Clostridium Botulinum

 

This bacterium is responsible for a neurointoxication, botulism. Most often the disease is secondary to the ingestion of preformed toxin in food contaminated by this species. This cosmopolitan disease is daunting.

Clostridium botulinum

Clostridium botulinum

HISTORY:

Botulism term is related to the fact that the first described case reports were due to the ingestion of sausage (ofbotulus Latin).

In 1895 Van Ermengen describes the germ and disease.Different serotypes have been described since then, Gimenez and Ciccarelli have shown that some strains could produce two toxins.

I – CLASSIFICATION:

C. botulinum includes various expèces producing a neurotoxin that is responsible for the same clinical syndrome botulism.

The strains of C. CG botulinum have between 26% and 28% against 22-55% for Clostridium.

The usual nomenclature has four groups of C. botulinum:

– Group I. proteolytic strains. Toxin types A, B, F

– Group II. Toxin type E and non-proteolytic strains of type B and F.

– Group III. Toxin types C and D, usually poultry.

– Group IV. Type G toxin; the name of C. argentinense was proposed instead of C. botulinum to designate these strains.

The situation is complicated by the fact that other Clostridium C. baratii and C. butyricum are capable of the production of botulinum toxin (Table I).

II – HABITAT – TRANSMISSION:

C. botulinum is a widespread telluric germ in nature, but the disease is rare, because the ingestion of the bacteria usually produces no effects.

The bacteria exist as spores in the soil, mud and water. The plants are contaminated with the spore (fruits, vegetables, fodder). The spore is present in the intestines of many animals: horses, sheep, and pigs. The fish are contaminated in contact with the mud.

For there to be disease, there must have ingested sufficient toxin. This toxin is only produced if the spore survives in the food to yield a toxin-producing vegetative form.

The meat contamination can occur in two ways:

– Slaughter by digestion period: postprandial bacteremia causes diffusion in the muscles,

– Due to contamination from the intestinal contents,

The persistence of spores is favored by:

– Improper storage of the food (smoking and curing defective)

– Insufficient sterilization (temperature below 100 ° C).

The transformation of the vegetative form in spore becomes possible in several conditions:

– Low salinity (NaCl lower than 10%, less than 2% acetic acid),

– Temperature near room temperature for the multiplication of the bacteria,

– Anaerobic performed in foods, in contact with the bone to the ham, under the fat layer (blocks), or because of sealed packaging,

– Presence of carbohydrates (canned fruit and vegetables).

Production of the toxin in the food takes some time, an average of eight days at 26 ° C. The release would be favored by microbial associations that lyse C. botulinum.

The toxin is heat labile, the food is dangerous when ingested raw.

Exceptionally botulism toxin can grow production at an infected wound or the intestine.

III – PATHOPHYSIOLOGY:

There are two forms of botulism: intoxication and poisoning. In some infants C. botulinum. is implanted in the intestine and releases the toxin synthesized in situ.

Toxin ingested bacteria pass without damage the gastric barrier. Toxin found in the intestine corresponds to the ingested toxin and the toxin released during bacterial lysis, this toxin therefore resistant to gastric acid and digestive juices.

Once in the intestine it passes into the lymph and blood, and will bind to the nervous tissue. Intoxication leads to a general paralysis (flange type) of neuromuscular and autonomic nervous system.

The action of the toxin is mainly exerted on the peripheral nervous system, unlike tetanus toxin that acts primarily on the central nervous system.

In humans, there is a decrease in the activity:

– The vagus nerve,

– Bladder contraction nerves, secretory organs, oculomotor,

– Motoneurons.

Mode of action:

The toxin prevents cholinergic transmission in which the mediator acetylcholine and acts primarily on nerve endings

– Nerve fibers preganglionic parasympathetic, sympathetic and autonomic nervous system

– Fibers of the parasympathetic nervous system,

– Fibers innervating motor neurons striated muscles.

Mechanism:

Acetylcholine is as shown in Figure 1 at the neuromuscular junction.

The toxin inhibits the release of acetylcholine, by preventing the activation of the ion release mechanism id “1” 1 “.

IV – PATHOGENICITY NATURAL:

A – For the animal:

Cattle contract the disease through ingestion of moldy fodder contaminated with dead rodents (botulinum A, B, D).

FIGURE 1: site of action of botulinum toxin at the neuromuscular junction

FIGURE 1: site of action of botulinum toxin at the neuromuscular junction

Sheep can also be affected. Equine botulism can decimate entire stables (botulism B). Avian botulism (botulinum C) causes flaccid paralysis of the neck muscles.

Experimentally if the guinea pig is the animal of choice, the mice are also very sensitive to the toxin, such as monkeys, cats and rabbits.

B – Human health:

It is often small family epidemics affecting guests at a meal.

1. It is essentially a “intoxination” related to the ingestion of preformed toxin:

The foods are typically involved:

– Family preserves, inadequately sterilized craft, especially pork meats: smoked hams, confit, foie gras (B botulism)

– Fruits and vegetables contaminated with soil, particularly in the USA (botulinum A)

– Sea or river fish canned, smoked or raw fish botulism managers E and F (USSR, Canada, Japan …).

After ingestion of contaminated food there are:

– The incubation period can be from 8 to 12 hours in case of hyperacute botulism, but up to 2 to 3 weeks.

– The period of invasion found in half of the cases with non-specific gastrointestinal symptoms (nausea, vomiting, diarrhea).

– The status phase characterized in typical forms by

a / Engines disorders:

Ocular: with the primary signs paralysis of accommodation with blurred near vision, mydriasis followed and extrinsic disorders (ptosis, strabismus, diplopia)

– Buccopharyngé: with painful dysphagia that may cause choking,

– Esophageal, colon, bladder … other paralysis may affect the muscles of the back, neck, members or diaphragm.

b / Secretaries disorders:

With drying lacrimation, salivary, the entire digestive tract, sweat, milk. The dry mucous membranes favors infections.

c / Negative signs:

No fever, no cardiovascular involvement, no loss of consciousness, and more biological CSF parameters are within the limits of normal.

– The evolution is usually favorable in France (4% of deaths), more severe in the United States (50-60% mortality).The deadly forms are often found among the short incubation forms and some serotypes (types A and E). Prognosis also varies with the amount of food ingested and with the degree of hydration of the foods. Highly hydrated foods contain more toxin.

2. This is rarely a “toxic infection”:

The seed is then isolated from the patient himself at wounds contaminated with gangrene or at the sites of parenteral injections (more than 40 cases in the US).

Recently cases of sudden infant death were attributed to C. botulinum.

Clostridium botulinumIngested germ, for example with milk foods containing honey, is implanted in the intestine and it secretes the toxin. There endogenous toxin production and development of constipation, respiratory problems and death (children to 12 weeks). The etiology was found in 4.7% of the 21 cases of sudden death, reported by Amon and would be due to rare toxin type (C, F and G) whose pathogenicity in humans is not always well documented . It is not clear why the germ can be implanted in the intestinal flora of children less than 1 year, but it should be noted that in a number of exceptional cases in adults this colonization may exist.

In some animals this implementation can be done in a very short period of life.

TABLE I: epidemiology next serotype Clostridium Botulinum

TABLE I: epidemiology next serotype Clostridium Botulinum

V – BACTERIOLOGICAL CHARACTERS:

A – vegetative form:

1. Body type:

C. botulinum is a gram positive bacillus (low) with rounded ends of 4-8 microns of 0.9-1.2 microns. It is mobile (6-20 peritrichous eyelashes). It is not encapsulated.

In young cultures, short chains can be observed. Unlike in older cultures, there vacuolar forms of involution and spores.

2. Crops:

It is a strict anaerobic organism for which the best conditions

growth are a temperature of 26-28 ° C and an alkaline pH (8 to 8.4).

– Aspect of colonies:

– Deep agar VF or VL added blood, egg yolk, are lenticular, the walls are smooth, sometimes spiny sea urchin (Figure 2).

– Surface colonies are circular and yellowish.

FIGURE 2: appearance of colonies of C. botulinum in semi-solid medium (smooth and spiny colonies)

FIGURE 2: appearance of colonies of C. botulinum in semi-solid medium (smooth and spiny colonies)

3. Biochemical Properties (Table II):

The main energy source is carbohydrates: glucose, maltose, levulose … releasing acetic, butyric acid (rancid butter smell characteristic) and lowest amount lactic acid. The D-type strains are more glucidolytiques; type C strains are not.

Proteolytic action: proteolysis more or less pronounced depending on the type. A and B are gelatin (+) decarboxylase (+), deaminase (+), …, C, D and E are not proteolytic. Type B produces a hemolysin.

Very marked lipolytic power for botu.linu.rn C. A, B and F.

High reducing power for A, low to B, C, E.

TABLE II: characteristics of Clostridium species producing neurotoxin

TABLE II: characteristics of Clostridium species producing neurotoxin

* Determined by gas-liquid chromatography

A. Ac. acetic, B. Ac.butyrique, ib. Ac. isobutyric, P. Ag. propionic, N. Ac.isovalérique

4. Vitality reduced:

Vegetative bacteria destroyed in 30 minutes at 60 ° C or 2 minutes at 80 ° C, but in these conditions the sporulation is fast.

B – Spore:

Sporulation is favored by the difficult living conditions and arginine.

The spore is ovoid, deforming, subterminal.

The spore is very heat resistant, and 8 minutes at 115 ° C 95% killing of the spores, the 5% remaining resist 5-10 minutes at 120 ° C.

Heat resistance varies serotypes, decreasing in the following order C, A, B, D, E; it is favored by low salinity (1-6% o) or lipids.

Spore resistant to antiseptics: it takes 24 hours to destroy the spores with 20% formalin.

C. botulinum serotype A is one of the more radioresistant bacteria.

The spores of C. botulinum are also quite resistant to various bactericidal agents: UV, hypochlorite, alcohol, quaternary ammonium compounds.

Germination is inhibited by NaCl (10 to 15%), some fatty acids and of the antibiotic substances produced by certain bacteria, such B. subtilis.

C – Toxin:

1. Production:

It is secreted by the bacteria in the exponential growth phase; she is endocellular portion and secondarily passes into the external environment upon lysis of the bacteria.

– It is sometimes secreted in an inactive form: protoxins A and E, and activated by proteolytic enzymes of the bacteria.

– The media used to obtain a good summary of the toxin are casein hydrolyzate or broth VF glucose 5% o. The optimum synthesis temperature is between 30 and 37 ° C, it is inhibited below 10 ° C, but not by a temperature “room” 25 ° C.

– The optimum production in crops is around the 6th day.

– The synthesis of the toxin, at least for types C and D, is in response to phage lysogens, non-lysogenic strains were not virulent.

Similarly the presence of the plasmids was correlated with in particular the production of the toxin G.

2. Properties:

The toxin is a protein nature, the molecular weight is high (Table III) ranging from 141 kDa to 170 kDa. We distinguish a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bridge. Toxin is initially synthesized as a single chain, which is then cleaved by a proteolytic enzyme of endogenous or exogenous origin. In type C can be seen a major neurotoxin called C2 and a second minor different C2 toxin has an effect on vascular permeability and lethal action.

It was long thought that the PM were much higher up to 900 kDa, but the toxins exist as polymers. The toxin could be obtained in crystalline form. Toxins are rich in asparagine and glutamine.

TABLE III: Molecular weight of botulinum toxin (from Sebald).

TABLE III: Molecular weight of botulinum toxin (from Sebald).

3. Toxicity:

This is the most active compound among the known organic: 1 mg of toxin A crystallized is 31 million MDA mouse and 1.2 million kg DMM guinea pig. The toxicity depends on:

– The route of administration. The oral route is less effective. Dehydrated, mere contact with the toxin conjunctiva caused the death of a researcher,

– Of the animal, mice and guinea pigs are very sensitive.

Toxin The assay is performed by study of the pathogenicity for mice (DMM, LD 50) by passive hemagglutination, precipitation (initial flocculation Ramon) … The toxins are antigenic, convertible into toxoid formalin and heat .

4. Stability:

Toxins are:

– Thermolabile destroyed at 80 ° C in 15 minutes, 10 minutes at 100 ° C, toxins C and D are the most heat-resistant;

– Sensitive to oxidants (bleach) to light;

– Stable at pH 3, and therefore resistant to gastric acidity in man; vultures possess digestive enzymes destroying the toxin in the bodies.

5. serotypes:

Now we know 7 serotypes designated by the letters A to G. The serotype distribution, habitat, geographical area of ​​each serotype, and mortality that they bear are listed in Table I. Type B is almost always involved in France.

6. Therapeutic use:

Type A neurotoxin is used in the treatment of neuromuscular disorders (dystonia and spasticity spasmodic torticollis) and ophthalmologic (strabismus, blepharospasm).

D – Other antigens:

Outside toxins there hemolytic factors, haemagglutinating, which do not seem related to the toxin. It also recognizes somatic antigens thermostable and thermolabile flagellar antigens. It was recognized 3 H subtypes for the A and 4 strains for strains B.

VI – LABORATORY DIAGNOSIS BOTULISM:

A – Search of the toxin in the serum of the patient:

This review great interest. It allows the diagnosis of crude forms or non digestive door entrance. The toxinémie appears near the 2nd day of illness and lasts 2-3 weeks. This examination should be performed before any administration in patient serum or anti-botulinic neurotropic medications. It requires about 20 ml of blood.

Toxemia is sought by inoculation by intraperitoneally patient serum in mice, using animals protected by the various antisera and unprotected animals.

In exceptional cases, the search for the toxin can be in the stool or vomit of products of the patient, gastric aspiration, or even in the serum or post-mortem viscera.

B – Research of the toxin in the suspect food:

It is essential in a suspected botulism to find food

suspects and send them together with the patient to the hospital.

1. Demonstration of the toxin:

It is done by looking pathogenicity test on animals and the protected animal.

– Is removed the suspicious area of ​​the food, for example for hams bone marrow, the area near the bone where the flesh has a pinkish appearance;

– Is ground tissue removed in saline or buffer, ground while maintaining a low temperature, low speed centrifuged to remove large particles, the germ can be sought on the base, the toxin will be sought on the supernatant;

– For research and titration of the toxin, we inoculated mice with growing volumes of supernatant by intraperitoneal route: from 0.1 to 0.25 – 0.5 to 1 ml (5 mice per dose). Sometimes used blindly, for a first screening 0.25 ml per mouse but the titration will be important for later successful neutralization;

– The serum neutralization test will verify that it is botulinum toxin that killed unprotected animal, but also to determine the serotype involved. For it is brought into a certain volume of supernatant (2-10 DMM) and the anti-A serum, the same volume of supernatant and anti B serum, etc. After allowing to stand for 30 minutes at 37 ° C, each mixture is injected in several animals intraperitoneally (Scheme).

The presence of toxin is shown by the death of the animal within 24 or 48 hours in a array of flaccid paralysis. Type toxin will be recognized by neutralizing the pathogenicity using specific antiserum.

This demonstration of the toxin, however, faces a number of challenges that must not be overlooked: the animal’s death can not be due to a toxin action. Indeed, the presence of heavy metals or other toxic hyperosmolarity created during inoculation may lead to misdiagnosis.

FIGURE typing the toxin test of the protected animal (eg toxin type B)

FIGURE typing the toxin test of the protected animal (eg toxin type B)

2. germ of Isolation:

For culture, we share is the pellet or supernatant if the centrifugation was slightly faster. Then the extract is treated by heat for 30 minutes at 75 ° C or 10 minutes at 100 ° C to select the spores. Is then inoculated into VF glucose medium, which is placed at 33 ° C for 7 to 8 days. Then search toxin production on culture by animal inoculation. It may take a strain isolation and identification seed a gallery.

A search of the germ could also be done directly on the food in immunofluorescence with anti-sera C. botulinum, but the specificity of this technique is questionable especially between serotypes and between species. False reactions were noted particularly with C. sporogenes.

An ELISA method has recently been described for detecting the toxins A and B in the food through monoclonal antibodies.

VII – TREATMENT:

A – Prophylactic treatment:

II relates to food.

1. Preparation:

Food for canning should be washed thoroughly to rid the earth (fruits, vegetables …)

– The animals used in the preparation of ham for example, should be fasted and rest at least 24 hours before slaughter;

– Fish for consumption or canning must be gutted.

2. Conservation:

Sterilization should be sufficient for canning (120 ° C in an autoclave)

– Salting, the use of acetic acid, ascorbic acid and Na nitrite can avoid anaerobic

– Packaging cellophane favor anaerobic

– Freezing does not destroy the toxin, but prevents its formation.

3. Consumption:

Avoid suspect food (butyric odor, rancid) meat with suspicious aspect, boxes bulging cans, or pressure at the opening (jet),

– Cooking destroys most toxins, which can not justify the ingestion of a single heated suspect food.

B – Curative treatment:

1. It is symptomatic:

Hospitalization in an intensive care unit, to prevent respiratory arrest (tracheal intubation, assisted respiration), and infections from mucosal impaired by the absence of secretions (disinfection).

2. The specific treatment or resting on physiopathoîogiques bases are more questionable effectiveness:

The versatile specific serum therapy before serotyping of the toxin and monovalent, has a neutralizing effect on the circulating toxin (in the absence of treatment, serum toxin can persist for several weeks), but it does not mobilize the toxin attached to the centers nervous. It should also avoid serum sickness (corticosteroids)

– The anatoxinothérapie can accommodate because of the prolonged course of the disease.

– Guanidine chloride reduce the neurological manifestations of botulism,

– The monoacetate guanine was also tried.

In fact a consensus seems to be to recognize that these treatments did not significantly influence the evolution of botulism.

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