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Chapter 13

Listeria monocytogenes

Learning Objectives

1. Use basic biochemical characteristics to identify Listeria monocytogenes

2. Understand what conditions in foods favor the growth of L. monocytogenes

3. Recognize, from symptoms and time of onset, a case of foodborne illness caused by L. monocytogenes

4. Choose appropriate interventions to prevent L. monocytogenes growth

5. Identify environmental sources of L. monocytogenes6. Understand the role of L. monocytogenes toxin(s)

and virulence factor(s) in causing foodborne illness

Outbreak1998-1999 Sporadic outbreaks• Spring summer 1998

– hot dog plant had unusually high numbers of Listeria

• July 1998 – air conditioning unit thought to be the

source of Listeria was removed• October 1998

– CDC investigated 4 listeriosis cases from Tennessee

- a 74-yr old woman who loved hot dogs died- additional cases came in from other states

Outbreak1998-1999• November 1998

- a 31-yr-old camp counselor died in Ohio - genetic fingerprints of the strains were identical

so they must have eaten a similar food• Early December 1998

- hot dogs were statistically implicated as the cause• Mid December 1998

- hot dogs from Sara Lee’s Bil Mar Foods Plant were implicated

- 4 people dead- plant stopped shipping the hot dogs

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Outbreak1998-1999• 22 December 1998

- Sara Lee announce the recall of all meat processed at the Bil Mar plant

• 23 December 1998- outbreak strain of Listeria monocytogenes

was isolated from an opened package of BilMar hot dogs

- This meets USDA criterion for a recall• 25 December 1998

- another victim dies in New York

Outbreak1998-1999• January 1999

- USDA issued its first press release on the recall

- Did not help a 27-yr-old pregnant woman who gave birth to stillborn twins

- Listeria monocytogenes was isolated from the placenta

• May 1999- USDA advises immunocompromised not to eat

hot dogs unless they are thoroughly heated

Outbreak

• USDA typically issues 30 recalls for L. monocytogenes contamination each year

• The number of recalls is increasing• Food safety is a critical component of

food-testing processes for many companies

• Items are recalled as a precaution or reaction to specific tests

Listeriosis• In the past 25 years, listeriosis has become a

major foodborne disease• Primarily foodborne and not waterborne• Outbreaks have been linked to coleslaw,

cheeses, lunch meats, milk, chicken nuggets, and fish

• 1600 cases per year• 266 deaths each year• 3rd leading cause of death from food poisoning• Lunch meat contamination cost the US about 1.1

billion dollars each year

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Primary factors contributing to listeriosis

1. Listeria monocytogenesthrives in the coldwhere it outcompetes other bacteria that normally outcompete L. m. at warmer temperatures

Primary factors contributing to listeriosis

2. Changing population: more at risk people such as elderly or immunocompromised. L. m. is an opportunistic pathogen.

Primary factors contributing to listeriosis

3. Changing food production practices4. Increased use of refrigeration to

preserve food – L.m. grows better in the cold than other organisms

5. Changing eating habits – greater demand for fresh, unprocessed food without preservatives

6. Changing awareness and ability to detect outbreaks

Listeriosis

• Sporadic and rare and severe• Causes meningitis, septicemia, and

abortion• 15.9% of people who get listeriosis die

from it• Long lag from consuming the food to

illness

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Listeria monocytogenes• Differs from most other pathogens• Widely distributed in nature• Resistant to adverse environmental

conditions• Psychotrophic and grows in human

macrophages• Survives for a long time in or on food,

soil, plants, surfaces• Unclear: the illness is severe but making

all food Listeria free is unrealistic

Characteristics of Listeria• Joseph Lister invented

medical disinfection andhad a mouthwash and Genusnamed after him

• Nonpathogenic species are isolated from the same foods and environments as L. monocytogenes

• Thirteen serotypes of L. monocytogenes• Three serotypes predominate in listeriosis

patients• PulseNet established by the CDC helps

identify strains by DNA fingerprints

Susceptibility to physical and chemical agents

• Listeria monocytogenes grows from 0oC to 45oC but grows slowly at cold temperature

• Freezing does not reduce the numbers • Killed at temps greater than 50oC

Temperature (oC ) Doubling time –growth (hours)

4 4310 6.637 1.1

Susceptibility to physical and chemical agents

• Survives but does not grow at a pH below 4.3

• Organic acids, such as acetic, citric and lactic acids, inhibit growth

• Grows best at water activity of greater than 0.97 and survives at water activity of 0.83

• Heat resistance increases as the water activity decreases

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Susceptibility to physical and chemical agents

• Grows well and survives in 6.5% salt• Lowering temperature increases survival

in high salt• Control of Listeria is best achieved by

multiple hurdles and environmental monitoring

Listeriosis and foodsReady-to-eat foods

• Pose a high listeriosis risk for susceptible populations

• 20% of refrigerators have temperatures greater than50oF

• Refrigeration cannot ensure the safety of ready-to-eat foods

• Refrigeration is an environment where Listeria can outcompete mesophilic pathogens

Listeriosis and foodsMilk

• Raw milk is a source of L. monocytogenes• Pasteurization reduces the numbers of

Lm• Post-process milk contamination is a

concern as Listeria grows faster in pasteurized milk than in raw milk due to fewer competitors

Listeriosis and foodsCheeses

• L. monocytogenes survives cheese manufacturing and ripening

• Temperature hardy, grows in the cold and salt tolerant

• Soft cheeses are a risk factor for listeriosis

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Listeriosis and foodsMeat and poultry products

• L. monocytogenes depends on the type of meat, pH, presence of other bacteria

• Grows best in poultry• Concentrates in the kidney, lymph nodes,

liver and spleen of animals• Meats packaged in brine favor growth• Once established in a food plant Lm can

persist for years

Listeriosis and foodsSeafoods

• Isolated from fresh, frozen and processed seafood

• High-risk foods are molluscs and salted, marinated, fermented and cold-smoked fish as well as heat-processed fish products

Environmental Sources

• Ubiquitous in the environment• Survive and grow in many water

environments• Pasture grasses and grass silages• Feces of health animals; animals can get

listeriosis• Patients of listeriosis and carriers of

Listeria shed the microbe

Food Processing Plants• Enters food-processing plants via shoes,

clothing, and vehicles• Enters facility via raw vegetable, animal

tissue, and human carriers• High humidity and nutrient levels

promotes growth – floor drains, stagnant water, floors, residues, equipment

• Attaches to surfaces, stainless steel, rubber, and glass

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Food Processing Plants• L. monocytogenes contaminates carcasses from

feces during slaughter• 11-52% of healthy animals are fecal carriers• Heavily contaminated areas

- cow dehiding- pig stunning- hoisting

• Turkey and poultry slaughterhouses found Lmassociated with drip water, chill water overflow, and recycled cleaning water

Food Processing Plants• L. monocytogenes does not survive heat

processing• Processed food is contaminated

primarily by post-process contamination• Lm is difficult to remove from

processing plants as it forms biofilms in hard to reach areas

• Sanitation is difficult• Lm is found in raw materials so it is

easily reintroduced into facilities.

Circulation of Listeria monocytogenes

Potential Routes of Transmission of Listeria monocytogenes

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Prevalence and the Regulatory Status of L. monocytogenes

• Contamination of food is widespread• Leading cause of recalls

Prevalence and the Regulatory Status of L. monocytogenes

• Contamination levels- 0% in bakery goods- 16% in ready-to-eat foods- 60% in raw chicken

• Preserved but not heat treated foods are more often contaminated

• Isolated from numerous vegetables• There is a zero tolerance for Lm in ready-

to-eat foods but no detectable method that is 100% accurate!

International Debate on Regulatory Status

• Because it is so common, regulatory agencies in some countries conclude that it is impossible to have Lm-free foods- tolerance levels are established- products that have caused listeriosis

are regulated more strictly- foods intended for susceptible

populations must be free of Lm- other foods less than 100 CFU/gr of

food

International Debate on Regulatory Status

• US and the UK have a zero tolerance level- infective dose is unknown therefore

an acceptable tolerance is impossible to set

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International Debate on Regulatory Status

• Citizens petition to change US regulations- Argument: If Listeria ……

1. cannot grow in the food2. is not held at temperatures that

support its growth3. has a low probability of making

someone sick4. then there is no justification for

zero tolerance

International Debate on Regulatory Status

• Two additional arguments against zero tolerance

1. FDA has legal authority based on sound science- incidence of listeriosis in

Europe with a low tolerance is the same as that in the US

2. International trade requires harmony with microbial specifics- Preventing the import of food

based on tolerance is not legal

Human Carriers

• Listeria monocytogenes is identified in feces of 6% of healthy people with no symptoms

• 21% of patients with listeriosis had >104

bacteria per gram of feces• The same strain was shed by 18% of

housemates of a patient• Fecal carriers amplify outbreaks

through secondary transmission

Foodborne OutbreaksFirst demonstrated outbreakin 1981 in Nova Scotia • 6-month period• Coleslaw was implicated

- cabbage fertilized with manure from sheep suspected to have meningitis

- cabbage was stored over the winter and spring in an unheated shed

- cold provided a growth advantage for Lm• 34 pregnant women, 7 non-pregnant adults• Lm isolated from unopened coleslaw packages

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Foodborne OutbreaksMexican-style cheesein 1985 in California • 8-month period• 142 cases

- 93 pregnant women- 42 non-pregnant adults- most had predisposing conditions for

listeriosis• One-third died• Inadequate pasteurization of milk and mixing raw

milk with pasteurized milk caused the outbreak

Foodborne OutbreaksSoft cheese in Switzerland • 4 year period• 122 cases in Switzerland• 300 cases in the UK• Controlled by warnings to the public and

prevention measures in processing facilitiesDeli turkey meat in 10-states in the US• 6-month period• 29 cases

- 8 perinatal- 21 non-perinatal cases

• 4 deaths• 3 miscarriages or stillbirths

Disease Characteristics

• Listeriosis occurs in well-defined high-risk groups- pregnant women- newborns- immunocompromised

adults

Disease Characteristics

• Mortality rate is 20-25%• In non-pregnant adults, Lm causes .......

- septicemia- meningitis- meningoencephalitis

• In pregnant women, Lm causes- flu-like symptoms- stillbirth or abortion of the fetus

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Disease Characteristics• Most cases are sporadic, rare - 7.5 in a

million people• Mode of infection for most sporadic

cases is unknown• Difficult to investigate due to long

incubation period – up to 5 weeks• Need a better understanding of the

epidemiology of sporadic cases to develop better control strategies

Disease Characteristics• Human listeriosis is poorly understood• Exposure to Lm is common but

listeriosis is rare- high human resistance- low pathogenicity of strains

• Although Listeria seems to target pregnant women, some pregnant woman have asymptomatic carriage of Lm and have normal pregnancies

Disease Characteristics• Liseria monocytogenes also causes

feverish gastroenteritis- affects people with no predisposing

risks- infection dose is higher (105 to 109

CFU per gram) than for the invasive listeriosis

- Symptoms appear within several hours as opposed to weeks later

Infectious Dose

• Depends on- the immunological status of the

human- the virulence of the strain- the food

• > 100 CFU/g in most foods responsible for outbreaks

• More epidemiological data are needed to accurately determine the dose

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Virulence factors and mechanisms of pathogenicity

• Unique to other foodborne pathogens that produce toxins or multiply in the blood

• L. monocytogenes enters the host cell, grows inside the host cell, and passes directly into neighboring cells

• Cell-cell transmission reduces exposure to antibiotics and circulating antibodies

Intracellular life cycle of L. monocytogenes

I. cell entry mediated by invasion factors InlA or InlBII. escape from phagolysosome by LLO and PlcAIII. actin recruitment and replication

IV. intracellular movement due to polarized actin-polymerization mediated by ActA

V. cell-to-cell spread by formation of listeriapodsVI. subsequent lysis of the two-membrane vacuole by LLO

and PlcB

Intracellular life cycle of L. monocytogenes Intracellular movement and cell-to-cell spread of L. monocytogenes cells (green) driven by the polarized polymerization of actin tails (red)

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Pathogenicity

• When mice are exposed, infection lasts for about 1 week

• 10 min following infection, bacteria are taken up by the liver and spleen

• During the first 6 hours, viable listeriae decrease in number

• Surviving listeriae infect susceptible macrophages and multiply exponentially

• After 3-4 days, the host may recover

Pathogenicity

• In humans, infection occurs via ingestion• L. monocytogenes

- crosses the intestinal barrier- internalized by macrophages- replicates within macrophages- transported to the lymph nodes via the

blood• Most are killed when they reach the liver

and spleen• Survivors travel to the brain or placenta

Specific genes mediate pathogenicity

• Internalin (inlA)- thermoregulated- expressed more at 37oC than at 20oC- plays a role in forming the initial

phagosome• hly gene encodes lysteriolysin (LLO), an

enzyme that breaks open the phagosome to release Listeria

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Specific genes mediate pathogenicity• actA gene codes for ActA

- located at one end of the bacterium- makes actin propel

the bacterium forward- actin pushes the cell

across the infected host’smembrane and into the membrane of the adjacent cell

- mimics Wiskott-Aldrich syndrome protein (WASP), a nucleation promoting factor present in host cells

• plcB gene encodes a membrane-hydrolyzing enzyme that helps lysteriolysin free the bacterium in the adjacent human cell

Summary• L. monocytogenes is a ubiquitous organism

that can grow at refrigeration temperatures

• Relatively few cases of listeriosis per year (5000/year) but there is a high fatality rate among those that have the disease

• L. monocytogenes is relatively resistant to dehydration, low pH, and low water activity

• Ready-to-eat foods preserved by refrigeration pose a special challenge with regard to L. monocytogenes

Summary• Asymptomatic and symptomatic people shed L.

monocytogenes in their feces• US has zero tolerance for L. monocytogenes in

food whereas Europe has some tolerance for its presence

• Outbreaks of listeriosis extend over a long period, caused by refrigerated foods, have high case fatality rates, and involve a high number of pregnant women

• L. monocytogenes grows inside the host cell and passes directly into neighboring cells

• Internalin, listeriolysin, ActA, and a membrane-hydrolyzing enzyme PlcA and PlcB are 5 major proteins that govern the pathogenicity