Anda di halaman 1dari 6

VMC 311, Veterinary Bacteriology, Notes compiled by Dr.

Gaurav Singhal

CLOSTRIDIUM
The members of the genus Clostridium are large anaerobic gram-positive
rods of variable length and breadth, ranging from long filamentous forms to
short plump bacilli. In an appropriate environment, individual bacilli of most
species produce a single spherical or ovoid spore that may be located
centrally, subterminally, or terminally within the vegetative cell. In most
instances the spores appear as swollen bodies since they are generally wider
than the diameter of the rods in which they develop. The shape and position
of the spore, as well as the swelling of the vegetative cell are characteristics
that may contribute to species identification as shown in table at the end.

Spores of Clostridium species are not stained by ordinary methods:


therefore, in gram- and methylene blue-stained smears, spores are
evidenced as unstained areas within the dark stained cytoplasm of
vegetative bacilli or as free hyaline bodies. The relatively impervious spore
bodies may be effectively stained using the Wirtz-Conklin technique.
CAUTION: Ordinarily heat fixation in preparing slide for staining may not
destroy all spores. Strict aseptic technique should be used; a bacteriological
hood may be useful in work with spore formers.

The majority of the clostridia are motile except Clostridium perfringens which
is non motile.

GROWTH REQUIREMENT OF CLOSTRIDIA


The clostridia are obligate anaerobes. Growth may be obtained over a wide
range of temperatures; however, 37º C is optimum for pathogenic species.
Although variations in nutritive requirements throughout the clostridia do
exist, they may be successfully isolated from specimens using blood agar
and thioglycollate broth. Anaerobic conditions may be provided by
incubating the inoculated blood agar plates in a Brewer anaerobic jar or
several other methods.

APPEARANCE OF CLOSTRIDIA
Stained smears of growth usually reveal spores, except when CI. Perfringens
is present. This species fails to sporulate on most media, especially those
media containing carbohydrates. On blood agar, after 48 hours anaerobic
incubation at 37º C, typical colonies of the various Clostridium species
appear as described in Table at the end. Most clostridia produce distinct beta
hemolysis on blood agar. Clostridium perfringens however, may exhibit a
"target" appearance or double zone of hemolysis. This is shown by a definite
narrow 1 to 2 mm zone, immediately around the colonies, which is
surrounded by a wide 4 to 5 mm zone of partial hemolysis. Although the
microscopic and colonial morphology of certain clostridia may appear quite
distinctive, final identification rests with the performance and interpretation
of biochemical tests.
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

PATHOGENICITY OF CLOSTRIDIA
Since the natural habitat of the clostridia is worldwide in soil or in the
intestinal tract of man and animals, pathogenic species are always present
and may cause disease when the opportunity arises. The pathogens are
those organisms responsible for botulism, tetanus, and gas gangrene.
a. Tetanus. Clostridium tetani causes tetanus. This disease results from the
introduction of spores (from soil or feces) of the organism into puncture
wounds, burns, surgical sutures, or other traumatic injuries. Spores of the
organism germinate to form the vegetative bacilli that multiply and produce
a powerful exotoxin at the expense of the necrotic tissue, created by
vascular destruction. The exotoxin spreads through rapid absorption and
acts particularly on the tissue of the spinal cord and peripheral motor nerve
endings. Toxemia is first evidenced by muscle spasms near the site of
infection with subsequent spasms of the jaw muscles (lockjaw). The
intoxication progresses to the nerves of other voluntary muscles causing
tonic spasms, convulsions and, ultimately, death. Prevention of disease rests
with active immunization of the animals with toxoid, or passive immunization
with antitoxin as soon as possible after injury.

b. Botulism.
(1) Clostridium botulinum is responsible for a fatal type of food poisoning,
botulism. The disease is an intoxication rather than an infection in that
outbreaks occur following ingestion of food in which C.botulinum has grown
and produced a highly potent exotoxin. Within the anaerobic environment of
the foodstuff, the spores germinate to form vegetative bacilli, which in turn,
produce toxin. Since the spores of C.botulinum will withstand a temperature
of 100º C for at least 3 to 5 hours, they present a definite hazard to canned
food. Toxin-containing foods may appear spoiled and rancid; cans may be
swollen due to gas formation by the organism. The toxin is destroyed by
heating the food at 100º C for 10 minutes.
(2) There are five antigenic types of C.botulinum toxin, designated A, B, C, D
and E. Type A toxin is one of the most poisonous substances known. After
approximately 18 to 48 hours following the consumption of toxic food,
neurotoxic manifestation is evidence in visual disturbances, inability to
swallow, and speech difficulty. Progressive signs of bulbar paralysis are
exhibited and these lead to fatal termination from respiratory failure or
cardiac arrest. Since the various toxin types are highly antigenic, potent
antitoxins may be obtained by injecting toxoids into animals. Polyvalent
antitoxin is administered intravenously to affected individuals.

c. Gas Gangrene.
(1) The Clostridium species most commonly associated with gas gangrene
are C. perfringens, C. novyi, and C. septicum. Clostridium perfringens is the
most frequent cause and is found either alone or mixed with other
anaerobes.
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

(2) Gas gangrene often develops as a complication of severe traumatic


injuries such as dirty, lacerated wounds, especially those accompanying
compound fractures. In these and other injuries, the circulation to a local
tissue area is often impaired or destroyed. The resulting necrotic tissue, void
of oxygen and rich in nutrients, affords an ideal anaerobic environment in
which the spores of gangrene organisms may germinate and multiply. The
organisms actively metabolize tissue carbohydrates to acid and gas. The
gangrenous process extends to other tissues primarily as a result of
exotoxins excreted by pathogenic clostridia. The exotoxins include
hyaluronidase, lecithinase, and collagenase.
(3) In addition, other enzymes may be present which exhibit hemolytic,
necrotizing and lethal effects on tissues. Gas gangrene is usually a mixed
infection composed of toxigenic and proteolytic clostridia and other aerobic
and anaerobic, grampositive organisms.

d. Black leg in cattle

It is caused by C. chauvoei and is characterized by a swelling edema and


emphysema of the muscle and the subcutaneous tissues of the infected part.
Infection occurs most commonly in shoulder and hind quarters. The swelling
increases rapidly in size and the emphysema is soon detected by the
crackling sound when the hand is drawn firmly across the affected part. He
body becomes distended with gas and the subcutaneous tissues of the
affected part become gelatinous. The underlying muscle are dark brown or
blackish. The disease usually results fatally in cattle.

e. Malignant edema

It is caused by C. septicum and is usually an acute febrile disease that


terminates fatally in two or three days. In man, death occurs in 24 hours. Gas
may not be present in great quantity, although gas bubbles are present. The
edema is blood stained and usually gelatinized. Muscle underlying
subcutaneous infection is generally dark red in colour soft in consistency.
Hemoglobin stained fluid is found in body cavities and pericardial sac.

f. Braxy
It is caused by C. septicum in sheep. In this disease, lesions are confined to
the tissues adjacent to the fourth stomach and the mucosa and sub mucosa
of stomach. Intense inflammation characterized by edema and hemorrhage
with some necrosis is found. Degenerative changes are found in the visceral
organs.

IDENTIFICATION OF CLOSTRIDIUM SPECIES


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

a. Specimens.
(1) Although bacteriological examination of swab specimens and tissues from
contaminated wounds may yield Clostridium tetani, such materials are rarely
submitted for tetanus.
(2) In cases of botulism, specimens from the patient are almost of no value,
since the disease is an intoxication rather than an infection. Laboratory
diagnosis is accomplished by demonstrating Clostridium botulinum toxin in
leftover food. The organism may be isolated from the food and identified on
the basis of biochemical tests and the demonstration of toxin production.
(3) From cases of gaseous gangrene, exudates from wounds may be
collected with a sterile swab or aspirated with a sterile syringe and needle.
Infections due to Clostridium species are readily recognized when typical
gaseous gangrene occurs.

b. Direct Examination. Gram-stained smears of specimens may reveal the


presence of large gram-positive rods with or without spores. Frequently,
specimens from gangrenous lesions are contaminated with gram-negative
rods and gram-positive cocci.

c. Examination of Cultures.
(1) If growth of large gram-positive bacilli is obtained in an anaerobic culture,
a member of the genus Clostridium should be suspected, provided aerobic
plates are negative.
(2) In addition to Clostridium species, gangrenous infections may contain
coliform bacilli, Pseudomonas species, or members of the genus Proteus.
Under such circumstances, the primary anaerobic blood agar plate may be
overgrown with these organisms, thereby making the isolation of
Clostiridium species difficult or impossible.
This may be overcome by incubating the sample in primary thioglycollate
broth cultures for 48 to 72 hours. Over this period of time, the gram-negative
bacilli will greatly decrease in numbers, allowing isolation of clostridia in
subculture.

d. Isolation Clostridium from Mixed Cultures.


The freshly inoculated broth is heated in a water bath at 80º C for 15 to 30
minutes. This will destroy all vegetative growth of bacteria, but will not
destroy the spores. The heated medium is then incubated for 24 to 48 hours,
resulting in a pure culture of the Clostridium species. When isolated colonies
are obtained on blood agar, the growth should be carefully picked and
transferred to thioglycollate broth. Following incubation, this pure culture is
used to carry out the confirmatory studies.
VMC 311, Veterinary Bacteriology, Notes compiled by Dr. Gaurav Singhal

e. Confirmatory Laboratory Tests. Cultural isolates may be distinguished


as belonging to the genus Clostridium on the basis of strict anaerobiasis, and
microscopic and colonial morphology. Final identification of a Clostridium
species is dependent upon the results of biochemical studies and/or the
demonstration of exotoxin production (C. tetani and C. botulinum).

Morphological and biochemical differentiation of clostridium species


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

Anda mungkin juga menyukai