The following notes are from 2003. Please see powerpoint for updated notes
Food-Borne Infections and Intoxications:
Background and History -
Great moments in food spoilage and food poisoning
Preservation by drying, adding salt, development of acid fermented foods, coating foods with honey, clay, olive oil.
Development of dietary laws -- India 1000 BC -- unclean foods included meat cut by sword, dog meat, human meat, meat of carnivorous animals, locusts, camels and hairless or excessively hairy animals. Rice which had turned sour, dishes which had been sniffed by a dog or a cat.
Jews and Muslims -- many dietary laws carrying the weight of religious sanctions but which are really laws of simple hygiene.
About 900 AD, Emperor Leo VI of Byzantium -- edict forbidding the making and eating of blood sausage prepared in pig stomachs and smoked --- threat of losing all property and exile. Chief magistrate of the city of the offense would be fined 10 pounds of gold --- equivalent to about $35,000 in todays value.
Spoiled grains recognized from at least Roman time.....over 40,000 deaths due to ergot poisoning (St. Anthonyâs fire) alone in France in 943 AD.
Canning discovered in 1805 by Francois (Nicholas) Appert induced by a 12,000 franc reward for the discovery of a practical method of food preservation --- preserved meats in glass jars kept in boiling water for varying periods of time.
50 years later Louis Pasteur demonstrated the role of microorganisms
in the spoilage of beer and wine. Who also demonstrated that the souring
of milk was due to microorganisms. This of course led to his use of heat
to destroy these spoilage organisms --- the discovery of pasteurization.
Minimum infective dose is unclear:
contaminated foods responsible for epidemic and sporadic cases indicate levels of at least 100 cfu per gram of food. But this is controversial some investigators say 10,000 to 100,000.
Entry and crossing of Host tissues
1. Crossing the intestinal barrier
the organism has to withstand stomach environment
there is some evidence that antacids and H2 blockers increase the risk for Listeriosis
organisms then enter the lumen of the small intestine
if the infectious dose was high, might experience gastroenteritis with febrile symptoms. (There is a question if organism actually multiplies in the lumen).
There are no gross lesions associated with the enteritis.
Translocation/Invasion of intestinal mucosa -- occurs quite rapidly -- in rats (ileal loop model) deep tissue invasion occurs within minutes-- which also implies that there is no multiplication necessary for this process.
Invade the apical epithelial cells of the villi and also the M-cells
And the organisms are quickly found within the macrophages
in the stroma and in the peyer's patches.
2. Multiplication in the liver
Organisms are carried by lymph and blood to lymph nodes,
spleen and liver. Some estimate that 90% end up in the liver.
This begins to happen within minutes of intestinal traversion.
Many localize to Kupffer cells and most organisms appear to die within these cells. (Induction of antigen specific T-cells and cell mediated immunity.) In most people, this is probably where the infection stops.
If some organisms survive this compartment, they begin to multiply within the Kupffer cells over the next 2-5 days.
During the course of the evolving infection, the organisms spread to hepatocytes -- which becomes the principle site of bacterial multiplication. Organisms spread cell-to-cell and never come in contact with humoral immunity.
Polys accumulate at distinct microabcesses around foci of infected hepatocytes over the next several days. These are gradually replaced by mononuclear cells and lymphs to form granulomas. Infection may clear at this point due to IFN-g activated macrophages and CD-8 antigen specific lymphs.
If infection not controlled here, will next get release of bacteria into circulation to get septecemia, multiple organ infections, with particular tropism for gravid uterus and CNS.
3. Colonization of gravid uterus and fetus
This has been reproduced in a variety of experimental animals including sheep, cattle, guinea pigs, rats and mice -- hematogenous penetration of the placental barrier:
Inflammatory infiltration of placental villi with microabcess formation and some necrosis. Eventual translocation across the endothelial barrier in the trophoblast and invasion of the fetal bloodstream.
Mother may be assymptomatic or exhibit flu-like symptoms. Mother is not at particular risk for systemic infection.
Get generalized infection, fetal death or premature birth of severely infected neonate.
Humans and other animals seem to be most susceptible late in pregnancy and it appears to be related to late pregnancy high physiological levels of estrogens -- these have been shown to depress aspects of cell mediated immunity.
4. Invasion of the brain
Organism has a well described predilection for the neural tissue. Seen most clearly in ruminants where disease presents as encephalitis (in humans its primarily a meningitis). In ruminants, often see focal infections in the pons, cerebellum, medulla oblongata and spinal cord.
How the organisms get into the brain is controversial.
Some say its hematogenous - since the organisms are known to be able to invade
endothelial cells and could hence be able to invade through the brain microvasculature.
Other investigators have shown that the organisms can invade neurons and
that intra-axonal transport is possible. Ruminants may get infected through
the trigeminal nerve after infection of the oral mucosa.
Intracellular Infectious Cycle and Virulence Factors:
In addition to professional phagocytes such as macrophages, these organisms can invade a number of cell types:
Neurons and possibly other neural cells
Organism adheres to surface of target cells and sinks into the target cell through a zipper-like mechanism. Doesn't involve the spectacular ruffling of Salmonella or any of the other membrane structures seen with other bacteria ie., pedestals in E. coli.
A variety of target cell receptors seem to be able to play a role including complement receptors, fibronectin and integrin, and E-cadherin
The most important Listeria adhesins appear to be Internalin A and Internalin B. -- 800 aa membrane anchored, cell surface proteins which play role in the internalization process.
Internalin A binds to E-cadherin (a calcium dependent intercellular adhesion glycoprotein whose cytoplasmic domain can trigger actin cytoskeleton rearrangements via a - and b -catenins)
Internalin B has recently been shown to have affinity for two eucaryotic surface proteins (1) Met - a receptor tyrosine kinase which normally functions as the receptor for hepatocyte growth factor. Binding to Met signal actin rearrangements through phosphatidylinositol-3 (PI3); and (2) globular C1-q receptor -- though this doesn't appear to have a signal domain so its significance is ??
p60 is a 460 aa, 60 kDa extracellular protein found associated with the cell wall and also in culture supernatants. It maybe a murein hydrolase important for proper cell division. The protein does bind to intestinal epithelial cells and mutants defective in the protein production are not invasive
Other adhesins: Ami, Lap, fibronectin binding protein (24.6 kDa).
Vacuole formation, proliferation and spread:
Listeria becomes engulfed into a phagosome and the vacuole becomes acidified soon after uptake and there is evidence that the phagosome is prevented from fusing with lysosomes.
Within thirty minutes of entry, the phagosomal membranes begin to lyse, a step essential to bacterial survival (and virulence) within the cell and a step mediated by hemolysin in combination with phospholipases:
The Listeria hemolysin Hly (also known as listeriolysin or LLO) is a streptolysin O related, cholesterol dependent, pore forming cytolysin. Mutants without Hly are avirulent · again underscoring its essential nature to the intracellular survival of this organism.
Active only at low pH. Has a narrow range: pH 4.5 - 6.5. This apparently insures that the toxin will lyse the phagosome but will not affect cell membranes once the bacterium is free in the cytoplasm. This hemolysin is noted for not being particularly cytotoxic.
Listeria phospholipases: PlcA and PlcB (phospholipase C A and B):These are responsible for the lecithinase activity also correlated with virulence. These also have a role in escape from the phagosome and also in escape from the double membrane vacuole which forms when cells spread cell-to-cell. Also there is evidence that these subvert host signalling pathways mediated by phospholipid hydrolysis products such as diacylglyceride, ceramide and inositol phosphates -- lipid metabolites that play key roles in important cell processes such as growth, apoptosis and the synthesis of cytokines and chemokines.
Once in the cytoplasm the bacteria multiply with a doubling time of ca. 1 hour.
Intracytoplasmic bacteria are immediately surrounded by a cloud of fine, fuzzy, fibrillar material composed of actin filaments which rearranges into a tail of up to 40 um at one pole of the bacterium. -
The polar assembly of the actin tail propels the bacterium in a random fashion through the cell.
Actin assembly is mediated by one protein, ActA, a 610 aa surface protein - distributed assymetrically on the bacterial surface.
Motile force is believed to be due to continuous deposition of actin monomers at the bacterial cell surface -- between the bacterial surface and the growing actin meshwork immobilized in the cytosol. The tangled actin meshwork, containing many actin cytoskeleton binding, crosslinking and regulatory proteins -- such as a -actinin, tropomyosin, talin, vinculin, fimbrin, filamin, villin, gelsolin, ezrin/radixin, cofilin, frofilin, coronin, Rac, CapZ, Arp3, and VASP.
Some bacteria eventually reach the periphery, contact the
cell's membrane, and push it out. This leads to finger-like protrusions which
penetrate neighboring cells and become phagocytosed by them. Resulting in
a secondary phagosome with a double membrane. Bacteria escape from these quickly
presumably using hemolysin and phospholipases.
Campylobacter jejuni is the leading cause of gastroenteritis in the US and probably world-wide. .
Properties of the organism
Reservoirs and epidemiology
Disease botulism named in 1793 after 13 people in Wildbad, Germany ate a large sausage and became ill --> 6 died. Botulus is sausage in Latin.
Organism isolated by Van Ermagen in 1896 after an outbreak of disease in members of a music club who had eaten salted ham --> 23 got sick and 3 died.
Gram positive rod
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
The species is also divided into 4 groups :
II = all E's and non-proteolytic B and F's - these are all non-proteolytic
III = C and D - don't cause human botulism but will in animals
IV = G (which is also called C. argentinense) - rarely form spores
refrigeration -- although growth can occur at low temp, toxin production is reduced
thermal inactivation (group I is most heat resistant)
pH -- acid foods donât allow growth
salt concentration -- affects water activity (aw)
(I) tolerates up to 10% salt = 0.94 aw
(II) tolerates up to 5% = 0.97 aw
These organisms need an anaerobic environment to grow but reducing substances in food will allow growth in the presence of O2.
Widely distributed in nature and in food
Home preserved vegetables --- "A and B"
Home cured meats --- "B"
Commercial products --- garlic in oil (now has to be acidified as an additional safety feature.) Roasted eggplant in oil responsible for several outbreaks.
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
abdominal pain and either diarrhea or constipation
(in general , cranial nerve first affected and then descend. With GBS its just the opposite --> extremities first and then ascend.)
Death would be due to respiratory failure....10% fatality rate
Can be confused with CO poisoning and GBS
constipation --- days to week after onset
generalized weakness and weak cry
poor feeding and sucking reflex
lack of facial expression
respiratory arrest may occur although death is rare.
0.1 ng/kg is lethal for mouse
active log "A" strains can produce 106 mouse lethal doses per ml.
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
A,B,E, F are chromosomally encoded
C, D are phage encoded
G is plasmid encoded
These exist in association with a number of nontoxic proteins
(hemagglutinin) which may protect the toxin from low stomach pH an proteases.
All serotypes block the exocytic release of acetylcholine from synaptic vessicles at perpheral motor nerve terminals.
H-chain binds to motor neurons, probably through glycoprotein receptor.
Receptor mediated endocytosis internalizes the toxin.
H-chain forms channel in endosome and L-chain dissociates and passes into cytoplasm
L-chain acts as zinc-dependent endopeptidase which reacts with components of the synaptic vessicle docking and fusion complex:
synaptobrevin (VAMP, vessicle associated membrane protein)
SNAP-25 (synaptosomal associated protein of 25 kDa)
Causes 2 distinct entities:
Necrotizing enteritis also known as Darmbrand or Pig-Bel (associated with Type C C. perfringes)
anaerobic but tolerant of some exposure to air
under optimal conditions, is capable of doubling every 10 minutes
ubiquitous distribution --- feces and soil
Temperature abuse during cooking, cooling or holding of food
Characteristics of the disease
Symptoms 8-24 hours after ingestion
Resolution 12-24 hours later
Diarrhea and cramps (severe)
May be serious in the elderly
have to eat alot --- 106 - 107 organisms per gm of food
CPE (Clostridium perfringes enterotoxin) is released during the sporulation process.
heat labile (destroyed by heating 5â at 60o C)
Binds to membrane receptor which involves 2 membrane proteins on the target cell --- 50 and 70 kDa.
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.
Causes two types of foodborne illness:
Emetic disease - food intoxication caused by toxin released into food.
spore former -- central spore or paracentral
grows aerobically and anaerobically
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 ---> soil and growing plants ---> spread to food is easy.
easy to cross contaminate meats
Rice, spices and dairy products are widely contaminated with this organism....
Turns out that it is a circular peptide, 1.2 kDa, called cereulide --- closely related to the potassium ionophore valinomycin.
(Like valinomycin, cereulide can disrupt mitochondrial function and at least one group has proposed using boar spermatozoa as a bioassay for the toxin --- the toxin uncouples ox-phos in the sperm stopping motility.) -- the mitochondrial toxicity may be related to reported liver toxicity of this toxin.)
Stimulates the vagus nerve leading to the emetic response.
At least three enterotoxins have been described, one of them has hemolytic activity the others do not.