinical isolates, n=342; food and food environments, n= 165; animals and farm environments, n=414

• 310 AscI and ApaI PFGE types (including 235 unique patterns) and 74 EcoRI ribotypes among 495 L. monocytogenes isolates collected over 3 years in New York State (Fugett et al., 2007, JCM)

• 83 ApaI PFGE types among 1028 L. monocytogenes isolates form 13 French pork salting plants (Thevenot et al. 2006, Intern J. Food Micro.)

• L. monocytogenes in different hosts and environments –presence, persistence, and diversity

• L. monocytogenes population genetics and evolution: evidence for distinct lineages that differ in virulence

L. monocytogenes L. monocytogenes lineages lineages -- summarysummary• Classification of L. monocytogenes into two common lineages as

well as one rare lineage (III) has been confirmed by almost all subtyping methods• MLEE• Ribotyping, PFGE, AFLP cluster analysis• MLST• Microarray-based gene presence/absence data

• Lineage I (or “division II”): highly clonal, includes serotypes 1/2b, 4b, 3b; includes EC I, EC Ia, EC II

• Lineage II (or “division I”): less clonal than lineage I, includes serotypes 1/2a, 1/2c, 3a; includes EC III

• Lineage III (“division III”): includes serotypes 4a, 4c, as well as atypical serotype 4b isolates

L. monocytogenes L. monocytogenes phylogeny (120 isolates)phylogeny (120 isolates)

0.01

B. subtilis concatenatedN429039CANE104339CAN

F236045BHSE104445AANE104639CANE112339CAN

F2632UNQHSF242146AHCF203245BFDE105245DANF255339CHC

F223762DFDN429230BAN

F214153AHCF263939BHSE105730BANE1047UNQAN

F272339CFDF251562AFDF266354CHSF237339CFDF202962DHS

F205530AFDF266930AHS

F2655UNQHCF266742BFD

F203044BHSF236642BHSF209142BHCF266142BHS

C140652AFDE105828AAN

F224338BHCE111942BANF229331AFDM202325AHC

E103942BANE104842BAN

F242744AHCF2140UNQHS

F264242BHSF249324AFD

E104142CANF252140AFDF260242AHC

F236942CFDF260142BHCF202242BHCF269342BHC

E104042BANF269561AHC

F252561AHC

//

72

65

80 66

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

80 66

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 IIINightingale et al. 2005, J. Bact. 187: 5537–5551

Neighbor Joining tree of prfA cluster (prfA, plcA, hly, mpl, actA, plcB)

0.01

R2142M1001F2086

J1208F2270

F2318NRRL33077

NRRL33105F2501

NRRL33177NRRL33184NRRL33188

NRRL33191f2695NRRL33183NRRL33182NRRL33229J2074NRRL33227

J1168NRRL33230

NRRL33115J2071NRRL33231

NRRL33185NRRL33190

R2128NRRL33092NRRL33181NRRL33187

f2667n4291f2898n4288e1119f2897n4289s4941e1124f2658

f2602f2493f2666

f2661f2637f2672e1125

f2601n4292

f2663e1123f2515

s4304s4766s4887f2373f2640f2723s4497s4821s4880

n4290n4293s4019

f2639f2590f2634

s6072s4295

NRRL33021NRRL33017

Lineage IIIBLineage IIIC

Lineage IIIA

Lineage I

Lineage II

100

96.9

100

99.5

100//

// //

“Lineage” III

100

100

L. ivanovii

Genomic microGenomic micro--array analysisarray analysis

• Analysis were performed using both a randomer serotype 1/2a and a randomer 4b chip

• Cluster analysis confirmed existence of three lineages

• 16 regions of difference (RD) were absent in lineage I strains, including 5 RDs with genes showing LPXTG cell wall-anchoring motifs and 3 RDs with genes showing leucine rich repeats

III

I

II

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

Recombination in Recombination in L. monocytogenes L. monocytogenes housekeeping and virulence geneshousekeeping and virulence genes

• Based on characterization of 120 isolates (J. Bacteriol. 187:5537 ff.) • sigB, gap: no evidence for recombination• prs: 1 single recombination event between lineages I and III• purM and ribC: 7 recombination events each; 10 within

lineages• inlA and actA: 6 and 1 recombination events, respectively; 5

between different lineages• inlB, InlG, inlC2, inlD, inlE, inlF: 1 – 3 recombination

events per gene; all but one between different lineages • Very limited recombination with lineage I as recipient (except

inlB)

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

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

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

Outbreak clonesOutbreak clones

• Three ribotypes have been responsible for 2 or more human listeriosis outbreaks• Ribotype DUP-1038B (EC I): Anjou (France, 1976),

Nova Scotia (Canada, 1981), Vaud (Switzerland, 1983-1987), and Los Angeles (U.S., 1985)

• Ribotype DUP-1042B (ECIa): Boston (U.S., 1979); Massachusetts (U.S., 1983); United Kingdom (1988-1990)

• Ribotype DUP-1044A (ECII): US (1998/99; hot dogs and deli meats); US (2002) that were linked to consumption of contaminated hot dogs and sliced turkey

Distribution of L. monocytogenes lineages and PFGE types among isolates from human clinical cases, ruminant farms, and urban and pristine environments.

No. of L. monocytogenes isolates froma PFGE type Human Food Farm Environment 7 4 1 7 3 22 15 (+) *** 4 0 (-) *** 0 38 1 0 0 (-) * 6 (+) *** 50 0 5 (+) *** 0 0 52 0 6 (+) *** 0 (-) * 0 189 0 0 8 (+) * 1 240 1 5 (+) * 3 0 300 0 0 7 (+) ** 0 315 0 0 7 (+) ** 0 336 2 7 (+) *** 0 (-) * 0 aOverall L. monocytogenes PFGE type distributions and PFGE type and genetic lineage prevalences that were significantly higher (+) or lower (-) from a specific source as determined by categorical analyses were labeled as * (indicating P ≤ 0.05), ** (indicating P ≤ 0.005), or *** (indicating P ≤ 0.0005).

PFGE typing of 495 isolates from across NY state (2001 – 2003)

EC IEC II

Ribotype DUP-1038B, epidemic clone (EC) IGeographically widely distributed across NYS (15 isolates), alsofound in Washington State (Borucki et al., 2004, JFP)Linked to previous outbreaks in LA (1985) and Switzerland (1983-87)

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 = HumanE = EnvironmentFm = FarmFd = Food

PFGE type 7 distributions in New York State

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

PFGE type 7

Plaque assay to test Plaque assay to test L. L. monocytogenesmonocytogenes invasion invasion and celland cell--toto--cell spreadcell spread

Average plaque areaLineage I (n=56) 110%

Lineage II (n=70) 93%DUP-1038B (n=8) 119%DUP-1042B (n=14) 126%

• Lineage I strains form larger plaques than lineage II strains (p

Caco-2 Invasion Screening Assay Results by Lineage

0

50

100

150

200

250FS

L R

2-31

1

FSL

L4-3

36

FSL

F2-0

31

FSL

L4-0

90

FSL

S4-2

68

FSL

F2-0

50

FSL

F2-0

06

FSL

N3-

034

FSL

R2-

081

FSL

N4-

231

FSL

N3-

163

FSL

F2-3

23

FSL

F2-1

17

FSL

N4-

239

FSL

F3-0

49

FSL

H4-

364

FSL

F2-4

15

FSL

F2-4

30

FSL

S4-8

21

FSL

R3-

001

FSL

H4-

110

FSL

E1-2

58

FSL

S4-6

58

FSL

F2-0

86

FSL

F2-5

25

FSL

E1-1

58

FSL

S4-1

54

FSL

N4-

501

FSL

F2-2

70

Isolate ID

%10

403S

Inva

sion

Lineage I Lineage II Lineage III U

Human virulence attenuation of Human virulence attenuation of ribotype DUPribotype DUP--1062A1062A

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

Wildtype

inlA (800 aa)

LM

Human intestinal epithelial cell

DUP-1062A

inlA (700 aa)

LM

Human intestinal epithelial cell

MA

Mutation type 6; DUP-1039C and DUP-1048B & Human fecal carriage strains from France (Olier et al., 2002)

NE S 492

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

SN 700M

575H

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

N S 606I Mutation type 1; DUP-1052A &

DUP-16635A

SN 656

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

ND S 460 Food isolate from France (NV8; Rousseaux et al.,

2002)

519NF S Food isolate from France (NV7; Rousseaux et

al., 2002)

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

SN 677K

SN 685L Food isolate from France (NV5;

Rousseaux et al., 2002)

NB

9 Mutation type 4 (frameshift); DUP-1039C

SS

LRR B RepeatsIRLPXTG

MA

29 357 462 650 711

N C

800

AEGD-e (Glaser et al., 2001)

NC S 189 Mutation type 5; DUP1029A

G563N Mutation type 7; DUP-1045B, DUP-

1062D

inlA premature

stop codons in different

LM strains

L. monocytogenesL. monocytogenes with with inlA inlA premature stop premature stop codonscodons• Found among food isolates from France, US, Portugal (poster P-19),

Italy (P-81)• inlA PMSC are common among food isolates, rare among human

isolates, and never found among isolates from pregnancy associated cases• 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)

• US: inlA premature stop codon strains represent >30% of food isolates and

Dose response differences among Dose response differences among L. L. monocytogenes monocytogenes lineages and subgroups lineages and subgroups

• Based on L. monocytogenes prevalence, contamination level, and subtype data for 31,707 RTE food samples (including 502 positive samples) (Gray et al., 2004; Gombas et al., 2003) and subtypes data for 492 human isolates from the US

• Isolates were grouped into (1) lineages I and II and (2) ribotype groups, including (i) DUP-1062A (inlA PMSC); (ii) DUP-1038B and DUP-1042B (ECI), and (iii) DUP-1039C (common ribotype; often found in high levels in foods)

Virulence parameter for selected Virulence parameter for selected L. L. monocytogenes monocytogenes lineages and subgroups lineages and subgroups

Molecular subgroup r-value (log) 95% confidence interval Lineage I -7.88 -8.64 to -7.12 Lineage II -10.3 -10.6 to -10.0 DUP-1062A -8.73 -9.04 to -8.42 DUP-1042B and 1038B -6.30 -6.52 to -6.09 DUP-1039C -11.0 -11.4 to -10.6 r-value = probability of contracting listeriosis from 1 cell of L. monocytogenes

Molecular subgroup r-value difference (log)

95% confidence interval

Lineage I vs. Lineage II 2.42 1.61 to 3.22 DUP-1062A vs. DUP-1042B and 1038B 2.42 2.08 to 2.76

Selected r-value differences

Chen et al., 2006. JFP 69: 335 ff.

Summary and conclusionsSummary and conclusions

• L. monocytogenes is found in many environments• Prevalence differs and may be highest in farm environments

and lowest in natural environments • While considerable L. monocytogenes diversity is found in

different hosts, certain L. monocytogenes subtypes are associated with specific sources and hosts

• L. monocytogenes can be separated into multiple distinct lineages• L. monocytogenes includes widely distributed virulence-

attenuated clonal groups (i.e., through inlA premature stop codons) as well as widely distributed and genetically stable epidemic clones

AcknowledgmentsAcknowledgmentsStudents and staff: K. Nightingale, R. Orsi, E. Fortes, A. Ho., B. Sauders, A. Hoffman, J. Thimothe, A. Roberts, D. Sue,, M. Chung,K. Evans, M. Kazmierczak, P. McGann, M. Garner, Y. Soyer, S. AlcaineCollaborators: K. J. Boor; Y. Grohn, Y. Schukken, J. Scarlett, P. McDonough, M. Smith (CU College of Veterinary Medicine), J. Bruce (Qualicon), W. Johnson, F. Rodgers, and D. Woodward (LCDC, Canada), J. Kiehlbauch, J. Hibbs, and D. Morse (NYSDOH), J. Schermerhorn, J. Corby (NYS Ag & Markets), J. Scott, D. Gombas (NFPA), P. Mead, B. Swaminathan (CDC), L. Kornstein (NYCDOH), T. Bannerman (Ohio DOH) and the Listeria Working GroupFinancial support: USDA-NRI, New York Sea Grant, USDA Special Research Grant, ILSI N.A., and NIH

Listeria monocytogenes includes common and widely distributed virulence attenuated clonal groups and epidemic clonesL. monocytogenes prevalenceFecal L. monocytogenes shedding in cattleL. monocytogenes ecology and persistence in processing plants Subtyping Results – seafood processing plant Subtyping Results - Plant II (cont.)L. monocytogenes persistence in plants2000 US outbreak - Environmental persistence of L. monocytogenes? Persistent L. monocytogenes environmental contaminationL. monocytogenes in retail environmentsL. monocytogenes diversity in different environmentsL. monocytogenes lineages - summary L. monocytogenes phylogeny (120 isolates)Neighbor Joining tree of prfA cluster (prfA, plcA, hly, mpl, actA, plcB)Recombination in L. monocytogenes housekeeping and virulence genesMolecular characterization of human, animal, and food isolatesOutbreak clonesPlaque assay to test L. monocytogenes invasion and cell-to-cell spreadHuman virulence attenuation of ribotype DUP-1062AL. monocytogenes with inlA premature stop codonsDose response differences among L. monocytogenes lineages and subgroups Virulence parameter for selected L. monocytogenes lineages and subgroups Summary and conclusionsAcknowledgments