Learning Objectives

INTRODUCTION
The genus Clostridium includes all anaerobic, grampositive bacilli capable of forming endospores. Spores
of clostridia are usually wider than the diameter of
the rods in which they are formed, giving the bacillus
a swollen appearance resemling a spindle (Clostridium
is Latin for ‘little spindle’). The name Clostridium is
derived from the word ‘Kloster’ (meaning a spindle).
Most of the species are saprophytes that normally
occur in soil, water and decomposing plant and animal
matter. They play an important part in natural processes
of putrefaction. A few species are opportunistic pathogens. The genus contains bacteria responsible for three
major diseases of human beings—gas gangrene, food
poisoning and tetanus.
GENERAL FEATURES OF CLOSTRIDIA
1. Morphology
The clostridia are gram-positive typically large, straight
or slightly curved rods, 3-8 × 0.6-1 μm with slightly
rounded ends. Gram-positive, gram-negative and pleomorphism forms are common. Most species of clostridia
are motile with peritrichous flagella except Cl. perfringens and Cl. tetani type VI which are nonmotile. All
clostridia are noncapsulated with the exception of Cl.
perfringens and Cl. butyricum.
All produce endospores. Spores of clostridia are usually wider than the diameter of the rods in which they
are formed. In the various species, the spore is placed
centrally, subterminally, or terminally. The position of
the developing sporoes within the vegetative cell is useful in identifying the species (Table 30.1).
2. Culture
Most species are obligate anaerobes. A few species grow
in the presence of trace amounts of air and some actually grow slowly under normal atmospheric conditions.
Clostridia grow on enriched media in the presence of
reducing agent such as cysteine or thioglycollate (to
maintain a low oxidation-reduction potential), or in an
O
2-free gaseous atmosphere provided by an air evacuated glove box, sealed jar, or other device.

Growth is relatively slow on solid media. Some

organisms produce large raised colonies with entire
margins (e.g. C. perfringens). Others produce smaller
colonies that extend in a meshwork of the fne flaments
(e.g. C. tetani). Many clostridia produce a zone of hemolysis on blood agar.
Liquid media like cooked meat broth (CMB) or thio
glycollate media (containing reducing agent thioglycollste and 0.1% agar) are very useful for growing clostridia.
A very useful medium is Robertson’s cooked meat
broth. It contains unsaturated fatty acids which take
up oxygen the reaction being catalyzed by hematin in
the meat, and also sulfhydryl compounds which bring
about a reduced OR potential. Clostridia grow in the
medium, rendering the broth turbid. Most species produce gas.

Clostridium
Chapter 30 ♦ Clostridium
291
 Table 30.1: A
morphological
and
biochemical
classifcation of
clostridia as
human
pathogens

Slightly pro Neither

Both Saccharolytic
Position teolytic but proteolytic
proteolytic and but not pro
of spores not nor
saccharolytic teolytic
saccharolytic saccharolytic

Proteolytic Saccharolytic
predominating predominating

Cl.
bifermentans
Cl. perfringens
Cl. botulinum Cl. fallax
Central or Cl. septicum
1. A.B.F. Cl. botulinum
subterminal Cl. chauvoei
Cl. histolyticum C.D.E
Cl. novyi
Cl. sordellii
Cl. sporogenes

Oval and Cl.

2. — Cl. diffcile — Cl. tertium
terminal cochlearium

Cl.
tetanomor
Spherical
3. — — CI. tetani phum
and terminal
Cl.
sphenoides

3. Biochemical Reactions
These organisms are biochemically active. On the basis
of biochemical reactions many clostridia can be divided
into: 1. Predominantly saccharolytic clostridia; 2. Predominantly proteolytic clostridia; 3. Slightly proteolytic
clostridia; (Table 30.1). Proteolytic clostridia turn
the meat black and produce foul odor and saccharolytic species turn the meat pink.
4. Resistance
The vegetative cells of clostridia do not differ from nonsporing bacilli in their resistance to physical and chemical
agents. Spores of Cl. botulinum may withstand boiling after 3 to 4 hours and even at 105°C may not killed
be completely in less than 100 minutes. Spores of most
strains of Cl. perfringens are destroyed by boiling for less
than fve minutes, but those of some Type A strains that
cause food poisoning survive for several hours. Cl. tetani
spores persist for years in dried earth or dust. All species are killed by autoclaving at 121°C within 20 minutes.
Among hospital disinfectants the greatest sporicidal activity is shown by alcoholic hypochlorite and
glutaraldehyde.
In general, clostridia are susceptible to metronidazole, penicillin chloramphenicol and erythromycin; less
so to tetracyclines, and resistant to aminoglycosides and
quinolones.
5. Diseases Produced
Clostridia are more commonly associated with skin and
soft tissue infections, food poisoning, and antibioticassociated diarrhea and colitis (Table 30.1).
CLASSIFICATION
The traditional method for classifying an isolate in the
genus Clostridium was based on a combination of diagnostic tests, including the demonstration of spores,
optimal growth in anaerobic conditions, a complex pattern of biochemical reactivity such as saccharolytic and
proteolytic capacities (Table 30.1) and the fndings yielded by gas chromatography analysis of the metabolic
by-products. With these methods, more than 130 species have been defned. Fortunately, most of the clinically
important isolates fall within a few species.
Clostridia of medical importance may also be considered under the diseases they produce classifcation
below).
CLOSTRIDIUM PERFRINGENS
(Cl. welchii, Bacillus aerogenes capsulatus,
B. phlegmonis emphysematosae)
The bacillus was originally cultivated by Achalme (1891)
but was frst described in detail by Welch and Nuttall
(1892) as Bacillus aerogenes capsulatus, who isolated it
from the blood and organs of a cadaver. It has been commonly known as C. welchii, especially in the UK.
Cl. perfringens is a normal inhabitant of the large
intestines of human beings and animals. The spores are
commonly found in soil, dust and air.
Morphology
It is a relatively large gram-positive bacillus (about 4-6 ×
1 μm) with straight, parallel sides and rounded or truncated ends, occurring singly or in chains or small bundles. It is
pleomorphic, and flamentous and involution
forms are common. It is capsulated and nonmotile.
Spores are typically oval, central or subterminal and
not bulging but are rarely seen in artifcial culture or in
material from pathological lesions, and their absence
is one of the characteristic morphological features of
Cl. perfringens. Special media normally must be used to
demonstrate sporulation.
Cultural Characteristics
It is an anaerobe but can also grow under microaerophilic conditions. It grows over a pH range of 5.5 to
8.0 and temperature range of 20°C to 50°C (optimum
temperature range 37-45°C). Robertson’s cooked meat
Section 3 ♦ Systemic Bacteriology
292
broth inoculated with mixtures of Cl. perfringens and
other bacteria and incubated at 45°C for 4 to 6 hours
serves as enrichment. Blood agar plates streaked after
that time and incubated at 37°C will have proportionally higher numbers of Cl. perfringens (yield pure or
predominant growth of Cl. perfringens). Good growth
occurs in Robertson’s cooked meat medium. The meat
is turned pink but is not digested. The culture has an
acid reaction and a sour odor.
It grows best on carbohydrate-containing media
such as glucose blood agar. Surface colonies are large,
smooth, regular, convex, slightly opaque disks. Colonies
of most strains demonstrate a ‘target hemolysis’ after
overnight incubation on rabbit, sheep, ox, or human
blood agar. It results from a narrow zone of complete
hemolysis due to theta toxin and a much wider darker
zone of incomplete hemolysis due to the α-toxin. On
longer incubation this double zone pattern of hemolysis
may fade.
C. perfringens also produces a characteristic pattern
of synergistic b-hemolysis when streaked alongside
Streptococcus agalactiae (the reverse CAMP test) (Fig.
30.1). Other types of colonies include one with a raised
opaque center and a flat radially striate transparent border. Rough flat colonies with an irregular edge resembling a
vine leaf may also occur. A variant occasionally
produces very mucoid broth cultures and tenacious
colonies on blood agar.
Biochemical Reactions
It is actively saccharolytic. Glucose, maltose, lactose and
sucrose are fermented with the production of acid and
gas. It is indole negative, MR positive and VP negative.
Hydrogen sulfde is produced abundantly; sulfte is
actively reduced; most strains reduce nitrates to nitrites.
In litmus milk medium, fermentation of lactose
leads to formation of acid, which is indicated by the
change in the color of litmus from blue to red. The acid
clots the milk—casein (acid clot) and the clotted milk is
disrupted due to the vigorous gas production. This is
known as ‘stormy fermentation or ‘stormy clot’ reaction that is produced by almost all strains of C. perfringens
but is not specifc for this organism.
Resistance
Spores are usually destroyed within fve minutes by
boiling but those of the ‘food poisoning’ strains of Type
A and certain Type C strains resist boiling for 1 to 3
hours. Autoclaving of 121°C for 15 minutes is lethal.
Spores generally resist routinely used antiseptics and
disinfectants with the exception of formaldehyde and
glutaraldehyde C. perfringens is sensitive to penicillin,
erythromycin, many cephalosporins and metronidazole. It is generally sensitive to clindamycin and typically
resistant to aminoglycosides.
Toxins
Cl. perfringens is one of the most prolifc of toxin-producing bacteria, forming at least 12 distinct soluble substances
or toxins, all of which are of protein in nature
and antigenic. Major lethal toxins include: α (alpha), b
(beta), e (epsilon) and i (iota) and minor lethal toxins
include: g (gamma), d (delta), k (kappa), l (lambda),
m (mu), h (nu), q (theta) and n (eta). The four ‘major toxins’, alpha, beta, epsilon and iota, are predominantly
responsible for pathogenicity (Table 30.2).
Classification
C. perfringens can be divided into fve types, A to E on
the basis of four major toxins (Table 30.3). Typing is
done by neutralization tests with specifc antitoxins by
intracutaneous injection in guinea-pigs or intravenous
injection in mice. Strains of C. perfringens type A that
produce enterotoxin are associated with a mild form of
food poisoning.
Alpha Toxin
The alpha (α) toxin is produced by all types of Cl. perfringens and most abundantly by Type A strains, is a
lecithinase (phospholipase C) that lyses erythrocytes,
platelets, leukocytes, and endothelial cells. It is lethal,
dermonecrotic and hemolytic for the red cells of most
species, except horse and goat. The lysis is of the hotcold variety, being best seen after incubation at 37°C followed
by chilling at 4°C. This toxin increases vascular
permeability, resulting in massive hemolysis and bleeding, tissue destruction, hepatic toxicity, and myocardial
Fig. 30.1: Reverse CAMP test

Table 30.2: Clostridia as

human pathogens
Cl. perfringens
A. The gas gangrene group:
Cl. septicum
1. Established pathogens
Cl. novyi

Cl. histolyticum
2. Less pathogenic
Cl. fallax

Cl. bifermentans
3. Doubtful pathogens
Cl. sporogenes

B. Tetanus: Cl. tetani

C. Food poisoning:
Cl. perfringens (Type A)
1. Gastroenteritis
Cl. perfringens (Type C)
2. Necrotizing enteritis
Cl. botulinum
3. Botulism

D. Acute colitis Cl. diffcile

Chapter 30 ♦ Clostridium
293
Fig. 30.2: Nagler’s reaction. Cl. perfringens colonies on the
right half of the plate are surrounded by haloes, while colonies
on the left half (containing antiserum to alpha toxin) have no
haloes around them
dysfunction. It is relatively heat stable and is only partially inactivated by boiling for fve minutes.
Nagler’s Reaction
Basis
The alpha (α) toxin is lecithinase C (or phospholipidase
C) splits lecithin into phosphoryl choline and diglyceride, in the presence of Ca++ and Mg++ ions because the
toxin is activated by Ca++ and Mg++ ions. This reaction
is seen as an opalescence in serum or egg-yolk media
and is specifcally neutralized by the antitoxin. This is
the basis of Nagler’s reaction.
Procedure
For rapid detection of C. perfringens, a culture plate containing 6 percent agar, 5 percent peptic digest of sheep
blood and 20 percent human serum or 5 percent eggyolk is prepared. The incorporation of neomycin sulphate in the
medium makes it more selective, inhibiting
coliforms and aerobic spore bearers. Human serum may
be replaced by 5 percent egg-yolk. Willis and Hobbs
medium also incorporates lactose and neutral red to
indicate lactose-fermenting organisms and milk to indicate proteolysis.
The plate is dried. On one half of the plate, 2 to 3
drops of C. perfringens antitoxin are spread and allowed
to dry. The plate is then inoculated with the test organisms or the exudate under investigation and incubated
anaerobically at 37°C for 18 hours.
Interpretation
On the section containing no antitoxin, C. perfringens
colonies show surrounding zone of opalescence, i.e.
Nagler’s reaction. There will be no opacity around the
colonies on the half of the plate with the antitoxin, due to
the specifc neutralization of the alpha toxin. (Fig. 30.2).
This reaction, however, is not totally specifc for
C. perfringens since the opalescence in the egg-yolk media
may be produced by other lecithinase forming bacteria
(Cl. novyi, Cl. bifermentans, some vibrios, some aerobic
spore bearers). The reaction produced by C. perfringens
is specifcally neutralized by C. perfringens antitoxin, but
serologically related phospholipases of C. bifermentans
and C. sordellii and some other phospholipases are also
inhibited. These organisms can be separated by other
tests.
Other Major Toxins (Table 30.4)
Beta (b), epsilon (e) and iota (i) toxins have lethal and
necrotizing properties.
Minor Toxins
Gamma and eta toxins have minor lethal toxins. Delta
toxin has a lethal effect and is hemolytic for the red cells
of even goat, pigs and cattle. Theta toxin is oxygenlabile hemolysin antigenically related to streptolysin O.
It is also lethal and a general cytolytic toxin.
Enterotoxin
C. perfringens type A strains produce a potent enterotoxin. Properties of the enterotoxin are erythema after
intracutaneous injection, fluid accumulation in ligated
rabbit ileal loop, lethality for mice, and diarrhea when
fed orally to monkeys, rabbits and human volunteers.

Table 30.3:
Toxins
produced
by Cl.
perfringens
types

Major Minor
Type Pathogenicity
toxins toxins
α b e i g d h q k l m n

Gas gangrene:
Wound
infections, +++
A -- -- -- -- -- - --
septicemia +++
Food
poisoning
 Lamb
B +++ +++ ++ - + - ++ - +++ +++ ++
dysentery

Enteritis in
animals
+++ +++ - +++ +++
C Enteritis -- -- -- -- -- -- +++
+++ ++ ++ - -
necroticans in
human beings

Enterotoxemia
D +++ - +++ - - - - ++ ++ ++
of sheep

Doubtful
pathogen of
E +++ - - +++ - - - ++ ++ ++
sheep and
cattle

Section 3 ♦ Systemic Bacteriology

294
Pathogenesis
1. Soft Tissue Infections
Soft tissue infections caused by C. perfringens are subdivided into: (1) cellulitis, (2) fasciitis or suppurative
myositis, and (3) myonecrosis or gas gangrene.
i. Cellulitis
Anaerobic cellulitis is a more serious form of wound
infection. Clostridial species can colonize wounds and
skin with no clinical consequences.
ii. Fasciitis or Suppurative Myositis
Cellulitis process can progress to suppurative myositis
characterized by an accumulation of pus in the muscle
planes, but muscle necrosis and systemic symptoms are
absent.
iii. Clostridial Myonecrosis or Gas Gangrene
The disease is characterized by rapidly spreading edema,
myositis, necrosis of tissues, gas production and profound toxemia occurring as a complication of wound
infection. The disease has been referred to in the past
as ‘malignant edema’. Other descriptive terms that have
been used are ‘anaerobic (clostridial) myositis’ and
‘clostridial myonecrosis.
Etiology
Generally, several species of clostridia are found in
association with anaerobic streptococci and facultative anaerobes such as E. coli proteus and staphylococci.
Amongst the pathogenic clostridia, Cl. perfringens is
the most frequently encountered (approximately 60%),
and Cl. novyi and Cl. septicum being the next common
(20-40%), and Cl. histolyticum less often. Other clostridia
usually found are Cl. sporogenes, Cl. fallax, Cl. bifermentans, Cl. sordellii, Cl. aerofoetidum and Cl. tertium. Since
anaerobic infections are due to wound contamination,
they are always polymicrobial.
Mechanism of Infection
Infection usually results from contamination of a wound
with soil, particularly from manured and cultivated
land. Clostridial spores are introduced into tissue, e.g.
by contamination with dirt, or by endogenous transfer
from the intestinal tract. After injury there is an incubation period may be as short as seven hours or as long as
six weeks, usually of 12 to 48 hours, before symptoms
suddenly appear.
The spores germinate and grow rapidly if the normal tissue oxidation-reduction potential is lowered, as
occurs when there is considerable cell injury or compromise of circulation. These lesions almost always involve
coinfection with several species of organisms, including
clostridia and other anaerobes, and facultative species
that use up available O2, thus protecting the anaerobes
from oxygen’s toxic effects.
Germination and outgrowth of clostridia spores
occurs. Alpha toxin and other exotoxins are secreted and
extensive cell killing ensues. The production of enzymes
that break down ground substance facilitates the spread
of infection. Fermentation of tissue carbohydrates yields
gas, and an accumulation of gas bubbles in the subcutaneous spaces produces a crinkling sensation on palpation
(crepitation), hence the name gas gangrene. The
exudates are copious and foul smelling. As the disease
progresses, increased capillary permeability leads to the
exotoxins being carried by the circulation from damaged tissue to other organs, resulting in systemic effects
such as shock, renal failure, and intravascular hemolysis. Untreated clostridial myonecrosis is uniformly fatal
within days of the initiation of gangrene.
2. Septicemia
The isolation of C. perfringens or other clostridial species in blood cultures can be alarming. Invasion of the
bloodstream may occur in association with malignancy
and may involve a localized myonecrosis in addition to
a fulminating clostridial septicemia.
3. Food Poisoning
The organisms usually involved are strains of type A
that produce heat resistant spores and minimal amounts
of theta toxin. Meat, chicken, fsh, and their by-products are the most common vehicles for clostridial food
poisoning.
Clostridial food poisoning, a relatively common but
underappreciated bacterial disease, is characterized by:

Table 30.4: Virulence

factors of Clostridium
perfringens

Virulence
Biologic activity
factors

Lethal toxin; phospholipase C

(lecithi
nase); increases vascular
α toxin
permeability;
hemolysin; produces
necrotizing activity

Lethal toxin; necrotizing

b toxin
activity
 Є toxin Lethal toxin; permease

Lethal binary toxin;

necrotizing activity;
d
adenosine diphosphate (ADP)
ribosylating

i toxin Hemolysin

Heat and oxygen-labile

q toxin hemolysin; cyto
lytic

Collagenase; gelatinase,
k toxin necrotizing ac
tivity

l toxin Protease

m toxin Hyaluronidase

Deoxyribonuclease;
n toxin hemolysin; necrotiz
ing activity

Alters membrane
Enterotoxin permeability (cytotoxic,
enterotoxic)

Alters cell surface

Neuramini ganglioside receptors;
dase promotes capillary
thrombosis

Chapter 30 ♦ Clostridium
295
(1) a short incubation period (8-24 hours), (2) a clinical presentation that includes abdominal cramps and
watery diarrhea but no fever, nausea, or vomiting, and
(3) a clinical course lasting less than 24 hours. The illness
is self-limited and recovery occurs in 24 to 48 hours. No
specifc treatment is indicated.
4. Enteritis Necroticans
(Necrotizing Jejunitis, Necrotic Enteritis)
This is a severe and often fatal enteritis known by different names in different countries: Germany (Darmbrand),
New Guinea (pigbel) East Africa, Thailand
and Nepal. b-Toxin-producing C. perfringens type C is
responsible for this disease.
5. C. perfringens Colitis
A sporadic diarrheal syndrome, usually occurring in
elderly patients during treatment with antibiotics, has
been described. An enterotoxin with a cytopathic effect
can be detected in the patient’s feces.
6. Clostridial Endometritis
This condition is a grave complication of incomplete
abortion, or the use of inadequately sterilized instruments. Gangrenous infection of uterine tissue is followed by
toxemia and bacteremia.
Laboratory Diagnosis
Gas Gangrene
Gas gangrene is a medical emergency. The diagnosis
of gas gangrene must be made primarily on clinical
grounds, and the function of the laboratory is only to
provide confrmation of the clinical diagnosis as well as
identifcation and enumeration of the infecting organisms.
1. Specimens
(1) Edge of the affected muscles; (2) Exudates from the
wound; and (3) Necrotic tissue and muscle fragments.
2. Microscopy
Gram stained flms give presumptive information about
the species of clostridia present and their relative numbers. If gas gangrene is present, gram-positive rods may
predominate. Thick, stubby, gram-positive rods suggest
C. perfringens or C. sordellii, ‘citron bodies’, boat- or leafshaped pleomorphic bacilli with irregular staining, may
indicate C. septicum; slender rods with round terminal
spores suggest C. tetani and large rods with oval subterminal spores indicate C. novyi.
3. Culture
Fresh and heated blood agar are used for aerobic and
anaerobic cultures. To prevent swarming by some species of clostridia, the use of plates containing increased
agar (5-6%) are considered. A plate of serum or eggyolk agar, with Cl. perfringens antitoxin spread on one
half is used for the ‘Nagler’s reaction’.
Four tubes of cooked meat broth are inoculated and
heated at 100°C for 5, 10, 15 and 20 minutes, incubated and subcultured on blood agar plates after 24 to 48
hours, to differentiate the organisms with heat resistant
spores. Blood cultures are often positive especially in
Cl. perfringens and Cl. septicum infections. However,
Cl. perfringens bacteremia may occur without gas gangrene.
4. Identification
Examine plates for typical colonies. The isolates are
identifed based on their morphological, cultural, biochemical and toxigenic characters.
5. Animal Pathogenicity
Laboratory Diagnosis of Food Poisoning
A diagnosis can be made from isolation of C. perfringens in higher than normal numbers from the feces of
infected patients and from samples of the ingested food.
CMB is inoculated and heated at 100°C for 30 minutes.
It is cooled and is incubated at 37°C for 18 hours and
subcultured on selective medium which is then incubated anaerobically at 37°C for 18 hours. Identifcation
of the bacterial isolates is done by morphology, cultural
characteristics, biochemical reactions and Nagler’s reaction. Isolation from feces, except in large numbers is not
meaningful as Cl. perfringens may be present in normal
intestines.
A more specifc diagnosis is possible by use of an
enzyme-linked immunosorbent assay (ELlSA) to detect
C. perfringens enterotoxin in the feces of affected persons.
Prophylaxis and Therapy
1. Surgery
All damaged tissues should be removed promptly and
the wounds irrigated to remove blood clots, necrotic tissue and foreign materials. In established gas gangrene,
uncompromising excision of all affected parts may be
life-saving.
2. Antibiotics
Penicillin, metronidazole and an aminoglycoside may
be given in combination. Alternatively, clindamycin
plus an aminoglycoside or a broad-spectrum antibiotic
such as meropenem or imipenem, may be considered.
3. Passive Immunization
A polyvalent antiserum used to be available but it has
now been replaced by intensive antimicrobial therapy.
4. Hyperbaric Oxygen
Hyperbaric oxygen may be benefcial in treatment and is
introduced in the depth of wound to reduce anaerobiosis.
5. Active Immunization
Toxoids induce antitoxic response experimentally but it
has not been come into use practically.
Section 3 ♦ Systemic Bacteriology
296
CLOSTRIDIUM TETANI
Clostridium tetani is the causative agent of tetanus, a disease that is now relatively rare in well-developed countries.
Tetanus has been known from very early times,
having been described by Hippocrates and Aretaeus.
Morphology
It is a gram-positive, slender bacillus, 2 to 5 × 0.4-1 mm
with rounded ends. The spores are spherical, terminal
and twice the diameter of vegetative cells giving them
typical drumstick appearance (Fig. 30.3). The spore does
not stain with the Gram stain and appears as a colorless
round structure. It tends to be pleomorphic and sometimes flamentous. It is noncapsulated and motile by
peritrichate flagella (except Cl. tetani type VI) with peritrichate flagella. Young cultures of the organism usually
stain gram-positive, but in older cultures and in smears
made from wounds, they are Gram variable and even be
gram-negative.
Cultural Characteristics
Cl. tetani is an obligate anaerobe. The optimal temperature for growth is 37°C, and the optimal pH is 7.4. It
can grow well in cooked meat broth (CMB), thioglycollate broth, nutrient agar and blood agar. In cooked meat
broth (CMB), growth occurs as turbidity and there is
also some gas formation. The meat is not digested but
becomes black on prolonged incubation. On blood agar
the bacilli produce a swarming (thin spreading flm)
growth. On horse blood agar, the colonies of Cl. tetani
are surrounded by a zone of α-hemolysis, which subsequently develops into b-hemolysis, due to the production of an
oxygen-labile hemolysin known as tetanolysin. On egg-yolk agar, it does not produce opalescence
or pearly layer.
In deep agar shake cultures, the colonies are spherical fluffy balls, 1-3 mm in diameter, made up of flaments
with a radial arrangement. In gelatin stab cultures a fr
tree type of growth occurs, with slow liquefaction.
Biochemical Reactions
Cl. tetani has feeble proteolytic but no saccharolytic
property. It does not attack any sugar. Gelatin liquefaction occurs very slowly. Coagulated serum is rendered
more transparent and softened but not liquefed. It is
indole positive and MR, VP, H2S and nitrate reduction
negative. A greenish fluorescence is produced on media
containing neutral red (as on MacConkey’s medium).
Resistance
The spores may be highly resistant to adverse conditions, but the degree of resistance varies with the strain.
Most are killed by boiling for 10 to 15 minutes but some
resist boiling for up to three hours. They can, however,
be killed by autoclaving at 121°C for 15 minutes.
Spores are able to survive in soil for years, and are
resistant to most antiseptics. They are killed by exposure
to iodine (1% aqueous solution), hydrogen peroxide (10
volumes) and glutaraldehyde (2%) within a few hours.
Antigenic Structure
Flagella (H), somatic (O), and spore antigens have been
demonstrated in C. tetani. The spore antigens are different from the H and O antigens of the somatic cell.
1. Flagella (H) Antigen
Ten serological types have been recognized based on
agglutination (types I to X) of which type I and III are the
commonest. This typing is on the basis of their flagellar
(H) antigens. Type VI contains nonflagellated strains.
2. Somatic (O) Antigen
There is a single somatic agglutination group for all
strains that permits identifcation of the organism by
use of fluorescein-labeled antisera.
Tetanus Toxin
Cl. tetani produces at least two distinct toxins - an oxygen-labile hemolysin (tetanolysin) and a powerful
plasmid-encoded, heat-labile neurotoxin (tetanospasmin). The two are antigenically and pharmacologically
distinct and their production is mutually independent.
A third toxin, a nonspasmogenic, peripherally active
neurotoxin, has been identifed. It is not known whether
this plays any role in the pathogenesis of tetanus.
1. Tetanospasmin
All of the symptoms in tetanus are attributable to an
extremely toxic neurotoxin, tetanospasmin, which is
an intracellular toxin released by cellular autolysis. The
toxin is a heat-labile, oxygen stable, powerful plasmidencoded neurotoxin that may be inactivated by heating
for 20 minutes at 60ºC. The toxin has been crystalized. It
gets toxoided spontaneously or in the presence of low
concentrations of formaldehyde. It is a good antigen
and is specifcally neutralized by the antitoxin.
Fig. 30.3: Cl. tetani, some with spores and some without spores
Chapter 30 ♦ Clostridium
297
Tetanus toxin is one of the most poisonous substances known. It is a simple protein composed of a
single polypeptide chain. On release from the bacillus,
it is autolyzed to form a heterodimer consisting of a
heavy chain (93,000 MW) and a light chain (52,000 MW)
joined by a disulfde bond. The purifed toxin is active
in extremely small amounts and has an MLD for mice
of about 50-75 × 10-6 mg. Its MLD for human beings is
about 130 nanograms.
2. Tetanolysin
Tetanolysin is a heat labile, oxygen labile hemolysin,
antigenically related to the oxygen labile hemolysins
produced by Cl. perfringens, Cl. novyi and Str. pyogens. It
is not relevant in the pathogenesis of tetanus.
Pathogenicity
Setting of the wound-oxidation reduction potential
must be properly poised-multiplication of the organism-toxigenesis.
The spores of Cl. tetani are ubiquitous. They occur in
the gastrointestinal tracts of man and animals. They are
also present in the soil especially in manured soil.
Tetanus develops following the contamination of
wound with C. tetani spores. The most typical focus of
infection in tetanus is a puncture wound caused, for
example, by a splinter. Introduced foreign bodies or
small areas of cell killing create a nidus of devitalized
material in which tetanus spores can germinate and
grow. The lowering of the oxidation-reduction potential
is associated with tissue necrosis after traumatic injuries or the injection of necrotizing substances. Germination of
spores is dependent upon the reduced oxygen
tension occurring in devitalized tissue. After germination of the spores, toxin is elaborated and gains entrance
to the central nervous system.
Cl tetani has little invasive power.Infection strictly
remains localized in the wound and the disease is due
to the effect of a potent diffusible exotoxin (tetanospasmin). It is transported from an infected locus by retrograde
neuronal flow or by blood. Tetanospasmin acts
by blocking the release of neurotransmitters (e.g. gamma-aminobutyric acid [GABA], glycine) for inhibitory
synapses, thus causing excitatory synaptic activity to
be unregulated (spastic paralysis). The abolition of spinal inhibition causes uncontrolled spread of impulses
initiated anywhere in the central nervous system. The
toxin exerts its effects on the spinal cord, the brain stem,
peripheral nerves, at neuromuscular junctions and
directly on muscles.
When tetanus occurs naturally, the tetanus bacilli
stay at the site of the initial infection and are not generally invasive. Toxin diffuses to affect the relevant level of the
spinal cord (local tetanus) and then to affect
the entire system (generalized tetanus). These stages,
including the intermediate one of ‘ascending tetanus’,
are demonstrable in experimental animals but the stages
tend to merge in their clinical presentation in man.
Clinical Manifestations of Tetanus
The incubation period is variable—from two days to
several weeks but is commoniy 6 to 12 days. The duration of the incubation period is directly related to the
distance of the primary wound infection from the central nervous system.
The onset of signs and symptoms is gradual, usually starting with some stiffness and perhaps pain in or
near a recent wound. Generalized tetanus is the most
common form. The initial complaint may be of stiffness
of the jaw (lockjaw) in some cases. Pain and stiffness
in the neck and back may follow. The stiffness spreads
to involve all muscle groups; facial spasms produce the
‘sardonic grin’, and in severe cases spasm of the back
muscles produces the opisthotonos (extreme arching of
the back). A severe case with a relatively poor prognosis
shows rapid progression from the frst signs to the development of generalized spasms. The autonomic nervous
system is involved in patients with more severe disease;
the signs and symptoms include cardiac arrhythmias,
fluctuations in blood pressure, profound sweating, and
dehydration.
In cephalic tetanus the primary site of infection is
the head and has high mortality. The most feared form
of tetanus, tetanus neonatorum, is a signifcant cause
of morbidity and mortality in developing nations. This
form of tetanus usually results from cutting the umbilical cord with unsterile instruments or from improper
care of the umbilical stump. The mortality in infants
exceeds 90 percent.
Epidemiology
Tetanus is more common in the developing countries,
where the climate is warm, and in rural areas where
the soil is fertile and highly cultivated, where human
and animal populations are substantial and live in close
association and where unhygienic practices are common and medical facilities poor. In rural India, tetanus
was a common cause of death, particularly in the newborn. But immunization of infants and expectant mothers has
reduced the incidence to a large extent.
Tetanus was a serious disease with a high rate of
mortality (80-90%), before specifc treatment became
available. The case fatality rate varies from 15 to 50 percent even with proper treatment. Tetanus neonatorum
and uterine tetanus have very high fatality rates (70-
100%), while otogenic tetanus is much less serious.
Laboratory Diagnosis
The diagnosis of tetanus is made on clinical grounds
because isolation of the organism can occur in the absence
of disease and also because it is possible to have the
disease but be unable to isolate the organism. Laboratory
Section 3 ♦ Systemic Bacteriology
298
tests only help in confrmation. Not infrequently, it may
not be possible to establish a laboratory diagnosis at all.
1. Specimen
Wound exudate or tissue removed from the wound.
2. Microscopy
Microscopy is unreliable and the demonstration of the
typical ‘drumstick’ bacilli in wounds itself is not diagnostic of tetanus. It may not also be possible to distinguish by
microscopy between Cl. tetani and morphologically similar bacilli such as Cl. tetanomorphum and Cl.
Sphenoides. Hence, microscopy is unreliable. Simple
light microscopy is often unsuccessful. Immunofluorescence microscopy with a specifc stain is possible but not
generally available.
3. Culture
The material is inoculated on one half of a blood agar
plate. Cl. tetani produces a swarming growth which
may be detected on the opposite half of the plate after
1 to 2 days incubation anaerobically. The incorporation
of polymyxin B, to which clostridia are resistant, makes
the medium more selective.
The material is also inoculated into three tubes
of cooked meat broth, one of which is heated to 80°C
for 15 minutes, the second for fve minutes, and the
third left unheated. The purpose of heating for different periods is to kill vegetative bacteria, while leaving
undamaged tetanus spores, which vary widely in heat
resistance. The cooked meat tubes are incubated at 37°C
and subcultured on one-half of blood agar plates daily
for up to four days. For identifcation and toxigenicity testing,blood agar plates (with 4% agar to inhibit
swarming), having tetanus antitoxin (1500 units per ml)
spread over one-half of the plate are used.
4. Toxigenicity Test
Toxigenicity is best tested in animals. Control mice are
protected with tetanus antitoxin. Two mice, one unprotected and other protected are used for each test. One
animal is protected by giving 1,000 units of tetanus antitoxin intraperitoneally 1 hour before the test. Inject 0.1
ml of a 48 hour CMB culture supernate of the organism
intramuscularly into the hind limb of one mouse (the
test) and the same amount in the another animal (control animals). The protected mouse remains well. Signs
of ascending tetanus develop in the unprotected animal
after several hours, they begin in the inoculated leg and
extend to the tail, then the other hind limb is affected
and then generalized signs appear. The animal dies
within 2 days but may be killed earlier as the appearance of ascending tetanus is diagnostic.
Prophylaxis
The available methods of prophylaxis are:
1. Surgical attention
2. Antibiotics
3. Immunization—passive, active or combined.
1. Surgical Prophylaxis
This includes prompt and adequate wound toilet and
proper surgical debridement of wound, removal of foreign material, necrotic tissue and blood clots to prevent
an anaerobic environment for the growth of C. tetani.
2. Antibiotic Prophylaxis
Antibiotics destroy or inhibit C. tetani and pyogenic
bacteria in the wound, so that the production of the toxin can be prevented. Long-acting penicillin injection is
the drug of choice. An alternative is erythromycin. Antibiotic prophylaxis does not replace immunoprophylaxis
but serves as a useful adjunct.
3. Immunoprophylaxis
It includes 3 types of immunization:
i. Active immunization
ii. Passive immunization
iii. Combined immunization
i. Active Immunization
Tetanus is best prevented by active immunization with
tetanus toxoid. Two preparations are available for active
immunization are:
a. Combined vaccine (DPT)
b. Monovalent vaccines
i. Plain or fluid (formol) toxoid
ii. Tetanus vaccine, adsorbed (PTAP, APT)
Tetanus toxoid (formol toxoid), which is available either as ‘plain toxoid’, or adsorbed on aluminum
hydroxide or phosphate (APT), is commonly used for
active immunization. Three doses of 0.5 ml tetanus
toxoid (APT) each are given intramuscularly, with an
interval of 4 to 6 weeks between frst two doses and 6
to 12 months between the second and third dose. A full
course of three doses confers immunity for a period of
at least 10 years. A ‘booster dose’ of toxoid is recommended after 10 years.
Tetanus toxoid is given along with diphtheria toxoid
and pertusis vaccine called DPT in children as triple vaccine. Pertusis vaccine acts as adjuvant. Three doses are
given intramuscularly at interval of 4-6 weeks, starting
at age as early as 6 weeks. Booster doses are given at age
of 18 months and then at fve years. Thereafter, booster
doses of TT (tetanus toxoid) are given at the age of 10
and 16 years. Subsequently, immunity to tetanus can be
maintained by booster doses of toxoid every 10 years.
ii. Passive Immunization
Passive immunity may be conferred by the administration of antitoxin. Temporary protection against tetanus can be
provided by an injection of human tetanus
hyperimmune globulin (TIG) or ATS.
Antitetanus Serum (ATS)
Antitetanus serum (ATS) from hyperimmune horses
was the preparation originally used. The dose employed
was 1500 IU given subcutaneously or intramuscularly
Chapter 30 ♦ Clostridium
299
in nonimmune persons soon after receiving any tetanus
prone injury. ATS gives passive protection for about 7
to 10 days.
Disadvantages of ATS
Equine ATS carried two disadvantages implicit in the
use of any heterologous serum—‘immune elimination
and hypersensitivity reactions. It is, therefore, mandatory to test for hypersensitivity before administration
of ATS. A ‘trial’ dose given subcutaneously would be
a better index of hypersensitivity. A dose of 0.5 ml of
ATS is given subcutaneously and the patient observed
for at least half an hour for general reactions. As even
this dose may precipitate anaphylaxis in some cases, a
syringe loaded with adrenaline (1/1000) must be kept ready.
The trial dose should be 0.05 ml of a 1/10 dilution of ATS
in persons with a history of any allergy.
Human Antitetanus Immunoglobulin (HTIG)
Passive immunity without risk of hypersensitivity can
be obtained by the use of human antitetanus immunoglobulin (HTIG). This is effective in smaller doses. The
prophylactic dose of HTIG is 250 units by intramuscular
injection.
iii. Combined Immunization
It consists of administering to a nonimmune person
ATS or HTIG at one site, along with the frst dose of a
course of active immunization with adsorbed toxoid at
the same time at another site, followed by second and
third doses of TT at appropriate intervals. The active
immunization course must be subsequently completed.
The purpose of antitoxin is for immediate temporary
protection, and the purpose of toxoid is for long-lasting
protection.
Treatment
1. The patient remains conscious and requires skilled
sedation and constant nursing.
2. Treatment of tetanus requires debridement of the
primary wound, use of metronidazole, passive
immunization with human tetanus immunoglobulin, and vaccination with tetanus toxoid.
3. Full wound exploration and debridement is
arranged, and the wound is cleansed and left open
with a loose pack.
4. The patient is given 10,000 units of human
tetanus immunoglobulin (RTIG) in saline by slow
intravenous infusion.
5. Penicillin or metronidazole is given for as long
as considered necessary to ensure that bacterial
growth and toxin production are stopped. The antitoxin and antibiotics are given immediately, and
preferably before surgical excision but delay must
be avoided.
6. Vaccination with a series of three doses of tetanus
toxoid followed by booster doses every 10 years is
highly effective in preventing tetanus.
CLOSTRIDIUM BOTULINUM
C. botulinum (from the Latin botulus, “sausage”) causes
botulism. Botulism is a severe, often fatal, form of food
poisoning characterized by pronounced neurotoxic
effects. The disease has been caused by a wide range
of foods, usually preserved hams, large sausages of the
German variety, home-preserved meats and vegetables,
canned products such as fsh, liver contact with the
organism itself is not required; hence the disease can be
a pure intoxication.
Morphology
C. botulinum is a strictly anaerobic gram-positive bacillus (about 5 x 1 mm). It is noncapsulated, motile with
peritrichous flagella and produces spores which are
oval, subterminal and bulging.
Cultural Characteristics
It is a strict anaerobe. Optimum temperature is 35°C but
some strains may grow even at 1 to 5°C. Good growth
occurs on ordinary media. Surface colonies are large,
irregular, semitransparent, with fmbriate border. On
horse blood agar, all strains except those of type G are
b-hemolytic. On egg-yolk agar (EYA) all types except G
produce opalescence and a pearly effect
Resistance: Spores are heat and radiation resistant, surviving several hours at 100°C and for up to 10 minutes
at 120°C. Spores of nonproteolytic types of B, E and F are
much less resistant to heat. The resistance of the spores
to radiation is of special relevance to food processing.
Classification
The species C. botulinum includes a very heterogeneous
group of strains that have been divided into eight serologically distinct types—A, B, C1, and C2, D, E, F, and
G—on the basis of the type of toxin produced. The toxins produced by different types are antigenically distinct but
pharmacologically similar. All types can cause
human disease but types A, B and E are most common.
The toxins produced by the different types of Cl. botulinum appear to be identical, except for immunological
differences. Toxin production appears to be determined
by the presence of bacteriophages, at least in types
C and D.
Botulinum Toxin
A powerful exotoxin produced by Cl. botulinum is
responsible for its pathogenicity. It differs, however,
from a classic exotoxin in that it is not released during
the life of the organism but appears in the medium only
after death and autolysis of the organism. It is believed
to be synthesised initially as a nontoxic protoxin or progenitor toxin. Trypsin and other proteolytic enzymes
activate progenitor toxin to active toxin. The production of botulinum toxin is governed by specifc bacteriophages.
Properties of Toxin
Botulinum toxin is one of the most potent toxins known.
It is isolated as a pure crystalline protein with MW
Section 3 ♦ Systemic Bacteriology
300
70,000. It has a lethal dose for mice of 0.000,000,033 mg
and lethal dose for human beings is probably 1 to 2 mg.
It is a neurotoxin and acts slowly, taking several hours
to kill.
The toxin is relatively stable being inactivated at
80°C for 30 to 40 minutes and at 100°C for 10 minutes.
Food suspected to be contaminated with botulinum toxin can be rendered completely safe by pressure cooking
or boiling for 20 minutes. It can be toxoided. It is a good
antigen and is specifcally neutralized by the antitoxin.
Botulinum toxin gains access to the peripheral nervous system, where it acts preferentially on cholinergic
nerve endings to block the release of the neurotransmitter, acetylcholine, from the nerve terminals of neuromuscular
junctions. A symmetric descending paralysis
is the characteristic pattern, ending in death bv respiratory paralysis. In minute doses, botulinum toxin preparations
have been used therapeutically to relieve spastic
conditions such as poststroke spasticity and. strabismus.
Pathogenicity
It is noninvasive and its pathogenicity is entirely due
to the toxin produced by it. The disease caused by this
organism is known as botulism. It is of 3 types: foodborne botulism, wound botulism and infant botulism.
1. Food-Borne Botulism
It is due to the ingestion of preformed toxin. The causative organism, C. botulinum, multiplies in the food before
it is consumed, and produces a powerful soluble toxin.
The source of botulism is usually preserved food such
as meat and meat products, fsh, and vegetables. Food
responsible for botulism is usually abnormal in appearance and odor. Bulging of tins and the presence of gas
bubbles on opening suggest contamination with C. botulinum. However, at times food may look normal.
Symptoms usually begin 18 to 36 hours after ingestion
of food and may include nausea, vomiting, thirst, constipation, double vision, diffculty in swallowing, speaking
and breathing. This may be followed by muscular weakness, blurred vision, and death as a result of respiratory
failure. Case fatality varies from 25 to 70 percent.
2. Wound Botulism
Wound botulism is a very rare condition resulting from
wound infection with Cl. botulinum. Toxin is produced
at the site of infection and is absorbed. The symptoms
are those of food-borne botulism except for the gastrointestinal components which are absent. Type A has been
responsible for most of the cases studied.
3. Infant Botulism
Infant botulism was frst recognized in 1976 and is now
the most common form of botulism in the United States.
This is a toxico-infection. Cl. botulinum spores are ingested in food, get established in the gut and there produce
the toxin. The disease typically affects infants younger
than 1 year (most between 1 and 6 months). The most
common food source in infant botulism is honey contaminated with botulinum spores.
Clinically, infant botulism is an acute flaccid paralysis. After a period of normal development, the infant
develops constipation, listlessness, diffculty in sucking
and swallowing, weak or altered cry, muscle weakness,
ptosis and loss of head control. Eventually the baby
appears ‘floppy’ (floppy child syndrome) and develops
respiratory insuffciency or respiratory arrest. Fulminant forms may resemble the sudden infant death syndrome
(SIDS or crib death). Patients excrete toxin and
spores in their feces. Toxin is not generally demonstrable in blood.
Management consists of supportive care and assisted feeding. Antitoxins and antibiotics are not indicated.
Degrees of severity vary from very mild illness to fatal
disease.
Laboratory Diagnosis
Botulism confrmed by isolating the organism or detecting the toxin in food products or the patient’s feces or
serum.
1. Specimens
Feces, food, vomitus, gastric fluid, serum, environmental samples and occasionally wound exudate.
2. Culture
For the isolation of C. botulinum, the specimen is inoculated on egg yolk agar (EYA), blood agar and CMB. Culture
of the heated specimen on nutritionally enriched
anaerobic media allows the heat resistant C. botulinum
spores to germinate. The strains of C. botulinum associated with human botulism are characterized by lipase
production (appears as an iridescent flm on colonies
grown on egg-yolk agar) as well as the ability to digest
milk proteins, hydrolyze gelatin and ferment glucose.
Presence of bacilli in food or feces in absence of toxin
is of no signifcance. Hence, toxin in culture fluid must
be demonstrated by toxigenicity test in mice.
3. Demonstration of Toxin
Demonstration of toxin production must be done with a
mouse bioassay. This procedure consists of the preparation of two aliquots of the isolate, mixing of one aliquot
with antitoxin, and intraperitoneal inoculation of each
aliquot into mice. Toxin activity is confrmed if the antitoxin treatment protects the mice, control animals protected
by polyvalent antitoxin remain healthy. Typing.
is done by passive protection with type specifc antitoxin. Samples of the implicated food, stool specimen and
patient’s serum should also be tested for toxin activity.
A retrospective diagnosis may be made by detection
of antitoxin in the patient’s serum but it may not be seen
in all cases.
Chapter 30 ♦ Clostridium
301
Diagnosis of Infant Botulism
The diagnosis of infant botulism is supported if (1) C.
botulinum is isolated from feces or (2) toxin activity is
detected in feces or serum. The organism can be isolated
from stool cultures in virtually all patients, because carriage of the organism may persist for many months even
after a baby has recovered.
Diagnosis of Wound Botulism
Wound botulism is confrmed by isolation of the organism from the wound or by detection of toxin activity in
wound exudate or serum.
Prophylaxis
Control can be achieved by proper canning and preservation. Infant botulism has been associated with the
consumption of honey contaminated with C. botulinum
spores, so children younger than 1 year should not eat
honey.
A prophylactic dose of polyvalent antitoxin should
be given intramuscularly to all persons who have eaten
food suspected of causing botulism.
Active immunization should be considered for laboratory staff who might have to handle the organism or
specimens containing the organism or its toxin. Two
injections of aluminum sulphate adsorbed toxoid may
be given at an interval of ten weeks, followed by a booster a year later.
Treatment
Patients with botulism require the following treatment
measures:
1. Elimination of the organism from the gastrointestinal tract through the judicious use of gastric lavage
and metronidazole or penicillin therapy.
2. The use of polyvalent antitoxin to neutralize unfxed toxin
3. Adequate ventilatory support.
CLOSTRIDIUM DIFFICILE
Cl. diffcile was frst isolated in 1935 from the feces of
newborn infants. It was so named because of the unusual diffculty in isolating It was not considered pathogenic till
1977 when it was found to be responsible for
antibiotic associated colitis. This organism was infrequently isolated in fecal cultures and rarely associated
with human disease.
Morphology
C. diffcile is a motile gram-positive rod with oval subterminal spores. Spores are large, oval and terminal. It is
nonhemolytic, saccharolytic and weakly’ proteolytic.
Toxins
The organism produces an enterotoxin (toxin A) and a
cytotoxin (toxin B). 1. Toxin A is an enterotoxin that is
primarily responsible for diarrhea. It is capable of producing fluid accumulation in ligated rabbit ileal loop
assay. 2. Toxin B is a potent cytotoxin capable of producing cytopathogenic effects in several tissue culture
cell lines.
Pathogenesis
It is a proven cause of antibiotic associated diarrhea
(AAD), and pseudomembranous colitis (PMC)—a lifethreatening condition. The disease develops in people
taking antibiotics because the agents alter the normal
enteric flora, either permitting the overgrowth of these
relatively resistant organisms or making the patient
more susceptible to the exogenous acquisition of C. diffcile. The disease occurs if the organisms proliferate in
the colon and produce their toxins there. Virtually all
antimicrobial drugs have been reported as predisposing to clostridial AAD and colitis. The three drugs most
commonly implicated are clindamycin, ampicillin
and the cephalosporins. The severity of disease varies
widely from mild diarrhea through varying degrees of
inflammation of the large intestine to a fulminant PMC.
It is postulated that in the immature large intestine, exotoxin receptors may not yet be present or accessible.
Laboratory Diagnosis
1. Isolation of Bacilli
C. diffcile can be isolated from the feces by enrichment
and selective culture procedures.
2. Demonstration of Toxin
Toxin B can be demonstrated in the feces of patients by
its characteristic effect on Hep-2 and human diploid
cell cultures or both toxins may be demonstrated by
immunological methods, e.g. enzyme-linked immunosorbent assay (ELISA).
Treatment and Prophylaxis
The disease is treated by discontinuing the antibiotic
that is presumed to have precipitated the disease and to
suppress the growth and toxin production of C. diffcile
by giving oral metronidazole or vancomycin.
) KEY POINTS
Clostridium
• The genus Clostridium includes all anaerobic, grampositive bacilli capable of forming endospores.
• Clostridia are more commonly associated with skin
and soft tissue infections, food poisoning and antibiotic-associated diarrhea and colitis.
• The shape and position of spores varies in different
species and is useful in identifcation of clostridia,
spores may be subterminal in Cl. perfringens and
drumstick appearance in Cl. tetani.
• Clostridia grow on enriched media in the presence of reducing agent, or in an O 2- free gasseoues
atmosphere. Clostridia grow well on blood agar
medium under anaerobic conditions. Liquid media
Section 3 ♦ Systemic Bacteriology
302
like cooked meat broth (CMB) is very useful for
growing clostridia.
Clostridium perfringens
• Toxins: Cl. perfringens is forming at least 12 distinct
soluble substances or toxins. The four ‘major toxins,
alpha, beta, epsilon. and iota, are predominantly responsible for pathogenicity. It produces lecithinase
(phospholipase C). Subdivided into 5 types (A-E)
on the basis of toxin production.
• Diseases: 1. Soft tissue infections (cellulitis, suppurative myositis, myonecrosis); 2. Food poisoning.
3. Septicemia.
• Nagler reaction and reverse CAMP test are useful
in identification Cl. perfringens, a causative a g e n t
of gas gangrene and food poisoning.
• Systemic infections require surgical debridement
and high-dose penicillin therapy; antiserum against
a toxin not used now.
Clostridium tetani
• Cl. tetani is the causative agent of tetanus.
• Tetanus toxin: Two distinct toxins—an oxygenlabile hemolysin (tetanolysin) and a neurotoxin
(tetanospasmin). Tetanospasmin blocks release
of neurotransmitters (i.e. gamma-aminobutyric
acid, glycine) for inhibitory synapses, thus causing
excitatory synaptic activity to be unregulated
(spastic paralysis).
• Treatment requires debridement, antibiotic therapy
(metronidazole), passive immunization with antitoxin globulin, and active immunization with tetanus
toxoid.Prevention through use of vaccination,
consisting of three doses of tetanus toxoid followed
by boosters every 10 years.
Clostridium botulinum
• Cl. botulinum forms a powerful exotoxin which is
responsible for the disease botulism. It can produce
one of eight distinct botulinum toxins (A-G).
• Diseases: The disease caused by this organism is
known as botulism. It is of 3 types: food-borne botulism, wound botulism and infant b