Post on 20-Feb-2022
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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