VMC 311, Veterinary Bacteriology, Notes compiled by Dr.

Gaurav Singhal

Bacillus

Bacillus is a genus of rod-shaped bacteria and a member of the division

Firmicutes. Bacillus species are obligate aerobes, and test positive for the
enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and
pathogenic species. Under stressful environmental conditions, the cells
produce oval endospores that can stay dormant for extended periods. They
are mainly found in soil and decaying organic matter and are important in
maintaining the fertility of soil. B. anthracis is non hemolytic while other
types are strongly beta hemolytic.

Two Bacillus species are considered disease causing: B. anthracis which

causes anthrax and B. cereus which causes a foodborne illness similar to
that of Staphylococcus. A third species, B. thuringiensis, is an important
insect pathogen, and is sometimes used to control insect pests. B. subtilis, is
a notable food spoiler, causing ropiness in bread and related food and
sometimes cause iridocyclitis, panophthalmitis and fatal septicemia. B.
coagulans is also important in food spoilage.

An easy way to isolate Bacillus is by placing non-sterile soil in a test tube

with water, shaking, placing in melted Mannitol Salt Agar, and incubating at
room temperature for at least a day. Colonies are usually large, spreading
and irregularly-shaped. Under the microscope, the Bacillus appears as rods,
and a substantial portion usually contain an oval endospore at one end,
making it bulge.

The cell wall

The cell wall of Bacillus is a structure on the outside of the cell that
withstands the pressure generated by the cell's turgor. The cell wall is
composed of teichoic acid. B. subtilis is the first bacterium for which the role
of an actin-like cytoskeleton in cell shapes determination and peptidoglycan
synthesis. The role of the cytoskeleton in shape generation and maintenance
is important.

Bacillus anthracis is a Gram-positive spore-forming, rod-shaped

bacterium, with a width of 1-1.2µm and a length of 3-5µm. It can be grown in
an ordinary nutrient medium under aerobic or anaerobic conditions. It is the
only bacterium with a protein capsule (D-glutamate), and the only
pathogenic bacteria to carry its own adenylyl cyclase virulence factor
(edema factor). It bears close genotypical and phenotypical resemblance to
Bacillus cereus and Bacillus thuringiensis. All form oval spores. Bacillus
anthracis spores in particular are highly resilient, surviving extremes of
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

temperature, low-nutrient environments, and harsh chemical treatment over

decades or centuries.

Historical background

Casimir Davaine first isolated this bacterium from the blood of sheep
suffering from anthrax. B. anthracis was the first bacterium conclusively
demonstrated to cause disease, by Robert Koch in 1877. The species name
anthracis is from the Greek anthrakis meaning coal and referring to the most
common form of the disease, cutaneous anthrax, in which large black skin
lesions are formed.

Distribution and Transmission

Anthrax is found all over the world. India has anthrax as a major livestock problem.
The organism is spread by soil contaminated feed and water. Hay from pasture land
transmits this disease. Carnivorous animals get infection by feeding on dead
carcasses. Birds also spread the infection by eating infected meat and eliminating
the spores in their faeces. Blood sucking flies also spread Anthrax.

Man contract the infection by contact with infected animals, by handling infected
hides, by sorting wool, from shaving brushes, from contaminated furs and hair
cushions and from insect bites.

Morphology and Staining

The bacilli are cylindrical rods. The capsule is not formed in the animal body. Spores
germinate at the pole young cell are gram positive but old cells may decolorize
easily.

Growth requirements and characteristics

B. anthracis grow readily on blood agar and is easily isolated from the blood or
internal organs of an infected animal. It is aerobic and the optimum temperature for
growth is 37o C. A slightly alkaline medium (pH 7.5 to 7.8) is most conducive to
good growth. On the agar surface, colonies are dull, opaque, grayish-white with an
irregular boarder from which long strands of cell are seen in parallel arrangement
giving the typical “medusa head” characteristics. In old colonies, vesicles appear on
the surface giving it a contoured surface. Broth is turbid with a floccular growth on
the surface which sinks to the bottom of the tube within 24 hours. In a gelatin stab
culture, filaments of growth radiate from the line of puncture and give the
appearance of “inverted fir tree”.

Resistance

The vegetative cells of B. anthracis are not resistant, however spores which
are resistant are rapidly formed by this organism. Spores will remain viable
for years and are very virulent. Hair, wool, bones and hides if not treated
sufficiently to kill spores may remain infective for years. The organism is
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

rapidly destroyed by decomposition in un-opened carcasses after the third

day at a temperature greater than 10 degree C. Repeated freezing and
thawing may kill vegetative cells but not spores. Mercuric chloride, Formalin,
Sodium Hydroxide and Hydrochloric Acid act as disinfectants for B.
antharacis.

Biochemical Properties

B. antharacis form acid but no gas from glucose, sucrose, maltose, fructose,
trehalose and dextrin. Indol and H2S are not produced. Nitrates are reduced
to nitrites. The organism is methyl red positive and voges-proskauer
variable. Litmus milk is coagulated, decolorized and slowly peptonized.

Antigenic Structure and Toxins

There are two antigenic substance and B. antharacis. One protein like
material is found in the capsule and the other is a somatic polysaccharide
antigen. Stanley and Smith found three factors in Anthrax Toxin. Factor I is
not toxic when give alone but when mixed with purified Factor II edema is
produced in the skin of the rabbit and mice are killed. Factor III is different
antigenically from I and II, it is present in anthrax toxin produced in vivo and
is non-lethal to mice when injected alone. It is lethal to mice when mixed
with II but not with I.

Pathogenesis

Three forms of anthrax disease are recognized based on their form of

inoculation.

• Cutaneous (Malignant carbuncle): the most common form (95%),

causes a localized inflammatory black necrotic lesion. Regional lymph
nodes are swollen and painful. If the organism enters the blood stream
septicemia results.
• Pulmonary (Wool sorters disease): highly fatal and characterized by
sudden massive chest edema followed by cardiovascular shock.
• Gastrointestinal: rare but also fatal (causes death to 25%) type results
from ingestion of spores

On postmortem examination, anthrax is characterized by edematous

infiltration in the subcutaneous tissues, hemorrhages in various parts of the
body, marked swelling of the spleen (raspberry-jam consistency). This
characteristic spleen has given rise to the terms “Splenic fever” and
“Milzbrand”. The blood is dark red, tarry and does not clot (hence also called
as “Charbon”). Hemorrhage is often observed from the natural body
openings

Immunity
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

Spore vaccine and hyper immune anti-anthrax serum are used for
prophylactic immunization.

Diagnosis

The postmortem should not be done as opening the carcass may spread
anthrax spores in the environment. Sending blood smears, cotton swabs, a
small vial of blood or tissue is preferable if sufficient caution is taken.
Laboratory diagnosis of anthrax is done by any of the following:
1. Direct microscopic smears from the suspected material stained with a
suitable capsule stain an observing capsule surrounding the bacteria.
2. Inoculation in blood agar where no hemolysis is produced and the
typical curly edge of the colony is seen
3. Subcutaneous inoculation of suspected material guinea pig in which
death is usually produced in 48 hours. Gelatinous edema and
hemorrhage are found at the inoculation site, and numerous bacilli are
seen in stained smears made from fluid in the area.

Treatment

Infections with B. anthracis can be treated with β-lactam antibiotics such as

penicillin, and others which are active against Gram-positive bacteria.

Bacillus subtilis

History: In 1835, the bacterium was originally named Vibrio subtilis by

Christian Gottfried Ehrenberg, and renamed Bacillus subtilis by Ferdinand
Cohn in 1872.

Distribution and Transmission: Bacillus subtilis, known as the hay

bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium
commonly found in soil. A member of the genus Bacillus, B. subtilis is rod-
shaped, and has the ability to form a tough, protective endospore, allowing
the organism to tolerate extreme environmental conditions. Unlike several
other well-known species, B. subtilis has historically been classified as an
obligate aerobe. They are transmitted by water, wind and feed and are
present all over the world.

Morphology and Staining: Gram positive, cylindrical rods with rounded

ends, singly or in chains and are motile with peritrichic flagella. Spores are
oval and centrally located.

Growth requirements and characteristics: Surface colonies on Agar are

small, grayish, and amoeboid with crenate margins. The surface is finally
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

granular and dull. The growth is membranous, slightly sticky and is

emulsified with difficulty. A thick ring pellicle which usually sinks within 24
hours forms on broth.

Resistance: It forms spores and hence is extremely resistant to heat. The

bacteria are killed at boiling temperature in 2 hours or at 120 oC in 15
minutes. Ordinary chemical disinfectant kills them on prolonged contact. B.
subtilis is sensitivce to penicillin.

Biochemical properties: Acid is formed from glucose, sucrose and

maltose, indol not formed, nitrates are reduced, it is methyl red negative and
Voges-Proskaeur positive, reduced methylene blue and coagulates litmus
milk.

Pathogenesis

B. subtilis is not considered a pathogen; it may contaminate food but rarely

causes food poisoning. B. subtilis spores can survive the extreme heating
that is often used to cook food, and it is responsible for causing ropiness — a
sticky, stringy consistency caused by bacterial production of long-chain
polysaccharides — in spoiled bread dough.

Reproduction

B. subtilis can divide symmetrically to make two daughter cells (binary

fission), or asymmetrically, producing a single endospore that is resistant to
environmental factors such as heat, acid, and salt, and which can persist in
the environment for long periods of time. The endospore is formed at times
of nutritional stress, allowing the organism to persist in the environment until
conditions become favorable.

B. subtilis is used as a soil inoculant in horticulture and agriculture. B. subtilis

has been used for a biowarfare simulant as it can convert explosives into
harmless compounds of nitrogen, carbon dioxide, and water and plays a role
in safe radionuclide waste disposal with the proton binding properties of its
surfaces

Enzymes produced by B. subtilis and B. licheniformis are widely used as

additives in laundry detergents.