Bacillus1

3/18/2015Dr. Yasser Al-Tarazi1
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).

• 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

• 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)

• 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.

• 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).

• 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.

• 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

•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.

• 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.

• 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.

• 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)

• 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

• 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.

• 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

Non-hemolytic Bacillus anthracis

Hemolytic Bacilli

• 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

• 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

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

• 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

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