2011 listeria handout

8/3/2019 2011 Listeria Handout

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Listeria monocytogenes:


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Required Reading Gandhi & Chikindas (2007) Listeria: A foodborne pathogen that knows how

to survive. International Journal of Food Microbiology, 113: 1-15.

Swaminathan & Gerner-Smidt (2007) The Epidemiology of human

listeriosis. Mixcrobes and infection, 9: 1236-1243.

Efstathios et al., (2007) Morphological changes in Listeria monocytogenes

subjected to sublethal alkaline stress. International Journal of Food

Microbiology, 120: 250258.

Thevenot et al., (2006). Serological and molecular ecology ofListeria

monocytogenes isolates collected from 13 French pork meat saltingcuring

plants and their products International Journal of Food Microbiology 112: 153-

161


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Mapel Leaf Outbreak statistics When: 2008 (June 3 Nov 22 08)

Who: elderly average age = 74 (29-98)

80% care home residents/ hospital patients41% in Ontario

Outcome: 57 cases; 22 deaths (40%).

Responsible: Maple Leaf Foods, Bartor Road, Ontario.

Actions: Recall > 220 products.


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Listeria monocytogenes Example of a zoonotic microbe

Gram +, facultatively anaerobic, catalase +,

oxidase -,

coccoid-rod shaped, motile (tumbling motilityat 20 25C)

Habitat: Ubiquitous in Environment


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FactorFactor

Temp (Temp (C)C) 0 42 (range) 30 35 (opt)

pHpH5.5 4.4 (min)


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A Biofilm is:

Community of cells attached to a surface and enclosed in a

matrix predominantly made up of polysaccharide material .

Monoculture or part of a mixed culture

Biofilm formation is a step-wise process involving:1. Adsorption

2. Attachment

3. differentiation.

It is affected by:

Type of surface,

Cell characteristics e.g. genetics and cell surface variation

Environmental parameters e.g. temperature and pH


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Quorum sensing (QS)

quorum sensing is a process of bacterial cell-to-cell

communication involving the production and detection of

extracellular signaling molecules called autoinducers.

Gram positive bacteria typically produce oligopeptides as

autoinducers, signaling between cells via a two-component

phospho-relay system.

Diffusible, low molecular weight autorepressor found inL.

monocytogenes restricts the expression of the PrfA virulence

regulon via a quorum sensing mechanism.


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1000 ppm free chlorine for 20 min reducedL. monocytogenes

biofilm by 2 Logs Planktonic cells were eliminated by an exposure to 10 ppm

free chlorine for 30 s

Enhanced production of extracellular polysaccharide inmultispecies biofilms, greater resistance to antimicrobial

agents than a monoculture biofilm.

Competitive-exclusion ofL. monocytogenesby organismswith anti-listerial activity gave a reduction of more than 5

log10 CFU


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Success ofL. monocytogenes as a pathogen

Survive passage through stomach (Ramalheira et al., 2010)

Evade the immune system hiding in macrophages

Form biofilms on food processing surfaces

Have shown the ability to become resistant to Antibiotics

Have shown the ability to withstand commercial sanitisers


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Disease progression

Incubation 1 70 d (median = 3 weeks) (YOPI)

Influenza-like symptoms including persistent fever

Septicemia, (50% MR)

*Headache

*Gastrointestinal (nausea, vomiting, diarrhoea )

*Intrauterine or cervical infections

*Spontaneous abortion (2nd/3rd trimester) (80% MR) *Stillbirth

13 34% MORTALITY RATE


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Incidence USA: 1,600 2,500 cases/ year 415 500 deaths/ year

IRELAND: 2-7 cases per year

Canada increasing year on year

Reported cases in Canada 2003-2008

0

50

100

150

200

250

300

2003 2004 2005 2006 2007 2008

year

Numbers

affected


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Implicated foods Raw milk, Cheeses (particularly soft-ripened varieties), ice

cream

Raw vegetables,

Raw and (under) cooked poultry, Raw meats (all types), fermented raw-meat sausages

Ready to Eat deli meats

Hot Dogs (served cold)

Raw and smoked fish

Pt and spreads


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Swaminathan and Gerner-Smidt (2007) Microbes and Infection, 9: 1236-43


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Examples of listeriosis outbreaks

Year Food No. Cases Place

2008 Sliced meat 57 (22d) Canada (ML)

2008 Cheese 38 (2d) Canada

2002 Cheese 30 British Columbia

1999 Pork tongue 26 (7d) France

1994 Chocolate milk 45 6 US states

1992 Pork tongue 279 (62d+22a) France

1981 Coleslaw 41 (18d) Nova Scotia


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Relative risk total US population per serving or per annum

Risk Per serving Per annum

High>5 cases/ billion

servings

100 cases/ annum

(VH)*

10-100 cases/ annum

(H)

Deli meats 1

Frankfurters (no reheat) 2

Pate + meat spreads 3

Unpasturised milk4

Smoked seafood 5RTE crustaceans 6

Deli meats* 1

Pasteurised milk2

High fat and other dairy prods. 3

Frankfurters (no reheat) 4

Moderate4 cases / billion servings

1-10 cases/annum

High fat + other dairy prods.7

Soft unripened cheese 8

Pasteurised milk9



Pate + meat spreads 6

Unpasturised milk7

RTE crustaceans 8Smoked seafood 9

Low


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Isolation and Identification

24 and 48 hours of enrichment (FDA)

Selective agar(Lithium chloride, phenyl ethanol, glycine, antibiotics)

Aesculin, Ferric ammonium citrate = Brown/Black

Sugar fermentation test

CAMP test (listeriolysin O, + S. aureus on blood agar)

ELISA PCR (specific non-radio labeled DNA probes)

Pulse Field gel electrophoresis

DNA microarray analysis

ISO 11290-1 - 5 D to confirm negative, 10D to confirm positive result.


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Strain Typing

Immunological typing / Serotyping

DifferentiatesListeria spp. into 13 serovars based on somatic

(O) and Flagellar (H) antigens.

Uses a series of polyvalent and monovalent antigens.

Serotypes 1/2a, 1/2b - most sporadic cases

Serotype 4b most outbreaks.

Not the most discriminatory method available.


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http://www.bio.davidson.edu/

Pulse Field Gel Electrophoresis (PFGE)


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Ecology ofL. monocytogenes during meat processing - France

French Pork meat implicated in outbreaks between 1992-2002

L. monocytogenes can grow at low temperatures

adapt to disinfectants/ antibiotics

form Biofilms

Persistent contamination in meat plants has been documentedover periods of months and years, in brine, knives and equipment.

Thevenot et al., (2006). International Journal ofFood Microbiology 112: 153-161


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13 dried Saucisson processing plants

1028 isolates ofL. monocytogenes characterised

1/2a 49.9%

1/2c 20.4%

1/2b 12%2b 8%

4e 0.8%

NT 8.7%

Serotype Results

= 83 pulse types

PFGE

Serotype distribution varied throughout processsing,

4b and 1/2b increased after processing 1/2 a and 1/2c decreased


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2, 1823, 2923238, 238, 12, 23, 73, 7512, 18, 23, 29, 43

-1, 6---1, 51

138---1, 2, 5, 8-

5. 323, 5, 9---1, 3, 5, 9, 10,16,

24, 32, 57

5, 5, 24

6, 411---6, 7, 8, 411

1, 7, 15, 28 1, 7, 287---1, 5, 7, 28

14, 22, 44, 59, 601, 6, 8, 53, 59, 62---1, 5, 6, 14, 15,

28, 40, 60, 63,

67

2, 5, 15. 28, 36, 25,

53,

23

-

-

-

-

-

-

During

Prodn

1, 5, 12, 23, 30,

37, 70, 73, 80,

74

1, 4, 32

1, 3, 6

1, 2, 5, 54

1, 2, 5, 24

1, 7, 8

1, 60

During

Prodn

23, 27, 71, 7623, 37, 51, 72--23, 29, 70H

2232---G

1, 3, 10, 16, 791, 3, 6--1, 3F

11---D

2, 5, 101, 2, 5--5, 16, 24C

7, 8----B

A 1, 28

Saucissons

1

Raw meat

-

Hands

-

Before

Prodn

Surface

(no meat)

1, 2, 25, 58

Before Prodn

Surface

( meat contact)

Plant

2, 1823, 2923238, 238, 12, 23, 73, 7512, 18, 23, 29, 43

-1, 6---1, 51

138---1, 2, 5, 8-

5. 323, 5, 9---1, 3, 5, 9, 10,16,

24, 32, 57

5, 5, 24

6, 411---6, 7, 8, 411

1, 7, 15, 28 1, 7, 287---1, 5, 7, 28

14, 22, 44, 59, 601, 6, 8, 53, 59, 62---1, 5, 6, 14, 15,

28, 40, 60, 63,

67

2, 5, 15. 28, 36, 25,

53,

23

-

-

-

-

-

-

During

Prodn

1, 5, 12, 23, 30,

37, 70, 73, 80,

74

1, 4, 32

1, 3, 6

1, 2, 5, 54

1, 2, 5, 24

1, 7, 8

1, 60

During

Prodn

23, 27, 71, 7623, 37, 51, 72--23, 29, 70H

2232---G

1, 3, 10, 16, 791, 3, 6--1, 3F

11---D

2, 5, 101, 2, 5--5, 16, 24C

7, 8----B

A 1, 28

Saucissons

1

Raw meat

-

Hands

-

Before

Prodn

Surface

(no meat)

1, 2, 25, 58

Before Prodn

Surface

( meat contact)

Plant

From: Table 3 from Thevenot et al 2006


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High pulsetype heterogeneity in each plant.

Increased throughput = increased variability

In some cases pulsetypes dissapeared, in others new pulsetypes

appeared during production.

Heterogeneity increased with time and processing

Persistence observed in several plants

A +F Knives contaminated with T21 + T8 respectively both days

C Knives and grinder contaminated with T24 + T5 respectively for21d.

H - mixing machine contaminated with T23 for >39d

Less frequent isolation from surfaces not in contact with meat

Main findings

2011 listeria handout

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