GENERAL:

The genus Bacillus includes rod-shaped bacteria, usually mobile, spore-forming. These bacilli are Gram-positive, facultatively aerobic or anaerobic strict.

Bacillus genus includes about twenty species, but is primarily concerned with B. anthracis, due to its pathogenicity (animals, man) and B. cereus (food poisoning).

However, in recent years, many publications make other Bacillus species responsible for infections in immunocompromised (bacteremia, meningitis, meningoencephalitis, pneumonia, endocarditis).

I – CLASSIFICATION AND NOMENCLATURE:

The Bacillus species are classified on their morphology and the position of their spores. This classification divides the genus Bacillus into 3 groups:

– Group 1 bacilli spores do not deform the microbial body,

– Group 2 deforming bacilli spores, oval,

– Group 3 deforming bacilli spores, round.

Within these groups, species and varieties are distinguished by morphological and physiological characteristics.

II – MAIN CHARACTERS OF THE GENUS BACILLUS:

A – Body type:

Mobile ciliature peritrichous, control from a broth (B. anthracis is still moving).

Gram stain positive theory can be negative (group 1 best guard staining).

The simple staining diluted fuchsin can better appreciate the forms, dimensions, presence of vacuoles cytosplasmiques due to lipid inclusions.

Bacillus rarely encapsulated except B. anthracis under certain conditions.

Without peptone agar is the most favorable environment for sporulation. The mature spore is oval or round. Its diameter is lower or higher than the bacillus. Its location is central, paracentral, subterminal or terminal. In some species there are parasporal inclusions. Sometimes we note the presence of protein crystals.

Resistance to physical and chemical agents is low for bacillary forms, easily killed by aging and heat (1 h at 55 ° C) and antiseptics.

It is higher for the spores (40 min at 120 ° C). B. subtilis is among the most resistant.

B – Cropping characters:

Most species grow best at 28-33 ° C than at 37 ° C, but many species tolerate marked thermal differences.

Determinations growth threshold temperatures were performed on agar slant incubated in a water bath.

Conventional culture media allow the growth of most species of the Bacillus genus.

Species each have their own requirements and often relatively homogeneous nitrogen use ammonia for some, amino acids for others, etc …

Useful favorable or hostile environments for identification are:

– Nutrient agar at pH 6,

– Potato: Culture or not,

– Hypersaline broth.

C – General biochemical characters:

Respiratory metabolism catalase (+) oxidase (±) strictly aerobic or anaerobic optional depending on the species.

Nitrate reductase

It is present or not depending on the species.

Attack of carbohydrates

In complex environments, acidification is often hidden. It is therefore necessary to use a mineral agar medium (Smith, Gordon, Clark), enriched with yeast extract.

Acidification or not glucose, arabinose and xylose are generally sufficient for identification.

Production of acetoin (acetyl-methyl-carbinol)

Its production on the usual medium of Clark and Lubs is not always good. It is better to use a glucose medium, peptone phosphate free. The temperature favorable for the production varies depending on the species (32 ° C, 37 ° C and 45 ° C).

Other characters to study

– Hydrolysis of starch, casein, gelatin,

– Hydrolysis of lecithin B. subtilis which has a phospholipase hydrolysis lecithin and diglycerides phosphorylcholine (clouding). Often the reaction is R (= Restricted), that is very localized in the culture. So sometimes you have to remove the colonies to highlight.

– Indole: rarely positive.

– Urease.

BACILLUS ANTHRACIS

HISTORY:

B. anthracis is responsible for the “coal”, known since antiquity.

1823: Barthélémy makes the first experimental transmission sheep.

1848 – 1850: Rayer and Davaine detect blood “smoky” of “small wiry body.”

1860 Delafond realize the culture of the bacteria.

1880 Shepherd, Roux and Chamberland attenuate the bacteria by heat.

1881 Experience Pouilly-le-Fort Pasteur proves the effectiveness of the anthrax vaccine.

I – HABITAT:

Spore gives a high resistance in outdoor environments. These are terrestrial organisms but which is also found in the water and in the air.

B. anthracis is widely distributed in the world. Sick animals disseminate the seeds; soil is the reservoir (fields, barns, meadows); corpses and animal products (skin, hair, bones, …) are involved in the pollution of land and can infect humans.

II – BACTERIOLOGICAL CHARACTERS:

A – Morphology and structure:

Wholesale bacillus (5-6 microns x 1 nm). Immobile (different from other Bacillus).

In pathological products, it is in the form of short chains.

Culture: long chains “bamboo cane” Gram positive. Square ends.

1. The capsule:

Virulent strains of B. anthracis are encapsulated. The capsule is produced in vivo or in culture under certain conditions (blood, serum, sodium bicarbonate, CO2). Non-encapsulated strains are avirulent.

2. The spore:

It is ovoid, non-deforming. B. anthracis belongs to group 1.

The sporogenesis is done in the presence of O ^, between 18 ° C – 42 ° C in a humid atmosphere in poor areas without peptone.

B – Cropping characters:

B. anthracis grows well on usual media.

Anaerobic, he prefers the presence of oxygen. Optimum temperature: 30 ° C – 35 ° C (15 ° C – 43 ° C); pH from 7 to 7.4 (6 to 8.5).

Slant nutrient agar, colonies are R with irregular contours. On serum agar, colonies are S (due to the production of a capsule).

• In ordinary broth and peptone water: flaking bottom of the tube with clear supernatant.

C – biochemical characters:

1. proteolytic capacity:

It is slightly marked.

Liquefaction of gelatin pellet after bite is slow.

It gives an appearance in “inverted tree”.

The milk is coagulated slowly digested slowly.

Liquid Serum: gelation and liquefaction.

Coagulated serum: slow digestion.

Hemolysis is light and appears slowly.

2. Power glucidolytique:

II ferments: glucose, maltose, sucrose, levulose, trehalose and dextrin.

Lactose (-), galactose (-), arabinose (-).

Other sugars are attacked inconstantly.

3. B. anthracis has nipénicillinase or urease:

Is H2S (-). Nitrate is reduced to nitrite.

D – Processed products:

B. anthracis is developing anthrax toxin. Is a proteinaceous toxin which is composed of three distinct proteins: Factor 1 = edema factor (adenylate cyclase), factor II = protective antigen, lethal factor = factor III (MW = 82 000).

II and III factors are combined to cause the death of the mouse, rat or guinea pig. The intradermal injection of factors 1 and II causes skin lesions in guinea pigs and rabbits.

The toxic action of various strains may vary according to the respective production of these three factors, and thus, some strains are very oedématogènes and other little.

The toxin production meets a plasmid determinism.

E – Antigenic Structure:

1. The capsule:

Polypeptide and it is involved in the virulence of the bacterium. It prevents phagocytosis, inhibits serum bactericidal and makes the anticoagulated blood.

The anti-capsular antibodies are not protective in humans.

2. Somatic antigens:

They can be studied by precipitation reaction (Ascoli reaction).

3. The toxin:

It causes the formation of neutralizing antibodies. There antigenic communities between B. and some other Bacillus anthracis, demonstrated by immunodiffusion (eg. with B. subtilis, B. megaterium and B. cereus).

F – Phage:

B. anthracis is sensitive to different bacteriophages. Phage typing can be used in the differential diagnosis betweenB. cereus and B. anthracis:

– Group A phages active on B. anthracis, B. Inactive cereus

– AC phage group active on B. anthracis and B. cereus

– Phages of inactive C Group B. anthracis, certain assets B. cereus.

III – PATHOGENICITY NATURAL:

A – In animals:

We distinguish subacute, acute and hyperacute. Coal can be “internal” or rarely “external”. The clinical forms vary according to the species:

Sheep: acute form (death within hours)

Horse and Beef: acute (often fatal in 1-2 days)

Pork: External coal subacute (swelling of the throat).

In the animal house coal, gross lesions are those of sepsis. The blood is black, sticky, incoagulable; splenomegaly and hematuria is observed. In the external coal, there is a focus on tumor lymph node package.

Microscopic lesions are represented by an accumulation of bacilli in the capillaries.

B – In men:

1. External Coal:

Coal is rarely found in men in France, but it is estimated that there are in the world every year between 20 000 and 100 000 cases of coal (Africa, Asia especially).

The typical lesion is the “malignant pustule”, which sits on the point of penetration of the bacillus (hands, arms, face).

This erythematous papule evolving into a vesicle and then a black eschar succeeded him. The evolution is usually favorable. Very rarely, there appeared a “malignant edema” invasive.

Cutaneous anthrax is an occupational disease (farmers, veterinarians, butchers, tanners …).

2. Internal coal:

It is rare. It can take different forms:

– Pulmonary form: Inhalation of dust after contact with wool,

– Gastrointestinal form: after ingestion of contaminated meat,

– Anthrax meningitis.

These forms have a much worse prognosis and often lead to death, sometimes before having established effective therapy.

IV – PATHOGENICITY METHODS:

The guinea pig is very sensitive to B. anthracis. After inoculation, death occurs in about two days.

V – PATHOGENESIS AND IMMUNITY:

After tissue penetration, the spores germinate and give rise to bacilli which encapsulate, multiply and enter the lymph nodes thereon. This lymph node retention is temporary and the bacilli then pass into the blood (bacteremia).The fixed rate bacilli. When its binding capacity is exceeded, the bacilli drain heavily in the blood and multiply (late sepsis).

Death is due to the action of anthrax toxin. The injection of anthrax toxin in guinea pigs produced similar disorders.

After healing, the subject has protective antibodies induced by the factor II. Non-encapsulated strains of moderate virulence, produce the toxin and are vaccinating.

VI – EPIDEMIOLOGY:

Coal is almost in all parts of the world but especially in Asia and Africa. There are seasonal variations in the incidence of the disease with a maximum in summer, especially during hot, dry summers.

A – The virulent material:

It is mainly the sick animals and their secretion products and excretion.

The bodies are sources of bacilli. The virulence of non-autopsied corpses disappear in a few days because the spores can not be formed.

Skin, hair, horsehair, wool are dangerous to humans.

Blood, muscles, bones (flours, meals) are dangerous to animals and humans.

The contaminated soil is an important and permanent reservoir (80 years).

Buried spores can be brought to the surface by earthworms or floods.

B – Responsiveness:

The animals do not have the same receptivity. In descending order of receptivity, we find the herbivores, omnivores, carnivores, birds.

In humans, the profession is critical. Coal is a professional zoonosis (tanners, butchers, dockers).

C – Mode of transmission:

1. Infection of animals:

It is mainly through the digestive tract (ingestion of grasses, forage, contaminated meat).

So there is contamination of local opportunities (meadows, fields cursed) or remotely (transmission by birds). Trade bone meal imported from countries where anthrax is common (India) can cause the appearance of animal charcoal in regions free.

2. Infection of man:

It is achieved through the skin. It reaches: farmers, veterinarians, slaughterhouse workers and rendering, butchers, tanners, dockers. Apart from this professional, it can occur accidental contamination (meat intake from sick animals).

VII – BACTERIOLOGICAL DIAGNOSIS:

It is often guided by epidemiology: Region (animals) or occupation (man).

A – samples:

1. Animal:

– Fragments of organs,

– Long bone,

– Crushed Bone, compound feed, skins, …

2. Man:

– Sampling of injury or pustule,

– Blood, CSF …

B – bacteriological examinations:

1. Smear blood or body:

Presence of large bacilli in small chains.

A Gram stain, B. anthracis bacilli appear as large, flat pieces (3-5 um / 1-1.25 pm). It appears as short chains in biological samples. Spores are rarely present.

2. Culture:

a / Collecting monomicrobien:

Usual media (blood culture broths, blood agar …)

b / Collection polymicrobial:

With polymicrobial samples (ex. Compound feed …) selecting the spores of B. anthracis by heating at 80 ° C, 5 min prior to seeding.

Selective media

– Medium Pearce and Powell nutrient agar at 40 ug / ml hematin and 60 ug / ml lysozyme, incubated at 40 ° C.

– Medium to Knisely: agar heart extract 30 U / ml polymyxin 40u.g / ml lysozyme, 300 ug / ml EDTA and 40 ug / ml of thallium acetate.

In culture, the chains are longer (bamboo sticks). The presence of a central spore non-deforming subterminal.

On blood agar, non-hemolytic colonies is observed. The colonies are of the type R on blood agar, nutrient agar or TSA-shaped “medusa head”.

They are not capped.

The subculture is to be made on a medium containing 0.5% sodium bicarbonate and incubated in 5% CO2. Virulent strains then produce a capsule, and the colonies become mucosa. This capsule formation is favored by adding to the medium of 0.7% bovine serum albumin.

Fluorescent antibody can be used to detect the bacilli encapsulated directly in tissues, on crops, or a blood smear.(Monoclonal antibodies and polyclonal anti-capsule are available CDC).

One can directly identify the colonies of B. anthracis on the agar. An anti-toxin antibody (Factor II) is deposited in a well at 5 mm of a suspicious colony. After one night at 4 ° C precipitation band is observed between the colony and the immune serum.

B. anthracis can also be identified using miniaturized identification galleries ready to use.

C – Differential diagnosis with other Bacillus:

1. One of the basic elements is the character of mobility:

B. cereus and B. thuringiensis are mobile. B. anthracis is stationary.

2. B. anthracis is sensitive to penicillin, not other Bacillus:

3. The rate of hydrolysis of para-nitrophenyl-glucopyranoside:

maltoside or B. anthracis is increased by the presence in the medium of 1% Triton X-100, whereas it is delayed for B.cereus, B. mycoides and B. thuringiensis.

4. The sensitivity to gamma bacteriophage:

Only B. anthracis is sensitive.

The differential diagnosis between B. anthracis and B. cereus may be supplemented by the following characters:

E – Animal inoculation:

The experimental pathogenicity is valuable to make a definitive diagnosis. Guinea pigs are used (600 g), which is shaved on the sides and which are made scarification. Sampling (polymicrobial, ie: blended food, piece of corpse …) is then deposited on the skin.

When the sample is monomicrobien (study of a strain), the injection is by subcutaneous injection.

The animal dies in a natural attitude, we will search for anthrax on organ smears (spleen).

F – Indirect diagnosis:

1. Reaction Ascoli:

Discloses thermostable polysaccharide antigens of B. anthracis in a sample by a precipitation reaction with an anti-anthrax serum.

Technique: The body part is ground in a mortar with 5 to 10 times its weight of water physiological, heated at 100 ° C for 5 minutes and filtered. Into a capillary is deposited anti-anthrax serum, and then the filtrate without mixing. A precipitate formed at the interface where the reaction is positive.

2. Search Bacillus anti-antibody:

In convalescents coal by precipitation or complement fixation

3. Allergy:

We can highlight an allergy condition B. anthracis by intradermal injection of 0.1 ml “of anthraxine”. A local reaction was observed after 24 hours.

VIII – TREATMENT:

Pet: penicillin

Man: penicillin + serum therapy in severe cases. Resistance to penicillin are exceptional.

Streptomycin and tetracycline were proposed.

In case of malignant edema or “internal coal” penicillin G is administered in high doses (20 million IU / day for adults).

IX – PROPHYLAXIS:

A – Health prophylaxis:

It is difficult and unrealistic because of the persistence of spores in contaminated soil. We can simply try not to create new dangerous areas by monitoring the imports of animals and animal products from infected countries or regions, or by treating them with different techniques (heat, gamma rays, sulfide sodium). In infected area, one must destroy anthrax carcasses by incineration or burial in pits containing quicklime. The prevention of human disease is linked to that of the animal disease.

B – Medical prophylaxis:

In animals, the prophylaxis of coal based on the annual injection of a suspension of spores of an attenuated non-encapsulated strain of B. anthracis in the presence of adjuvant.

In humans, vaccine trials have not passed the experimental stage.

The success of the fight against animal anthrax vaccine based on a regularly applied to sensitive species in polluted areas, sanitary measures associated with the destruction of corpses at the onset of the disease. This disease appears in France in the list of diseases deemed contagious and in the list of occupational diseases compensable for exposed professions.

BACILLUS CEREUS

General:

Bacillus cereus belongs to the genus Bacillus Group 1 Gram (+), optional aero-anaerobic, motile (±), nitrate (+), catalase (+), mannitol. (-) (See B. anthracis) He grows between. 10 and 45 ° C with an optimum at 30-35 ° C (see general characteristics of the genus Bacillus).

This germ is widespread in nature, in the air and the soil, it can contaminate food through its spores.

B. cereus is responsible for food poisoning after massive proliferation of the organism in contaminated food.

Two types of poisoning are described:

– The first, corresponding to the enterotoxin production in vivo, like a poisoning caused by Clostridium perfringens:after incubation for 8-16 h, it appears profuse diarrhea with abdominal pain, nausea. Symptoms resolve in 24 h.

– The second syndrome, corresponding to the ingestion of preformed enterotoxin in food, is characterized by violent vomiting: incubation can be short (30 min to 5 h). The regression of clinical signs is also fast.

I – TOXINS AND enzymes secreted:

B. cereus produces toxins and various enzymes, including a hemolysin is the céréolysine, proteases, phospholipases. Enzymes explain the character of some fulminant ocular infections due to B. cereus. B. cereus also produces more specific toxins as to their action on the gastrointestinal tract.

A – enterotoxin A protein of 50 kDa, also called “toxin diarrhéigène”:

“This toxin acts directly on the gastrointestinal tract. It changes the vascular permeability and causes fluid accumulation in the intestinal lumen. It has a necrotic action and cytotoxic action. It is antigenic and thermolabile.

B – A toxin, also called emetic toxin 5kDa:

It is not antigenic and thermostable. Its production is characteristic of certain strains, related to the stage of sporulation and not produced in excess of 40 ° C.

II – DIETARY RISK:

A – The sources of germs:

B. cereus is a commensal of human and animal. It is common in the soil, on the plant, the spied, cereals (especially rice).

B – incriminated foods:

Many foods can cause poisoning: meat, raw or cooked vegetables, meat preparations and vegetables, pasta, sauces (instant sauces tomato), soups, milk, vanilla cream, apple puree ground dried (eaten after a more or less long shelf). In the case of diarrheal syndromes, food contamination levels B. cereus are close to 107 to 109 seeds / gram.

This multiplication of germs is due to inadequate storage conditions. The prepared rice in advance, heated to a temperature too low to kill spores after a room temperature storage period of up to 2 to 3 days is considered characteristic food originally vomiting (hot season Eastern cuisine).

Note that in France, over 611 episodes of food poisoning identified in 1989, B. cereus has been identified once, but its place in this pathology is probably underestimated. It nevertheless represents 5% of cases of food poisoning in the US.

III – DIAGNOSIS:

Isolation of B. cereus in stool is not enough.

The laboratory diagnosis is based on the enumeration of microorganisms in the food and the identification of the species B. cereus.

Enumeration and isolation environments using the most characteristic properties of the bacteria: haemolytic properties, presence of gelatinase, a Lecithinase, polymyxin resistance, lack of action on mannitol. These properties are used jointly in an egg yolk-based medium, mannitol and polymyxin. These tests are sufficient for a presumptive diagnosis.

Confirmation of B. cereus must be made ​​by other characters: mobility germ, hemolysis (alpha or beta), and resistance to penicillin (B. anthracis is sensitive to penicillin).

B. cereus is differentiated B. mycoides by agglutination tests lectins: B. cereus is agglutinable by soybean lectin, whereas B. mycoides is also agglutinated by lectins Helix pomatia.

Finally serological classification, based on the specificity of flagellar antigens allows to highlight the predominance of certain serotypes in case of poisoning, serovar 1 is most often isolated, probably because of its greater resistance to heat .

IV – TREATMENT AND PREVENTION:

Treatment is not justified, at most an anti-emetic may be necessary.

The prevention of this poisoning is firstly a problem of collective food hygiene where the use of dehydrated products grows (kitchens, restaurants) and also industrial hygiene in the agro-food sector.

Opportunistic BACILLUS

Other Bacillus species can sometimes act as opportunistic pathogens and we know that in recent years, a significant increase of nosocomial infections was noted. Traditionally, in a bacteriology laboratory, these were Bacillus, perhaps hastily assimilated to skin contaminants or air. Currently, we must sometimes give them importance in a wide variety of infections (Table III) where, however, it should be clear the field (immunosuppressed, cancer) and demonstrate the responsibility of Bacillus, especially when repeated isolations from a normally sterile product. Have been involved in various events: B. brevis, B. circulans, B. macerans, B. licheniformis, B. coagulans, B. pumilus, B. sphaericus, B. thuringiensis, B. subtilis.

Recently American authors have placed particular emphasis on the growth of Bacillus endophthalmitis (B. cereus, B. subtilis …).