Gram positive spore forming bacilli

Bacillus

Clostridium

Bacillus

Bacillus species are aerobic, sporulating, rod-shaped bacteria that are ubiquitous in nature.

Bacillus anthracis, the agent of anthrax, is the only obligate Bacillus pathogen in vertebrates.

Structure and Classification

The family Bacillaceae, consisting of rod-shaped bacteria that form endospores, has two principal subdivisions:

- the anaerobic spore-forming bacteria of the genus Clostridium,

- and the aerobic or facultatively anaerobic spore-forming bacteria of the genus Bacillus frequently known as ASB (aerobic spore-bearers).

Structure and Classification

Bacterial cells of Bacillus cultures are Gram positive when young, but in some species become Gram negative as they age.

Bacillus anthracis - Gram staining

Bacillus anthracis – culture smear (spores and chains arrangement)

Bacillus anthracis – culture smear (spores and Gram negative appearance)

Bacillus anthracis – spore’s structure

Structure and Classification

Most Bacillus species are saprophytes. Classification – using characteristics of

some of the species most likely to be encountered by the physician.

Classi-fication

Pathogenisis

The pathogenicity of B. anthracis depends on two virulence factors:

- capsule, which protects it from phagocy-tosis by the defensive phagocytes of the host,

- toxin produced in the logarithmic phase of growth.

Pathogenisis

This toxin consists of three proteins:- protective antigen (PA), - lethal factor (LF), - edema factor (EF).

Pathogenisis

Host proteases in the blood and on the eukaryotic cell surface activate protective antigen (PA), exposing a binding site for LF (lethal factor) and EF (edema factor).

An active polypeptide binds to specific receptors on the host cell surface, thereby creating a secondary binding site for which LF and EF compete.

Pathogenisis

The complex (PA+LF or PA+EF) is internalized by endocytosis and, following acidification of the endosome, the LF or EF cross the membrane into the cytosol via PA-mediated ion-conductive channels.

This is analogous to the A-B structure-function model of cholera toxin with PA (protective antigen) behaving as the B (binding) moiety.

Pathogenisis

Pathogenisis EF, responsible for the characteristic

edema of anthrax, is a calmodulin-dependent adenylate cyclase.

(Calmodulin is the major intracellular calcium receptor in eukaryotic cells)

The only other known bacterial adenylate cyclase is produced by Bordetella pertussis, but the two toxins share only minor homologies.

LF appears to be a zinc-dependent metalloprotease (though its substrate and mode of action have yet to be elucidated).

Pathogenisis

The toxin and capsule of B. anthracis are encoded on two large plasmids called

pXO1 and pX02, respectively. Strains lacking either of these plasmids have

greatly reduced virulence.

Pathogenisis

The attenuated live vaccine strain developed by Sterne in 1937, which is still the basis of most anthrax vaccines for livestock, lacks pX02 and is therefore Cap- Tox+.

The protection afforded by such vaccines apparently is related primarily to antibodies specific for the protective antigen component of the toxin.

Pathogenisis

In contrast, the attenuated vaccine strains developed by Pasteur (in 1881) were inadvertently cured of pXO1 (by subculturing at 42° to 43°C).

These Pasteur strains are therefore Cap+ Tox-. Strains of this type do not induce protective immunity. The partial effectiveness of Pasteur's vaccines is now

believed to have been due to the residual uncured (Cap+ Tox+) cells they contained, and this would also explain the partial virulence of these strains.

Host Defenses

Anthrax has been documented in a wide variety of warm-blooded animals.

Some species, such as rats, chickens, and dogs, are quite resistant to the disease, whereas others (notably herbivores such as cattle, sheep, and horses) are very susceptible.

Humans have intermediate susceptibility. The specific mechanisms of resistance in the

more resistant species are not known.

Host Defenses

Protective immunity against anthrax requires antibodies against components of anthrax toxin, primarily protective antigen.

Both the noncellular human vaccines and live-spore animal vaccines confer protection by eliciting antibodies to protective antigen.

The poly-D-glutamic acid capsule of B. anthracis is poorly immunogenic, and antibodies to the polysaccharide and other components of the cell wall are not protective.

Host Defenses

Nothing is known about immune responses to food poisoning or other types of infections with Bacillus species other than B. anthracis.

These types of infection are rare, and effective vaccines against them have not been developed.

Clinical Manifestions

Although anthrax remains the best-known Bacillus disease, in recent years other Bacillus species have been increasingly implicated in a wide range of infections including:

abscesses, bacteremia/septicemia, wound and burn infections, ear infections, endocarditis, meningitis, ophthalmitis, osteomyelitis, peritonitis, and respiratory and urinary tract infections.

Clinical Manifestions

Most of these occur as secondary or mixed infections or immunodeficient or otherwise immunocompromised hosts (such as alcoholics and diabetics), but a significant proportion are primary infections in otherwise healthy individuals.

Clinical Manifestions

Some of these infections are severe or lethal. Of the Bacillus genus species (so called

antrachoids), most frequently implicated in these types of infection is B. cereus, followed by B. licheniformis and B. subtilis.

Bacillus alvei, B brevis, B circulans, B coagulans, B macerans, B pumilus, B sphaericus, and B thuringiensis cause occasional infections.

Clinical Manifestions

As secondary invaders, Bacillus species may exacerbate preexisting infections by producing either tissue-damaging toxins or metabolites such as penicillinase that interfere with treatment.

Clinical Manifestions

Bacillus cereus is well known as an agent of food poisoning, and a number of other Bacillus species, particularly B. subtilis and B. licheniformis, are also incriminated periodically in this capacity.

Anthrax

Anthrax is primarily a disease of herbivores.

Humans acquire it as a result of contact with infected animals or animal products.

In humans the disease takes one of three forms, depending on the route of infection.

Anthrax

cutaneous anthrax, which accounts for more than 95 percent of cases worldwide, results from infection through skin lesions;

intestinal anthrax results from ingestion of spores, usually in contaminated meat;

pulmonary anthrax results from inhalation of spores.

Anthrax

Cutaneous anthrax usually occurs through contamination of a cut or abrasion, although in some countries biting flies may also transmit the disease.

After a 2- to 3-day incubation period, a small pimple or papule appears at the inoculation site. A surrounding ring of vesicles develops.

Over the next few days, the central papule ulcerates, dries, and blackens to form the characteristic eschar.

18.03

Anthrax (cutaneous eschar)

Anthrax (cutaneous)

Anthrax (cutaneous)

Anthrax

The lesion is painless and is surrounded by marked edema that may extend for some distance.

Pus and pain appear only if the lesion becomes infected by a pyogenic organism.

Similarly, marked lymphangitis and fever usually point to a secondary infection.

In most cases the disease remains limited to the initial lesion and resolves spontaneously.

Anthrax

The main dangers are that a lesion on the face or neck may swell to occlude the airway or may give rise to secondary meningitis.

If host defenses fail to contain the infection, however, fulminating septicemia develops.

Approximately 20% of untreated cases of cutaneous anthrax progress to fatal septicemia.

However, B. anthracis is susceptible to penicillin and other common antibiotics, so effective treatment is almost always available.

Anthrax

Intestinal anthrax is analogous to cutaneous anthrax but occurs on the intestinal mucosa.

As in cutaneous anthrax, the organisms probably invade the mucosa through a preexisting lesion.

Organisms spread from the mucosal lesion to the lymphatic system.

Anthrax

In pulmonary anthrax, inhaled spores are transported by alveolar macrophages to the mediastinal lymph nodes, where they germinate and multiply to initiate systemic disease.

Gastrointestinal and pulmonary anthrax are both more dangerous than the cutaneous form because they are usually identified too late for treatment to be effective.

As complication in both: meningitis (haemorrhagic CSF, and fatal in all cases!!!)

Pulmonary anthrax

Pulmonary anthrax

Diagnosis

The clinical diagnosis of anthrax is confirmed by directly visualizing or culturing the anthrax bacilli.

Fresh smears of vesicular fluid, fluid from under the eschar, blood, lymph node or spleen aspirates, or (in meningitic cases) cerebrospinal fluid are stained with polychrome methylene blue (M'Fadyean's stain) and examined for the characteristic square-ended, blue-black bacilli surrounded by a pink capsule.

(It should be remembered that B. anthracis organisms are not invariably detected in stained blood smears of humans dying of anthrax.)

Gram staining

Diagnosis

Alternatively, the bacilli may be cultured from these specimens and checked for sensitivity to the anthrax gamma phage, for penicillin sensitivity, and for capsule formation.

Colonies grown overnight at 37°C on blood agar are gray or white, nonhemolytic, with a dry, ground-glass appearance; they are at least 3 mm in diameter and sometimes have tails.

Colonies (R type and tails)

R type non-hemolyitic colonies

Colonies (R type and tails)

Diagnosis

Capsules can be seen in polychrome methylene blue-stained smears of cultures (cultivated in special conditions: grown on nutrient agar containing 0.7 percent sodium bicarbonate

and incubated overnight under CO2 (e.g., in a candle jar); encapsulated colonies are mucoid.

Mucoid colonies (capsulated strains)

Capsule (clear region around bacilli)

Polychrome methylene blue-stained smears

The lack of capsule

Diagnosis Alternatively, 2 ml of blood (such as commercial defibri-

nated horse blood) inoculated with a pinhead quantity of material from a suspected colony and incubated at 37°C yields readily demonstrable encapsulated bacilli in 6 hours.

Culturing may be unsuccessful if the patient has been treated with antibiotics.

Treatment

Bacillus anthracis is susceptible to penicillin and to almost all other broad-spectrum antibiotics.

Because it is easily recognized, cutaneous anthrax is almost always treated early and cured.

Prophylaxy: ciprophloxacine Gastrointestinal and pulmonary anthrax

infections are difficult to identify before the fulminant phase and therefore carry a high mortality.

Treatment

Tetracyclines (tests in animals indicate doxycycline is good), chloramphenicol, gentamicin, or erythromycin may be used if the patient has penicillin hypersensitivity.

The fluoroquinolone, ciprofloxacin, has also been shown to be effective in monkeys and guinea pigs and would be expected to be effective in treatment of cases of human anthrax.

Epidemiology

Source: animals, soils (spores).

Bacillus Food Poisoning Bacillus cereus can cause two distinct types of

food poisoning. * The diarrheal type is characterized by diarrhea

and abdominal pain occurring 8 to 16 hours after consumption of the contaminated food.

- It is associated with a variety of foods, including meat and vegetable dishes, sauces, pastas, desserts, and dairy products.

* In emetic disease, on the other hand, nausea and vomiting begin 1 to 5 hours after the contaminated food is eaten.

Bacillus Food Poisoning Boiled rice that is held for prolonged periods at

ambient temperature and then quick-fried before serving is the usual offender, although dairy products or other foods are occasionally responsible.

The symptoms of food poisoning caused by other Bacillus species (B. subtilis, B. licheniformis, and others) are less well defined.

Diarrhea and/or nausea occurs 1 to 14 hours after consumption of the contaminated food.

A wide variety of food types have proved responsible in recorded instances.

Bacillus Food Poisoning

A Bacillus food poisoning episode usually occurs because spores survive cooking or pasteurization and then germinate and multiply when the food is inadequately refrigerated.

Bacillus Food Poisoning

The symptoms of B. cereus food poisoning are caused by a toxin or toxins produced in the food during this multiplication.

Toxins have not yet been identified for other Bacillus species that cause food poisoning.

Microscopy: Bacillus spp.

B. cereus - spores

B. cereus - diagnosis

Hemolytic colonies Lecithinase (+) Resistant to penicillin The lack of pathogenecity for mice.

B. cereus - Hemolytic colonies

B. cereus (left) and B. anthracis (right)

B. cereus - lecithinase (+)

B. cereus

Vancomycine – first choice.

Clostridium

Anaerobic, spore forming Gram positive bacilli.

The clostridia are relatively large, Gram-positive, rod-shaped bacteria.

All species form endospores and have a strictly fermentative mode of metabolism.

Most clostridia will not grow under aerobic conditions and vegetative cells are killed by exposure to O2, but their spores are able to survive long periods of exposure to air.

Introduction

Of the anaerobes that infect humans, the clostridia are the most widely studied.

They are involved in a variety of human diseases, the most important of which are

- gas gangrene, - tetanus, - botulism, - pseudomembranous colitis and - food poisoning.

Introduction

In most cases, clostridia are opportunistic pathogens.

All pathogenic clostridial species produce protein exotoxins (such as botulinum and tetanus toxins) that play an important role in pathogenesis.

Introduction Most generalizations about Clostridium

have exceptions. The clostridia are classically anaerobic

rods, but some species can become aerotolerant on subculture; a few species (C. carnis, C. histolyticum, and C. tertium) can grow under aerobic conditions.

Introduction

Most species are Gram-positive, but a few are Gram-negative.

Also, many Gram-positive species easily lose the Gram reaction, resulting in Gram-negative cultures.

Introduction

The clostridia form characteristic spores, the position of which is useful in species identification; however, some species do not sporulate unless exposed to exacting cultural conditions (C. perfringens).

Many clostridia are transient or permanent members of the normal flora of the human skin and the gastrointestinal tracts of humans and animals.

Unlike typical members of the human bacterial flora, most clostridia can also be found worldwide in the soil.

Position of spore

Position of spore

Clostridium spp.

Clostridium spp.

C. botulinum (spores and flagella -EM)

C. tetani

C. tetani

C. perfringens

C. tetani

Clostridium tetani is the causative agent of tetanus.

The organism is found in soil, especially heavily-manured soils, and in the intestinal tracts and feces of various animals.

Carrier rates in humans vary from 0 to 25%, and the organism is thought to be a transient member of the flora whose presence depends upon ingestion.

C. tetani

The organism produces terminal spores within a swollen sporangium giving it a distinctive “drumstick” appearance.

Although the bacterium has a typical Gram-positive cell wall, it may stain Gram-negative or Gram-variable, especially in older cells.

C. tetani (G)

Gram staining (terminal spore)

Tetanus

Tetanus is a highly fatal disease of humans. Mortality rates reported vary from 40% to 80%. The disease stems not from invasive infection but from a potent neurotoxin (tetanus toxin or tetanospasmin) produced when spores germinate and vegetative cells grow after gaining access to wounds.

The organism multiplies locally and symptoms appear remote from the infection site.

Tetanus

Because of the widespread use of the tetanus toxoid for prophylactic immunization, the disease become very rare in some regions (fewer than 150 cases occur annually in the U.S., but the disease is a significant problem world-wide where there are > more than 300,000 cases annually).

Most cases in the U.S occur in individuals over age 60, which is taken to mean that waning immunity is a significant risk factor.

Pathogenesis

Most cases of tetanus result from small puncture wounds or lacerations which become contaminated with C. tetani spores that germinate and produce toxin.

The infection remains localized often with only minimal inflammatory damage.

The toxin is produced during cell growth, sporulation and lysis.

It migrates along neural paths from a local wound to sites of action in the central nervous system.

Clinical picture

The clinical pattern of generalized tetanus consists of severe painful spasms and rigidity of the voluntary muscles.

Spasms and rigidity of the voluntary muscles.

Spasms and rigidity of the voluntary muscles.

Clinical picture

The characteristic symptom of "lockjaw" involves spasms of the masseter muscle.

It is an early symptom which is followed by progressive rigidity and violent spasms of the trunk and limb muscles.

Spasms of the pharyngeal muscles cause difficulty in swallowing.

Death usually results from interference with the mechanics of respiration.

Neonatal tetanus

Accounts for about half of the tetanus deaths in developing countries.

In a study of neonatal mortality in Bangladesh, 112 of 330 infant deaths were due to tetanus.

Neonatal tetanus follows infection of the umbilical stump in infants born to nonimmune mothers (therefore, the infant has not acquired passive immunity).

It usually results from a failure of aseptic technique during the birthing, but certain cultural practices may contribute to infection.

Tetanus Toxin

There have been 11 strains of C. tetani distinguished

primarily on the basis of flagellar antigens. They differ in their ability to produce

tetanus toxin (tetanospasmin), but all strains produce a toxin which is identical in its immunological and pharmacological properties.

Tetanus Toxin

Tetanospasmin is encoded on a plasmid which is present in all toxigenic strains.

Tetanus toxin is one of the three most poisonous substances known, the other two being the toxins of botulism and diphtheria.

Tetanus Toxin

The toxin is produced by growing cells and released only on cell lysis.

Cells lyse naturally during germination the outgrowth of spores, as well as during vegetative growth.

After inoculation of a wound with C. tetani spores, only a minimal amount of spore germination and vegetative cell growth are required until the toxin is produced.

Tetanus Toxin

The bacterium synthesizes the tetanus toxin as a single 150kDa polypeptide chain (called the progenitor toxin), that is cleaved extracellularly by a bacterial protease into a 100 kDa heavy chain (fragment B) and a 50kDa light chain (fragment A) which remain connected by a disulfide bridge.

Tetanus Toxin

Tetanus toxin is produced in vitro in amounts up to 5 to 10% of the

bacterial weight. Because the toxin has a specific affinity

for nervous tissue, it is referred to as a neurotoxin.

Tetanus Toxin

The toxin has no known useful function to C. tetani.

Why the toxin has a specific action on nervous tissue, to which the organism naturally has no access, may be an anomaly of nature.

The toxin is heat labile, being destroyed at 56°C in 5 minutes, and is O2 labile.

The purified toxin rapidly converts to toxoid at 0°C in the presence of formalin.

Toxin Action

Tetanospasmin initially binds to peripheral nerve terminals.

It is transported within the axon and across synaptic junctions until it reaches the central nervous system.

There it becomes rapidly fixed to gangliosides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis.

Toxin Action

The effect of the toxin is to block the release of inhibitory neurotransmitters (glycine and gamma-amino butyric acid) across the synaptic cleft, which is required to check the nervous impulse.

If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus.

Tetanospasmin

Tetanospasmin appears to act by selec-tive cleavage of a protein component of synaptic vesicles, synaptobrevin II, and this prevents the release of neurotrans-mitters by the cells.

The receptor to which tetanospasmin binds has been reported as ganglioside GT and/or GD1b, but its exact identity is still in question.

Tetanospasmin

Binding appears to depend on the number and position of sialic acid residues on the ganglioside.

Isolated B fragments, but not A fragments will bind to the ganglioside.

The A fragment has toxic (enzymatic) activity after the B fragment secures its entry.

Binding appears to be an irreversible event. Recovery depends on sprouting a new axon

terminal.

Immunity

Unlike other diseases, such as diphtheria, recovery from the natural disease usually does not confer immunity, since even a lethal dose of tetanospasmin is insufficient to provoke an immune response.

Prophylactic immunization is accomplished with tetanus toxoid, as part of the DPT (DTP) vaccine or the DT (TD) vaccine.

Immunity

Three injections are given in the first year of life, and a booster is given about a year later, and again on the entrance into elementary school.

Whenever a previously-immunized individual sustains a potentially dangerous wound, a booster of toxoid should be injected.

Wherever employed, intensive programs of immunization with toxoid have led to a striking reduction in the incidence of the disease.

C. botulinum

Is a large anaerobic bacillus that forms subterminal endospores.

It is widely distributed in soil, sediments of lakes and ponds, and decaying vegetation.

Hence, the intestinal tracts of birds, mammals and fish may occasionally contain the organism as a transient.

Seven toxigenic types (A - G) of the organism exist, each producing an immunologically distinct form of botulinum toxin (A, B and E are most often isolated from humans).

Clostridium

C. botulinum

Toxins The toxins are designated A, B, C1, D, E, F, and

G. In the U.S. type A is the most significant cause of botulism, involved

in aprox. 60% of the cases. Not all strains of C. botulinum produce the

botulinum toxin. Lysogenic phages encode toxin serotypes C and

D, and non lysogenized bacteria (which exist in nature) do not produce the toxin.

Type G toxin is thought to be plasmid encoded.

Pathogenesis of Botulism

Food-borne botulism In food-borne botulism the botulinum toxin

is ingested with food in which spores have germinated and the organism has grown.

The toxin is absorbed by the upper part of the GI tract in the duodenum and jejunum, and passes into the blood stream by which it reaches the peripheral neuromuscular synapses.

Pathogenesis of Botulism

The toxin binds to the presynaptic stimulatory terminals and blocks the release of the neurotransmitter acetylcholine which is required for a nerve to stimulate the muscle.

Food-borne botulism is not an infection but an intoxication since it results from the ingestion of foods that contain the preformed clostridial toxin.

Pathogenesis of Botulism

In this respect it resembles staphylococcal food poisoning.

Botulism results from eating uncooked foods in which contaminating spores have germinated and produced the toxin.

C. botulinum spores are relatively heat resistant and may survive the sterilizing process of improper canning procedures.

Pathogenesis of Botulism

The anaerobic environment produced by the canning process may further encourage the outgrowth of spores.

The organisms grow best in neutral or "low acid" vegetables (>pH 4.5).

Pathogenesis of Botulism

Clinical symptoms of botulism begin 18-36 hours after toxin ingestion with weakness, dizziness and dryness of the mouth.

Nausea and vomiting may occur. Neurologic features soon develop: blurred vision,

inability to swallow, difficulty in speech, descending weakness of skeletal muscles and respiratory paralysis.

usually no fever no loss of sensation no loss of awareness

Pathogenesis of Botulism

Botulinum toxin may be transported within nerves in a manner analogous to tetanospasmin, and can thereby gain access to the CNS.

However, symptomatic CNS involvement is rare. There may also be autonomic signs with - dry mouth, fixed or dilated pupils - cardiovascular, gastrointestinal and urinary

dysfunction

Infant Botulism

Infant botulism is due to infection caused by C. botulinum.

The disease occurs in infants 5 - 20 weeks of age that have been exposed to solid foods, presumably the source of infection (spores).

It is characterized by constipation and weak sucking ability and generalized weakness.

Infant Botulism

C. botulinum can apparently establish itself in the bowel of infants at a critical age before the establishment of competing intestinal bacteria (normal flora).

Production of toxin by bacteria in the GI tract induces symptoms.

This "infection-intoxication" is restricted to infants.

Infant Botulism

C. botulinum organisms, as well as toxin can be found in the feces of infected infants.

Almost all known cases of the disease have recovered.

Infant Botulism

The possible role of infant botulism in "sudden infant death syndrome-SIDS" has been suggested and is under investigation.

C. botulinum, its toxin, or both have been found in the bowel contents of several infants who have died suddenly and unexpectedly.

The Botulinum Toxins

The botulinum toxins are very similar in structure and function to the tetanus toxin, but differ dramatically in their clinical effects because they target different cells in the nervous system.

Botulinum neurotoxins predominantly affect the peripheral nervous system reflecting a preference of the toxin for stimulatory motor neurons at a neuromuscular junction.

The Botulinum Toxins

The primary symptom is weakness or flaccid paralysis.

Botulinum toxin is synthesized as a single polypeptide chain with a molecular weight around 150 kDa.

In this form the toxin has a relatively low potency.

The Botulinum Toxins The toxin is nicked by a bacterial protease (or

possibly by gastric proteases) to produce two chains: a light chain (the A fragment) with a

molecular weight of 50 kDa; and a heavy chain (the B fragment), with a mw of 100kDa.

As with tetanospasmin, the chains remain connected by a disulfide bond.

The A fragment of the nicked toxin, on a molecular weight basis, becomes the most potent toxin found in nature.

Toxin Action

The botulinum toxin is specific for peripheral nerve endings at the point where a motor neuron stimulates a muscle.

The toxin binds to the neuron and prevents the release of acetylcholine across the synaptic cleft.

The heavy chain of the toxin mediates binding to presynaptic receptors.

Toxin Action

The toxin (A fragment) enters the cell by receptor mediated endocytosis.

Once inside a neuron, the toxin types probably differ in mechanisms by which they inhibit acetylcholine release, but a mechanism similar to or identical to tetanospasmin has been reported (i.e., proteolytic cleavage of synaptobrevin II).

Toxin Action

The affected cells fail to release a neuro-transmitter, thus producing paralysis of the motor system.

Once damaged, the synapse is rendered permanently useless.

The recovery of function requires sprouting of a new presynaptic axon and the subsequent formation of a new synapse.

Toxin Action

As stated above, the mechanism by which acetylcholine release is prevented is not known.

However, recent evidence suggests that both botulinum toxin as well as tetanus toxin are zinc-dependent endopepti-dases that cleave specific proteins that are involved in excretion of neurotrans-mitters.

Toxin Action

Both toxins cleave a set of proteins called synaptobrevins.

Synaptobrevins are a set of proteins found in synaptic vesicle of neurons, the vesicles responsible for release of neurotransmitters.

Presumably, proteolytic cleavage of synaptobrevin II would interfere with vesicle function and release of neurotransmitters.

Immunity

On the average there are about 25 cases of botulism annually in the U.S.

Prior to the advent of critical care, the case fatality rate exceeded 60%, but currently it is about 20%.

The first (or only) patient in an outbreak has a 25% chance of death, whereas subsequent cases which are diagnosed and treated more quickly, carry only a 4% risk.

Immunity The toxins that cause botulism are each

specifically neutralized by its antitoxin. Botulinum toxins can be toxoided and make

good antigens for inducing protective antibody.

As with tetanus, immunity to botulism does not develop, even with severe disease, because the amount of toxin necessary to induce an immune response is toxic.

Repeated occurrence of botulism has been reported.

Immunity

Once the botulinum toxin has bound to nerve endings, its activity is unaffected by antitoxin.

Any circulating ("unfixed") toxin can be neutralized by intravenous injection of antitoxin.

Individuals known to have ingested food with botulism should be treated immediately with antiserum.

Immunity

A multivalent toxoid evokes good protective antibiody response but its use is unjustified due to the infrequency of the disease.

An experimental vaccine exists for laboratory workers.

Prevention The most important aspect of botulism

prevention is proper food handling and preparation.

The spores of C. botulinum can survive boiling (100 degrees at 1 atm) for more than one hour although they are killed by autoclaving.

Because the toxin is heat-labile boiling or intense heating (cooking) of contaminated food will inactivate the toxin.

Prevention

Food containers that bulge may contain gas produced by C. botulinum and should not be opened or tasted.

Other foods that appear to be spoiled should not be tasted.

Clostridium perfringens

Produces a huge array of invasins and exotoxins, causes wound and surgical infections that lead to gas gangrene, in addition to severe uterine infections.

Clostridium perfringens

Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive process.

Clostridium perfringens also produces an enterotoxin and is an important cause of food poisoning.

Usually the organism is encountered in improperly sterilized (canned) foods in which endospores have germinated.

C. perfringens

C. perfringens

Double hemolysis zone (largest external, clearer and smallest, inside)

Lecithinase activity

Gas gangrene

Muscles necrosis (microscopy)

C. perfringens - treatment

Surgery Penicilline or metronidazole (before surgery)

and for minimum 3 days (after) Serotherapy Prevention: deep wound must be rigorously

cleaned!

C. difficile

Clostridium difficile

C. difficile

Pseudomembranous colitis in humans is caused by overgrowth of Clostridium difficile in the colon, usually after the normal flora has been disturbed by antimicrobial chemotherapy (ampicilline, clindamycine, cephalosporines, etc)

C. difficile produces two toxins: - toxin A is referred to as an enterotoxin

because it causes fluid accumulation in the bowel;

- toxin B is an extremely lethal (cytopathic) toxin.

C. difficile - Pseudomembranous colitis Colonos-copy

C. difficile - colitis

Diagnosis

Diagnosis of C difficile disease includes the presence of diarrhea associated with antibiotic therapy in the preceding 4 to 6 weeks, and the recovery of C. difficile organisms and/or toxin from the stool.

Diagnosis

Diagnosis of pseudomembranous colitis requires demonstration of pseudomem-branes by colonoscopy, and C. difficile can be isolated from the stools of almost all patients with this disease.

Treatment

In many cases, symptoms resolve 1-14 days after the offending antibiotic is discontinued, and antibiotic treatment is not needed.

Vancomycin or metronidazole are the antibiotics of choice to treat active disease.