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Bacillus

Family: Bacillaceae

Genus: Bacillus

General characteristics:

They are aerobic (some are facultative anaerobe) grow

best under aerobic condition, large Gram positive or

Gram variable rods with size range (0.5x1.2µm to

2.5x10 µm).

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• Endospore forming bacilli, most of species are catalase positive,

fermentative or respiratory or both. Some of them do not attack

sugar and most are motile.

• According Bergey,s manual the number of Bacillus species exceed

200. The most important of which are:

• Bacillus anthracis

• Other Bacillus spp. :

• B. alvei, B. brevis, B. cereus, B. coagulans,

• B. circulans, B. subtilis, B. licheniformis,

• Some of them are reported to cause disease in human and animals

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• Habitat:

• Most of bacilli spp. are saprophytic, found on vegetation and in soil,

water, air and dust. The bacilli are among the most common

laboratory contaminants

• Bacillus anthracis

• Its highly pathogenic for man and animals, cause disease called

anthrax, primarily of domestic animals.

• Human anthrax are rare but occasionally has been contracted by

farmers, Veterinarian, slaughterhouse workers, and workers at plant

processing imported goat hair, wool, or hides (industrial anthrax)

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• History:

• Discovery of the bacillus that cause anthrax is by Davaine and

Rayer (1863-68). Koch fulfilled his postulates with B. anthracis in

1876-77.

• Distribution:

• B. anthracis is found worldwide and considered always as

pathogen.

• Mode of infection

• The organism is acquired by ingestion, inhalation, wounds,

scratches and through skin. Mechanical transmission of B. anthracis

by blood feeding insects has been reported.

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• Pathogenesis

• Animals died of toxemia, exotoxin was found in the

plasma of dead animals. The anthrax toxin is a complex

consisting of three protein components 1, 11, and 111.

• Component 1 is the edema factor (EF)

• Components 11 is the protective antigen (PA)

• Components 111 is the lethal factor (LF)

• EF and PA form edema toxin that cause edema

• PA with LF form lethal toxin

• Edema toxin with lethal toxin cause maximum mortality

• The three components act synergistically to produce

toxic effects seen in anthrax

• Genome structure

• B. anthracis has a single chromosome which is a circular

DNA molecule. It also has two circular,

extrachromosomal, double-stranded DNA plasmids,

pXO1 and pXO2. Both the pXO1 and pXO2 plasmids are

required for full virulence

•pXO1 plasmid

• The pXO1 plasmid (182 kb) contains the genes that encode for

the anthrax toxin components:

• pag (protective antigen, PA), lef (lethal factor, LF),

and cya (edema factor, EF). These factors are contained within

a pathogenicity island (PAI).

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• The lethal toxin is a combination of PA with LF and the

edema toxin is a combination of PA with EF. The PAI

also contains genes which encode a transcriptional

activator and the repressor, both of which regulate the

expression of the anthrax toxin genes.

• pXO2 plasmid

pXO2 encodes a five-gene operon which synthesizes a

poly-γ-D-glutamic acid (polyglutamate) capsule. This

capsule allows B. anthracis to evade the host immune

system by protecting itself from phagocytosis.

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• Expression of the capsule operon is activated by the

transcriptional regulators, located in the pXO2

pathogenicity island (35 kb). Interestingly, transcriptional

regulators expression are under the control of pXO1.

• In presence of serum the organism produce capsule

and colonies are smooth while in absence of serum

no capsules are produced and colonies are rough.

• Only encapsulated, toxigenic strains are virulent

• The capsule is antiphagocytic

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•Pathogenesis B. anthracis possesses an antiphagocytic capsule

essential for full virulence. The organism also produces

three plasmid-coded exotoxins: edema factor, a

calmodulin-dependent adenylate cyclase, causes

elevation of intracellular cAMP, and is responsible for the

severe edema usually seen in B. anthracis infections;

lethal toxin is responsible for tissue necrosis; protective

antigen (so named because of its use in producing

protective anthrax vaccines) mediates cell entry of

edema factor and lethal toxin.

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• The spores usually enter through the skin or mucous

membranes and germinate at the site of entry.

• In the septicaemic form, the vegetative bacilli spread via the

lymphatic to the blood stream. Deaths is attributed to

respiratory failure and anoxia caused by the toxin, blood fail to

clot and enlargement of the spleen (splenomegaly) is produced

• In localized form lymph nodes of head and neck are infected.

In terminal stage large numbers of bacilli are shed from natural

orifices and animal die.

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• Pathogenicity:

• The organism is obligate pathogen. Peracute, acute, subacute,

chronic and cutaneous forms of the disease were observed.

• The acute form mostly seen in cattle, sheep, horses and mules.

• The cutaneous form occasionally seen in horses and cattle

• In humans: the form of the disease depends on the route of entry

e.g. pulmonary anthrax, cutaneous form (malignant carbuncle or

pustule) and intestinal anthrax.

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• Direct examination:

• Smear from tissues or blood are made and stained with

Grams, Giemsa or wrights stains.

• The microorganism measure 1-1.5x3-8 µm in length,

arranged singly or in short chains. Large square-ended,

Gram positive rods suggest anthrax (Clostridia may be

isolated from tissue and blood but it does not grow

aerobically, have no capsule, and have round ends)

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• The capsule is demonstrated by Giemsa stain were its

appear as reddish-mauve surrounded large square

ended rods known as M, Fadyean reaction and the

capsule is unique consist of poly D-glutamic acid.

• It does not form spores in the living animals. Spores are

formed under conditions unfavorable for multiplication of

vegetative form.

• B. anthracis grow well on blood agar with no haemolysis

in contrast to other saprophytic bacilli

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• Specimens:

• Blood smear, swabs from exuded blood or blood taken by syringe

• In localized form swabs or fluid aspirated from affected lymph nodes

• Isolation and cultivation

• The organism grows well on all laboratory media

• Quinea pigs and mice inoculated with blood or suspension begin to

die within 24 hrs

• Surface colonies appear after 24 hrs incubation and are gray-white,

rough and have a curled edge and under hand lens or stereo -

microscope they exhibit "medusa head" or curled hair-lock and its

non-hemolytic.

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• Identification:

• Based on:

• 1. Pathogenic to quinea pigs and mice

• 2. Characteristic colony morphology; Gram positive rods, spore

• formers, spores centrally located

• 3. Non-motile and aerobic. Other anthracoids are motile

• 4. Virulent strains are encapsulated with square ends

• 5. In presence of 5% CO2 the organism form capsule and the

colonies are smooth and mucoid. At the end of logarithmic phase of

growth, spores begin to appear in the cultures and are numerous

after 48 hours of incubation at 37o C

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Non-hemolytic Bacillus anthracis

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Hemolytic Bacilli

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• The oval spores are visible at the center of the bacilli and stained

with spore stain

• Antigenic nature:

• All strains appear to be antigenically identical. Antigens are

polypeptide capsule, somatic antigen (polysaccharides) and the

toxins

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• Resistance:

• Spores are more resistant to physical and chemical influences than

are the vegetative form. It may survive for 22 years in dried cultures,

remain viable in soil for years and did not affected by freezing

• Destroyed by boiling for 10 minutes and by exposure to dry heat at

140o C for 3 hours

• Disinfectants must be used in high concentration over long period of

time, spores destroyed by 10-20% formalin in 10 minutes and by

autoclaving at 121o C for 15 minutes

• Wool, hides and horse hair should be sterilized by gas sterilization

•Prevention and treatment

A number of anthrax vaccines have been developed for

preventive use in livestock and humans. Infections

with B. anthracis can be treated with β-

lactam antibiotics such as penicillin, and others which

are active against Gram-positive bacteria. Penicillin-

resistant B. anthracis can be treated with

fluoroquinolones such as ciprofloxacin or tetracycline

antibiotics such as doxycycline.

In human the skin form usually treated but the pulmonary form are not

responded. Sick animals with fever should be treated and healthy

animals in the flock should be immunized.

Immunity: By annual vaccination of animals in epidemic areas

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B. cereus

• has been incriminated as the cause of gangrenous bovine mastitis

and abortion in cows and ewes. In human it has been implicated in

food poisoning. Spores germinates in different food where

enterotoxin or emetic toxin may be produced. Two syndrome, the

emetic and the diarrheal are seen

•

• B. subtilis

• claimed to cause conjunctivitis, septicemia, endocarditis, respiratory

infections and food poising in humans

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• B. licheniformis

• cause bovine, ovine and porcine abortions, occasionally may cause

septicemia, peritonitis and food poisoning in humans

• B. stearthermophilus

• Spores are used to test the efficacy of autoclaving and other

sterilizing procedures