Food-Borne Infections and Intoxications:

• Listeria monocytogenes

• Campylobacter jejuni

• Clostridium botulinum

• Clostridium perfringes

• Bacillus cereus

• Vibrio cholera

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Background and History 3,000,000,000 years ago First Bacteria

3,000,000 years ago First Humans

20,000 years ago Agricultural societies

10,000 BC Soups, breads, beerWinecooking, drying,smoking, freezing,marinating, salting,and pickling

5,000 BC Yogurt, cheesesDietary Laws

2,000 BC Popcorn Chocolate

900 AD Sausage Outlawed 1202 Assize of Bread 1850 – 1900 Handwashing

PasteurizationGerms discovered

1904 Typhoid Mary 1916 Refrigeration 1960 Hazard Analysis and

Critical Control Point(HACCP)

1984 Rajneeshees1993 Jack in the Box E.

coli 2005 Staph and money

Overview of foodborne illness case #'s, illnesses, hospitalizations and deaths -

(Table 3)

Listeria monocytogenes

• Gram positive rods– Faculative anaerobe– No spores– No capsule– Motile 10-25o C– Closely related to Bacillus, Clostridium,

Enterococcus, Streptococcus and Staphylococcus

– Genus has 6 species-- L. monocytogenes and L. ivanovii are pathogenic

Listeria monocytogenes

• Major public health concern because:

– Severe, non-enteric nature of the disease:

– High case fatality rate can be as high as 20-30%

– Long incubation time

Listeria monocytogenesRisk groups

Pregnant women and neonates

Elderly

Immunocompromised or debilitated people:Malignancy, antineoplastic treatment, immunosuppressed, chronic liver disease, collagen diseases (lupus), diabetes, AIDS

Listeria monocytogenes

• Properties of the Organism:

• Habitat and sources -- widely distributed! - (next slide)

• Bottom line Many foods have been implicated but foods marketed as refrigerated and ready to eat are the ones that have been associated with most of the outbreaks.

• Human Carriage

Listeria monocytogenes• Habitat and sources

Listeria monocytogenes

• The organism is beta hemolytic and is easily confused with hemolytic streptococci. Listeria may also grow in short chains. Do catalase test!

Listeria monocytogenes

• Growth and Laboratory Characteristics– Facultative anaerobe – The organism is -hemolytic.– It is catalase positive– It is a gram positive rod– It is psychrotropic– The organism is motile

Listeria monocytogenes

• Clinical signs are similar in all hosts:– Perinatal listeriosis– Adult listeriosis

• Both are disseminated infection often with CNS involvement

Listeria monocytogenes

• Neuromeningeal listeriosis in sheep -- Circling Disease

Listeria monocytogenes

• Listeriosis in sheep -- pyogranulomatous lesions

Listeria monocytogenes

• Human stillborn -- Granulomaosis infantiseptica

Listeria monocytogenes• The Disease Entity:

Listeria monocytogenes

• Brain lesions in sheep

Listeria monocytogenes

In addition to professional phagocytes such as macrophages, these organisms can invade a number of cell types:

• Epithelial cells

• Fibroblasts

• Hepatocytes

• Endothelial cells

• Neurons and possibly other neural cells

Listeria monocytogenesInternalization:

• Host factors– E-cadherin, – C-Met– Globular C1-q receptor (complement receptors),– glycosaminoglycans– fibronectin and integrin

• Listeria adhesins: – Internalin A – Internalin B. – p60 – Ami – Lap – fibronectin binding protein (24.6 kDa).

Listeria monocytogenes

• Internalin A (InlA)– Binds to E-cadherin in the adherens junction, triggering actin cytoskeleton

rearrangements via association with catenins

Listeria monocytogenes

• Internalin A (InlA)– 800 aa with 14 N-

terminal leucine rich repeats

– Leucine-rich repeat structure on a serine/threonine receptor kinase

– Binds to E-cadherin in the adherens junction,

Listeria monocytogenes

• Internalin A (InlA)– 800 aa with 14 N-

terminal leucine rich repeats

– Binds to E-cadherin in the adherens junction, triggering actin cytoskeleton rearrangements via association with catenins

Listeria monocytogenes

• Vacuole formation, proliferation and spread:

• phagosome formation, lysis and release

• Listeria hemolysin Hly (also known as listeriolysin or LLO)

• Listeria phospholipases: PlcA and PlcB (phospholipase C A and B):

Listeria monocytogenes

• Phagocytic Vacuole formation– Phagosome acidifies

– Lysosome fusion inhibited

Listeria monocytogenes

• Escape from the Phagocytic Vacuole– Listeria hemolysin,

(Hly, Listeriolysin, LLO),

• Cholesterol dependent, pore forming weak cytolysin

Listeria monocytogenes

• Escape from the Phagocytic Vacuole– Listeria phospholipases: PlcA

and PlcB (phospholipase C A and B)

– Role in escape from the phagosome

– Escape from the double membrane vessicle in cell to cell spread

– Subvert host cell signalling pathways

Listeria monocytogenes

Listeria invasion

Listeria invasion

Gastroenteritis

Gut Associated Lymphatic Tissue

Peyer’s Patch

M Cell

Campylobacter jejuni

• Campylobacter jejuni is the leading cause of gastroenteritis in the US and probably world-wide. .

• Hawai'i has the highest incidence in the country about 900 reported cases a year with an incidence of 75/100,000, but the thinking is that infections are grossly underreported.

Organism Reservoirs Human Disease

C. jejuni Humans, birds, other mammals

Diarrhea, systemic illness, GBS

C. coli Pigs, birds Diarrhea

C. lari Birds, dogs Diarrhea

C. fetus Cattle, sheep

Systemic illness, diarrhea

Other related organisms include:

C. sputorum, C. concisus, C. curvus, C rectus, C. showae. Arcobacterium butzleri, A. cryaerophilus, A. skirrowii

Campylobacter jejuni

Properties of the organism• Curved s-shaped gram (-) rods, motile with a single

polar flagellum at one or both ends.• Defined "viable but not culturable" state.• respiratory metabolism, microaerophilic. • Grow with 10% CO2 / 5% O2 . Some species /

strains require additional H2 in the atmosphere• C. jejuni will grow at 42o C and this is used as a

selection criterion.• The organism is unusually thin (0.2 - 0.9 )

Campylobacter jejuni

Campylobacter jejuni

Reservoirs and epidemiology

• Human cases are associated with:

• Poultry - especially eating chicken out

• Pets - especially young puppies

• Water supply

• Raw milk

• Most cases occur in the summer months -- late spring to early autumn -- this is also true in Hawai'i.

Campylobacter jejuni

Campylobacter jejuni

Pathogenesis and Disease Characteristics– Low infectious dose– Two disease entities:

• Diarrhea

• Dysentery

– Associated with Guillaine-Barrè syndrome

Campylobacter jejuni

• Virulence factors• Cj can invade intestinal epithelial cells.• Cj secretes a number of novel proteins upon cultivation with

enterocytes:– CiaB

• pVir, present only in some strains of Cj appears to be important for invasion.

• Microtubule mediated endocytosis occurs• Cj apparently stays within vacuole

• adenyl cyclase activating cholera toxin-like enterotoxin

• cytotoxin

Campylobacter jejuni

• Virulence factors• Microtubule mediated endocytosis occurs and microfilament mechanism may be

involved too

• Microtubules aggregate into finger-like protrusions with C.j. at the tips

Campylobacter jejuni

• Virulence factors– Cytolethal Distending Toxin

• Irreversable cell cycle arrest

• All three CDT genes need to be expressed for activity

– Adenyl cyclase activating enterotoxin?

Campylobacter jejuni

• Guillaine-Barrè Syndrome– Ascending muscle weakness or paralysis,

rapidly progressing– 40% of GBS patients have evidence of

Campylobacter infection– Penner 1,2,8,17,19,41 are disproportionately

represented– LPS oligosaccharides structurally related to

human motor neuron gangliosides

Bacillus cereus

• Causes two types of foodborne illness:

• Diarrheal disease

• Emetic disease

Bacillus cereusSpecies Colony

morph.Hemolysis Motility Crystal

Inclusions

B. cereus white + + -

B. anthracis

white - - -

B. thuringensis

White or gray

+ + +

B. mycoides

Rhizoid (+) - -

Bacillus cereus•Gram positive large (width > 1 um) rod,•spore former -- central spore or paracentral•grows aerobically and anaerobically•beta hemolytic •usually motile•may be present in stools of healthy individuals•grown out of food samples after heat shock --> treat sample at 70o C for 10 minutes; or after ethanol shock --> mix 1:1 with absolute ethanol for 1 hour•Widely disseminated in nature

•Rice, spices and dairy products

Bacillus cereus

• Disease entities:

• Two types of food borne illness:

• Emetic --- emetic toxin, food intoxication» circular peptide, 1.2 kDa, called cereulide

» Stimulates the vagus nerve leading to the emetic response.

• Diarrheal --- enterotoxin, food infection» At least three enterotoxins have been described

Bacillus cereus

Diarrheal vs.Emetic Disease

Characteristic Diarrheal Emetic

Infective Dose 105 - 107 cells 105 - 108 per g

Toxin produced: In small intestine

In food

Toxin type Protein-enterotoxin

Cyclic peptide

Incubation period

8-16 hours 30 min-5 hours

Duration 1 to several days 6-24 hours

Bacillus cereusDiarrheal vs.Emetic Disease

Characteristic Diarrheal Emetic

Symptoms Abd pain, watery diarrhea, some nausea

Nausea, vomiting,malaise, diarrhea if enterotoxin also produced

Foods Meat, soups, vegetables, puddings, sauces, milk

Fried rice, cooked rice, pasta and noodles, pastry

Clostridium botulinum

• Gram positive rod• anaerobic• spore former• seven types based on serologic specificity of

neurotoxin named A through G

– A, B, E and sometimes F --> causes of human botulism

– C and D ---> animal botulism, contaminated feed.

– G ---> no clear association with disease

Clostridium botulinum

Clostridium botulinumReservoirs

Clostridium botulinumFood Sources

Clostridium botulinum

• Disease Characteristics:

• Symptoms hit 12-36 hours after ingestion (sometimes sooner, sometimes weeks later!)

– nausea and vomiting (B and E)– visual impairment: blurred, ptosis, dilated pupils– loss of mouth and throat function (A and B)– dry mouth, throat, tongue, sore throat– fatigue and loss of coordination– respiratory impairment– abdominal pain and either diarrhea or constipation

Clostridium botulinum

• Infant Botulism:

– constipation --- days to week after onset

– generalized weakness and a weak cry

– poor feeding and sucking reflex

– lack of facial expression

– floppiness

– respiratory arrest may occur although death is rare.

Clostridium botulinum• Virulence Factors• Neurotoxin (BoNT)

– water soluble– produced as a single polypeptide --- 150,000 MW (progenitor)– cleaved by a protease to form two polypeptides which then

become S-S bonded : 100,000 and 50,000 MW– There are differences in serotypes:

• A=dimer, trimer, and can be larger• E= monomer and dimer

– B= dimer– A,B,E, F are chromosomally encoded

• C, D are phage encoded• G is plasmid encoded

Clostridium botulinum- architecture of the motor end

plate

Synaptic cleft

Muscle cell

Motor neuron

Clostridium botulinum

Synaptic vessicle

Synaptobrevin (VAMP):BoTox B, D, F, G

Synaptic cleft

SNAP-25:BoTox A, E

Syntaxin (HPC-1):BoTox C

Motor end plate of the muscle cell

Clostridium perfringes

• Type A food poisoning

• Necrotizing enteritis (Enteritis necroticans) also known as Darmbrand or Pigbel - caused by Type C

• Gram positive rod• Spore forming

– anaerobic but tolerant of some exposure to air– under optimal conditions, is capable of doubling every 10

minutes

• ubiquitous distribution --- feces and soil

Clostridium perfringes

• Type A

• Symptoms 8-24 hours after ingestion• Resolution 12-24 hours later

• Diarrhea and cramps (severe)• No vomiting• No fever• May be serious in the elderly

Clostridium perfringes

• Infection --- organisms multiply and then sporulate in the small intestine.

• CPE (Clostridium perfringes enterotoxin) is released during the sporulation process.

• CPE is a single polypeptide, 35000 Da– heat labile (destroyed by heating 5’ at 60o C)

– Binds to membrane receptor which involves 2 membrane proteins on the target cell --- 50 and 70 kDa -- (Fig 7 and 8).

– Inserts into the membrane and is believed to cause a membrane lesion which then alters permeability -- fluid and electrolyte loss and damage to the epithelium. Glucose is still absorbed.

Clostridium perfringes

Clostridium perfringes

Vibrio cholera

Whoops !

sorry

Overview of foodborne illness case #'s, illnesses, hospitalizations and deaths -

(Tables 1 and 2)

Overview of foodborne illness case #'s, illnesses, hospitalizations and deaths -

(Tables 1 and 2)