3. o The genus clostridia consists of gram-positive ,
anaerobic , spore-forming bacilli.
o The genus contains bacteria responsible for gas
gangrene, food poisoning, tetanus and botulinum.
o Some of common pathogens found in human and
animal intestines.
o Intestinal clostridia rapidly invade the blood and
tissues of the host after death and initiate
decomposition of the cadaver. Most species are
saprophytes found in soil, water, decomposing plant
and animal matter.
General introduction:
4. � Molecular :
� Currently based on 16SrRNA gene sequence, 19
clusters are identified of which clinically relevant
species belong to cluster J.
� Phenotypic:
� However, conventional classification based on
morphology, culture and bio chemicals is
commonly used in laboratories.
Classification:
5. � Highly pleomorphic, usually 3-8
× 0.4-1.2 µm in size, gram-
positive rods.
� Long filaments of evolution
forms are common.
� Motile with peritrichate flagella
(C.perfringens and C.tetani type
IV are non motile.)motility is
slow i.e., ‘stately’.
� C.perfringens and C.butyricum
are capsulated while others are
not.
Morphology:
6. Spores:
❑ Wider than bacillary bodies, giving bacillus a swollen
appearance, resembling a spindle.
❑ Spore formation occurs with vary frequency in different
species, most spores being wider than bacillary bodies.
❑ Some like C.sporogenes sporulate readily while others like
C.perfringens do so inconstantly.
❑ Sporulation also takes place in human body.
❑ Shape and position of spores vary in different species and
used for identification and classification on clostridia.
7. � Resistance in spores:
❑ Spores exhibit a pronounced but variable resistance to heat, drying and
disinfectants.
❑ The vegetative cells of clostridia do not differ from those of non-sporing
bacilli in their resistance to physical and chemical agents.
❑ Spores of C.botulinum survive boiling after 3-4 hours and even at 105°C,
they do not killed completely in less than 100 minutes.
❑ Spores of most strains of C.perfringens are destroyed by boiling for less than
5 minutes but some type A strains that cause food poisoning survive for
several hours.
❑ C.tetani persist for years in dried earth. spores of some strains of C.tetani
resist boiling for 15-19 mins. ,though in most cases, they are destroyed
within 5 mins.
❑ All species are killed by autoclaving at 121˚C within 20 mins.
Disinfectants: particularly resistant to phenolic disinfectants.
Formaldehyde is not vary active and spores may sometimes survive
immersion in a 2% solution for up to five days.
❑ Halogens are effective and 1% aqueous iodine solution kills spores within 3
hours. Glutaraldehyde (2% at pH 7.5-8.5) is very effective in killing spores.
8. Clostridia are anaerobic. The sensitivity to oxygen varies
in different species. Some (for example, C.novyi) are
exacting anaerobes and die on exposure to oxygen, while
others (for example, C.histolyticum) are aero tolerant
and may even grow aerobically. of greater importance
than the absence of oxygen, is the provision of
sufficiently low redox potential (Eh) in the medium. This
can be achieved by adding reducing substances such
as unsaturated fatty acids, ascorbic acid, glutathione,
cysteine, thioglycolic acid, alkaline glucose, sulphites. Or
metallic iron. A small concentration of CO2 appears to
enhance growth.The optimum temperature for
pathogenic clostridia is 37°C.Some saprophytic clostridia
are thermophilic while others are psychrophilic. The
optimum pH required for growth is 7-7.4.
Growth is relatively slow on solid media. Colonial
characteristics are variable. Some species are hemolytic
on blood agar.
Cultural characteristics:
9. Robertson’s cooked meat broth (useful medium)
• Contains unsaturated fatty acids which take up oxygen, the reaction
being catalysed by hematin in the meat and also sulphydryl
compounds which bring about a reduced oxidation (OR) potential.
• Clostridia grow in the medium, rendering the broth turbid. Most species
produce gas. The saccharolytic species turns meat pink, while
proteolytic species turn it black, producing foul and pervasive odours.
10. Clostridia can produce disease only when the conditions
are appropriate. Their invasive powers are limited:
• Exotoxin: Pathogenic clostridia form powerful exotoxins.
C.botulinum bacilli are virtually non-invasive
and non-infectious. Botulism is caused by ingestion of preformed toxin
in food .
• Invasive : C.tetani has little invasive property and is
confined to the primary site of lodgement. Tetanus
results from the action of the potent exotoxin it
produces.
• Invasive toxin: C.perfringens, besides being toxigenic,
is also invasive and can spread along tissues and even
cause septicemia.
Pathogenicity:
11. C.perfringens is a normal inhabitant of the
large intestine of humans and animals. It is found
in the feces and contaminates the skin of the
perineum, buttocks and thighs. The spores are
commonly found in soil, dust and air.
� The bacillus was originally cultivated by Achalme(
1891) but was first described in detail by Welch and
Nuttall (1892),who isolated it from the blood and
organs of a cadaver.
� Most important of the clostridia causing gas
gangrene. It also produces food poisoning and
necrotic enteritis in human beings and many serious
diseases in animals.
C.perfringens
12. Spores: Spores are usually destroyed within five
minutes by boiling but those of the 'food
poisoning' strains of type A and certain type C
strains resist boiling for1-3 hours. Autoclaving at
121°C for 15 minutes is lethal. Spores are resistant
to the antiseptics and disinfectants in common
use.
13. � Toxins: C.perfringens form at least 12 distinct toxins, besides
many other enzymes and biologically active soluble substances.
The four ‘major toxins’ alpha, beta, epsilon and iota are
predominantly responsible for pathogenicity.
Pathogenicity:
Virulence factors : C.perfringens strains are classified into
five types A to E, based on the toxins they produce. Typing is
done by neutralization tests with specific antitoxins by
intracutaneous injections in guinea pigs or intravenous injection
in mice.
14. Major toxins: The alpha (α) toxin is produced by all types of C.perfringens and
most abundantly by type A strains. This is the most important toxin biologically and
is responsible for the profound toxemia of gas gangrene. It is lethal dermonecrotic
and hemolytic. It is a phospholipidase (lecithinase C) which, in the presence of
Ca++ and Mg++ ions, splits lecithin into phosphoryl choline and diglyceride. This
reaction is seen as opalescence in serum or egg yolk media and is specially
neutralised by the antitoxin as described as Naegler’s reaction.
Beta (β), epsilon (ε), and iota (t) toxins have lethal and necrotising properties.
Minor toxins:
Enterotoxin: C. perfringens enterotoxin, a small, heat-labile
protein, acts in the lower portion of the small intestine. The
molecule binds to receptors on the epithelial cell surface and
alters the cell membrane, disrupting ion transport (primarily in
the ileum), and leading to loss of fluid and intracellular proteins. Interestingly,
enterotoxin-producing strains are unusually heat resistant, the spores remaining
viable for over an hour at 100°C, thus enhancing their threat as enteropathogens.
Degradative enzymes: C. perfringens is a metabolically vigorous organism that
produces a variety of hydrolytic enzymes including proteases, DNases,
hyaluronidase, and collagenases, which liquefy tissue and promote the spread of
infection.
15.
16. ▪ Myonecrosis (gas gangrene): Clostridial spores are introduced into tissue, for example,
by contamination with dirt, or by endogenous transfer from the intestinal tract. Severe and
open wounds, such as compound fractures and other necrotizing injury (for example, in car
accidents) are a prime predisposing condition. If the normal tissue oxidation-reduction
potential is lowered, as occurs when there is considerable cell injury or compromise
circulation, the spores germinate and grow rapidly. These lesions almost always involve
coinfection with several species of organisms, including clostridia and other anaerobes, and
facultative species that use up available , thus protecting the anaerobes from oxygen's toxic
effects. 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, although this finding is not unique to clostridial infections. 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.
� Oakley (1954) defined gas gangrene as a rapidly spreading, edematous myonecrosis,
occurring characteristically in association with severe wounds of extensive muscle
masses contaminated with pathogenic clostridia, particularly C.perfringens. The
disease has been referred to in the past as malignant edema . Other descriptive
terms are anaerobic (clostridial) myositis and clostridial myonecrosis.
Clinical manifestations:
17. Polymicrobial etiology:
The bacteriology of gas gangrene is
varied. Rarely is this due to infection
by a single clostridium. Generally,
several species of clostridia are found
in association with anaerobic
streptococci and facultative anaerobes
such as E.coli, proteus and staphylococci.
Among the pathogenic clostridia,
C.perfringens is the most frequently
encountered (approximately 60 per
cent), with C.novyi and C.septicum next
(20-40 per cent) and C.histolyticum less
often. Other clostridia usually found
are C.sporogenes, C.fallax, C.bifermentans,
C.sordellii, C.aerofoetidum and C.tertium.
These may not be pathogenic by
themselves.
18. � Food poisoning: C. perfringens is a common cause of food
poisoning in the United States. Typically, the onset of nausea,
abdominal cramps, and diarrhea occurs eight to eighteen hours after
eating contaminated food. Fever is absent and vomiting rare. The
attack is usually self-limited, with recovery within one to two days.
Meat, chicken, fish, and their by-products are the most common
vehicles for clostridial food poisoning, and the occurrence of clinical
symptoms requires a large inoculum of 10⁸ organisms or greater.
Therefore a typical episode of clostridial enterotoxin food poisoning
involves cooking that fails to inactivate spores, followed by holding the
food for several hours under conditions that allow bacterial
germination and several cycles of growth.
� Enteritis necroticans: Outbreaks of a necrotizing bowel disease
with high mortality (greater than fifty percent) caused by C.
perfringens have been sporadically reported. The disease is
known as pigbel in New Guinea and darmbrand in Germany, and
a clinically similar illness is known, but unusual, in the United
States.
19.
20. � Biliary tract infection:
� C.perfringens has been reported to produce two rare
but serious infections of the biliary tract: acute
emphysematous cholecystitis and post-
cholecystectomy septicemia.
� Brain abscess and meningitis:
� Brain abscess and meningitis due to C.perfringens have
been reported very rarely .
� Panophthalmitis:
� Panophthalmitis due to C.perfringens has occasionally
followed penetrating eye injuries.
� Thoracic infections:
� Clostridial infection of the chest cavity may follow
penetrating wounds of the thorax. This is more often
seen in battle casualties than in civilian situations.
� Urogenital infections:
� Infection of the urinary tract may occasionally follow
surgical procedures such as nephrectomy. Clostridial
infection of the uterus is a serious and not infrequent
condition, commonly associated with septic abortion.
Septicemia is common in this condition.
21. � Clinical presentations:
� The incubation period may be as short as seven hours or as long as six
weeks after the wound was created, the average being 10- 48 hours with
C.perfringens,2-3 days with C.septicum and 5-6 days with C.novyi infection.
The disease develops with increasing pain, tenderness and edema of the
affected part along with systemic signs of toxemia. There is a thin, watery
discharge from the wound, which later becomes profuse and
serosanguinous. Accumulation of gas makes the tissues crepitant . In
untreated cases, the disease process extends rapidly and inexorably.
Profound toxemia and prostration develop and death occurs due to
circulatory failure.
22. 3. Culture:
� It is an anaerobe but can grow under microaerophilc conditions.
pH range= 5.5-8.0 , temperature range =20-50°C
Usually grown at 37°C. Optimum temp.= 45°C (for many strains).
� Gram-staining: shows plump, Gram-positive bacillus with straight, parallel
sides and rounded or truncated ends, about 4-6 x 1 μm in size, usually
occurring singly or in chains or small bundles . It
is pleomorphic, and filamentous and involution forms are common. It is
capsulated and non-motile. Spores are central or subterminal but are rarely
seen in artificial culture or in material from pathological lesions, and their
absence is one of the characteristic morphological features of
C.perfringens.
� A plate of serum or egg yolk agar ,
with C.perfringens antitoxin spread on
one half, is used for the Naegler's
reaction .
Gram- staining
23. Note – The mere presence of clostridia in wounds does not constitute
gas gangrene.
1. Specimens: films from the muscles at the edge of affected area, from
tissue in necrotic area and deeper in the wounds
• From depths of wound may be collected with capillary pipette or swab
(must be soaked in exudate).
• Blood cultures may be required as they are more often positive especially
in C.perfringens and C.septicum infections. However, C.perfringens
bacteraemia may occur without gas gangrene.
2. Microscopic examination:
� The presence of large numbers of regularly brick-shaped, gram-positive
bacilli without spores is strongly suggestive of C.perfringens infection.
� ‘Cistron bodies’ and boat or leaf shaped pleomorphic bacilli with irregular
staining suggest C.septicum.
� Large bacilli with oval, sub terminal spores indicate C.novyi.
� Slender bacilli with round, terminal spores may be C.tetani or
C.tetanomorphum.
Laboratory diagnosis:
24. � Naeglar’s reaction:
1. When C.perfringens is grown on a medium containing 6 % agar, 5%
Fildes' peptic digest of sheep blood and 20% human serum, with the
antitoxin spread on one half of the plate, colonies on the other
half without the antitoxin will be surrounded by a zone of opacity.
2. There will be no opacity around the colonies on the half of the plate with the
antitoxin, due to the specific neutralization of the alpha toxin.
3. This specific lecithinase effect, known as the Nagler's reaction , is
a useful test for the rapid detection of C.perfringens in clinical specimens.
The incorporation of neomycin sulphate in the medium makes it more
selective, inhibiting coliforms and aerobic spore bearers.
25. � reverse CAMP test: used to identify
C.perfringens
� In this test, C.perfringens is streaked over the centre
of the plate Streptococcus agalactiae is streaked at
right angles to it. A positive reverse
CAMP test shows the presence of an arrow-shaped
zone of enhanced hemolysis pointing towards
C.perfringens.
� Biochemical reactions: In C.
perfringens , glucose, maltose,
lactose and sucrose are fermented
with the production of acid and gas .
It is indole-negative, MR positive
and VP-negative . H2S is formed
abundantly. Most strains reduce
nitrates.
26. � Surgery is the most important prophylactic and therapeutic
measure in gas gangrene. All damaged tissue 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 .
Where facilities exist, hyperbaric oxygen may be beneficial in
treatment.
� The drug of choice is metronidazole given intravenously before
surgery and repeated every eight hours for 24 hours. As mixed
aerobic and anaerobic infections are usual , it should be combined with
clindamycin and cephalosporins.
� Passive immunization with 'anti- gas gangrene serum' (equine
polyvalentantitoxin in a dose of 10,000 IU C.perfringens , 10,000 IU
C.novyi and 5,000 IU C.septicum antitoxin given IM or in emergencies
IV) used to be the common practice in prophylaxis.
Prophylaxis and treatment
27. � Causative organism of tetanus.
C.tetani is widely distributed in soil and in the intestines of humans and
animals. It is ubiquitous and has been recovered from a wide variety of other
sources, including street and hospital dust, cotton, wool plaster of Paris,
bandages, catgut, talc, wall plaster and clothing. It may occur as an
apparently harmless contaminant in wounds.
Epidemiology :C. tetani spores are common in barnyard, garden, and other
soils. 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.
Special circumstances may also lead to infection, for example, after
severe burns or surgery. Illicit drugs often contain spores that are
introduced by injection. In less developed countries, tetanus is a common
complication of birth, where the umbilical wound becomes infected from
unclean cutting instruments or dressings.
C.tetani
28. � It is a Gram-positive, slender bacillus ,
about 4-8 x 0 .5 μm in size , though
there may be considerable variation in
length. It has a straight axis, parallel
sides and rounded ends. It occurs singly
and occasionally in chains. The spores
are spherical, terminal and bulging,
giving the bacillus the characteristic
‘drumstick 'appearance.
� Morphology of spores depend on the
stage of development an the young spore
may be oval rather than spherical. It is
non-capsulated and motile by
peritrichate flagella. Young cultures are
strongly gram-positive but older cells
show variable staining and may even be
gram-negative.
� Spores can survive in soil for years.
Morphology :
29. Resistance of spores:
The resistance of tetanus spores to heat appears to be subject
to strains differences. Most are killed by boiling for 10-15
minutes but some resist boiling for up to three hours. When
destruction of spores is to be ensured,
autoclaving at 121 °C for 20 minutes is recommended . On the
other hand, when heat is applied to free cultures of C.tetani
from non-sporing contaminants, it is important not to exceed
80°C for 10 minutes, as even
this mild treatment can cause considerable destruction . They
are resistant to most antiseptics.
Disinfectants
They are not destroyed by 5% phenol or 0.1 % mercuric
chloride solution in two weeks or more. Iodine (1 % aqueous
solution) and hydrogen peroxide ( 10 volumes) kill the spores
within a few hours.
30. C.tetani has little invasive power. Washed spores injected into
experimental animals do not germinate and are destroyed by
phagocytes. Germination and toxin production occur only if favorable
conditions exist, such as reduced OR potential, devitalized tissues,
foreign bodies or concurrent infection. The toxin produced locally is
absorbed by the motor nerve endings and transported to the central
nervous system intra-axonally . The toxin is specifically and avidly
fixed by gangliosides of the grey matter of the nervous tissue.
� C.tetani produces at least two distinct toxins- a hemolysin
(tetanolysin) and a powerful neurotoxin (tetanospasmin).
� A third toxin, a non-spasmogenic, peripherally active neurotoxin, has
been identified. It is not known whether this plays any role in the
pathogenesis of tetanus .
Pathogenicity :
31. � Tetanospasmin: This is the toxin responsible for
tetanus. It is oxygen-stable but relatively heat labile,
being inactivated at 65 °C in five minutes. It is plasmid-
coded. It gets toxoided spontaneously or in the presence
of low concentrations of formaldehyde . It is a good
antigen and is specifically neutralized by the antitoxin.
The toxin has been crystallized. It is a simple protein
composed of a single polypeptide chain. On being
released from the bacillus, it autolysis to form a
heterodimer consisting of a heavy chain (93,000 MW)
and a light chain (52,000 MW) joined by a disulphide
bond. The tetanus and botulinum toxins resemble each
other in their amino acid sequences.
� The heavy fragment (B) mediates binding to
neurons and cell penetration of the light
fragment (A). The A fragment blocks
neurotransmitter release at inhibitory synapses,
and thus causes severe, prolonged muscle
spasms . The A fragment has been shown to be
a protease; it cleaves a small synaptic vesicle
protein, synaptobrevin, and abolishes the flow of
inhibitory neurotransmitters. [Note: Patients
who recover from tetanus do not have an
antibody response, presumably because the
dose of toxin was too small to be immunogenic.]
32. Lethal dose: The purified toxin is active in
extremely small amounts and has a minimum
lethal dose (MLD) for mice of about 50-75 x 10-⁶
mg. Its MLD for human beings is about 130
nanograms. There is considerable variation in the
susceptibility
of different species of animals to the toxin. The
horse is the most susceptible. Guinea pigs, mice,
goats and rabbits are susceptible in that
descending order. Birds and reptiles are highly
resistant. Frogs,
which are normally insusceptible, may be
rendered susceptible by elevating their body
temperature.
� Tetanolysin: This is a heat labile , oxygen-
labile hemolysin, antigenically related to
the oxygen-labile hemolysins of
C.perfringens, C.novyi and S.pyogenes. It is not
relevant in the pathogenesis of tetanus.
33. Tetanus
Tetanus is characterized by tonic muscular spasms, usually
commencing at the site of infection and, in all but the mildest cases,
becoming generalized, involving the whole of the somatic muscular
system.
Clinical significance: Tetanus has an incubation period varying from
four days to several weeks. A shorter period is usually associated with
more severe disease and wounds closer to the brain. Tetanus presents
as a spastic paralysis, in which muscle spasms often first involve the
site of infection. In the early stages of the disease, the jaw muscles are
affected, so that the mouth cannot open (trismus or "lockjaw").
Gradually other voluntary muscles become involved , and any external
stimulus (for example, noise or bright light) precipitates a painful
spasm, and sometimes convulsions. Death, which occurs in fifteen to
sixty percent of cases, is usually the result of paralysis of chest
muscles leading to respiratory failure.
Clinical manifestations:
34. � Mortality: Tetanus was a serious disease with a high rate of
mortality, 80-90 per cent, before specific treatment became available.
Even with proper treatment, the case fatality rate varies from 15 to 50
per cent. Tetanus neonatorum and uterine tetanus have very high
fatality rates (70-100 per cent) , while otogenic tetanus is much less
serious.
� Incidence: Tetanus is common in the developing countries, warm
climate, and in rural areas where the soil is made fertile with organic
manure, where human and animal populations live in close
association and where unhygienic practices are common and medical
facilities poor.
� Neonatal tetanus: In rural India, tetanus was a common cause of
death, particularly in the newborn. However, universal immunization
of infants and expectant mothers has reduced the incidence of
maternal and neonatal tetanus to a large extent.
35. � Cultural characteristics: C. tetani has feeble proteolytic but no
saccharolytic properties. It does not ferment any sugar. It is indole-
positive, MR- and VP-negative and does not produce H2S. Nitrate is
not reduced. Gelatin liquefaction occurs very slowly. A greenish
fluorescence is produced on media containing neutral red (as on
MacConkey medium). The diagnosis of tetanus should always be
made on clinical grounds. Laboratory tests are not usually helpful.
Laboratory diagnosis may be made by demonstration of C. tetani by
microscopy, culture or toxigenicity tests.
� Specimens : necrotic tissue may be collected from site of injury.
1. Microscopy : It is unreliable and demonstration of the typical
'drumstick' bacilli in wounds in itself is not diagnostic of tetanus. The
bacilli may be present in some wounds without tetanus developing. It may be
indistinguishable from C. tetanomorphum and C.sphenoides.
Laboratory diagnosis:
36. 2. Culture : obligate anaerobe Optimum temp. 37°C and pH 7.4
Grows on ordinary media but growth is improved by blood and serum
but not by glucose.
� Swarming: C.tetani produces swarming growth. An extremely
fine, translucent film of growth is produced that is practically
invisible, except at the delicately filamentous advancing edge.
This property enables the separation of C. tetani from mixed
cultures, which may be detected on the opposite half of the plate
after 1-2 days of anaerobic incubation. The incorporation of
polymyxin B, to which other clostridia are resistant, makes the
medium selective for C.tetani.
� Fildes technique: If the water of condensation at the bottom
of a slope of nutrient agar is inoculated with the mixed bacterial
culture, after incubation anaerobically for 24 hours, subcultures
from the top of the tube will yield a pure growth of the tetanus
bacillus.
� Deep agar shake cultures: The colonies appear as spherical
fluffy balls, 1-3 mm in diameter, made up of filaments with a
radial arrangement.
37.
38. In gelatin stab cultures, a fir tree type of growth occurs , with
slow liquefaction. It grows well in Robertson's cooked meat broth,
with turbidity and some gas formation. The meat is not digested
but is turned black on prolonged incubation. On blood agar, a
hemolysis is produced, which later develops into β hemolysis, due
to the production of hemolysin (tetanolysin).
� Toxigenicity testing:
• In vitro: blood agar plates (with 4% agar to inhibit swarming) with tetanus
antitoxin ( 1500 units per ml) spread over one half of the plate are used.
The C.tetani strains are stab-inoculated on each half of the plate, and
incubated anaerobically for two days. Toxigenic C. tetani strains show
hemolysis around the colonies, only on the half without the antitoxin.
Hemolysis is inhibited by the antitoxin on the other half. This indicates the
production only of tetanolysin and not necessarily of tetanospasmin, which
is the pathogenic toxin.
• In vivo: In vivo toxigenicity is best tested in animals. A 2-4-day-old
cooked meat culture (0.2 ml) is inoculated into the root of the tail of a
mouse. A second mouse that has received a dose of tetanus antitoxin
(1000 units) an hour earlier serves as the control. Symptoms develop in the
test animal (non-immunised) in 12-24 hours, beginning with stiffness in the
tail . Rigidity proceeds to the leg on the inoculated side, the opposite leg,
trunk and forelimbs, in that order. The animal dies within two days .
39.
40. � Prevention: Active immunization with tetanus toxoid (formalin inactivated
toxin) prevents tetanus. It is usually administered to children as a triple
vaccine with diphtheria toxoid and pertussis antigens, called DPT. The
currently recommended regimen calls for injections of infants at two, four,
six, and eighteen months of age, followed by a booster upon entering
school, at about age five years. Recent studies have confirmed that
circulating antibody levels gradually decline, and that many older individuals
lose protection. Therefore booster immunizations with a preparation of
diphtheria and tetanus toxoids given every ten years throughout life are
recommended. Tetanus immune globulin can be used to give immediate
passive immunity to injury victims with no history of immunization. Active
immunization should also be started. Antitoxin and toxoid, administered in
different areas of the body, can be given simultaneously.
Prophylaxis and treatment:
41. � Passive immunization is by injection of tetanus antitoxin. Anti-
tetanus serum (ATS) from hyperimmune horses was originally
used. However, equine ATS carried two disadvantages common in
the use of any heterologous serum: 'immune elimination‘ and
hypersensitivity. The half-life of ATS in human beings is
normally about seven days but in persons previously injected with
horse serum, it is eliminated much more quickly because it
combines with preexisting antibodies. Prior sensitization also leads
to type III hypersensitivity reactions , which may range from mild
local reactions to serum sickness. Infrequently, fatal anaphylaxis
(type I hypersensitivity) may also occur. It is obligatory that a test
for
hypersensitivity be done prior to administration of ATS.
� Human anti-tetanus immunoglobulin (TIG) provides passive
immunity without the risk of hypersensitivity. This is effective in
smaller doses (250 units) and has a longer half-life (3-5 weeks). As
TIG is prepared by immunization of human volunteers, its availability
is limited .
Passive immunization is an emergency procedure
to be used only once.
42. � Treatment:
Tetanus patients should be treated in hospitals, preferably in special
units. Isolation is necessary to protect them from noise and light
which may provoke convulsions .
• Supportive therapy consists of ensuring quiet environment,
controlling spasms and autonomic dysfunctions with sedatives and
muscle relaxants, maintaining airway by tracheostomy with intermit
tent positive pressure respiration and attention to feeding.
• Human TIG: 10,000 IU suitably diluted may be given by slow IV
infusion, followed , if needed, by another 5,000 IU later. Even though
TIG may not neutralize the toxin already bound to the nervous tissue,
it can inactivate the unbound toxin and any further toxin that may be
produced.
• Antibacterial therapy with penicillin or metronidazole should be
started at once and continued for a week or more. Patients
recovering from tetanus should receive a full course of active
immunization, as an attack of the disease does not confer immunity.
Second attacks of tetanus have been recorded.
43. C. botulinum causes botulism, which occurs in several clinical
forms. Botulism is caused by the action of a neurotoxin that
is one of the most potent poisons known. It causes a flaccid
paralysis. Contact with the organism itself is not required;
hence the disease can be a pure intoxication.
Epidemiology: C. botulinum is found worldwide in soil and
aquatic sediments, and the spores frequently contaminate
vegetables, and meat or fish. Under appropriate
conditions, including a strictly anaerobic environment at
neutral or alkaline pH, the organism germinates, and toxin
is produced during vegetative growth. Because the toxin is
often elaborated in food, outbreaks frequently occur in
families or other eating groups.
C.botulinum
44. C.botulinum is motile by peritrichate flagella, producing
subterminal, oval, bulging spores.
� Spore : Spores are heat- and radiation-resistant,
surviving several hours at 100°C and for up to 10
minutes at 120°C. Spores of the non-proteolytic types B,
E and F are much less resistant to heat.
Morphology:
45. � C.botulinum is non-invasive and virtually non-infectious.
� Its pathogenicity is due to the action of its toxin, the
manifestations of which are collectively called botulism.
Toxins:
� Exotoxin: C.botulinum produces a powerful exotoxin that is
responsible for its pathogenicity. The toxin differs from other
exotoxins in that it is not released during the life of the organism.
It is produced intracellularly and appears in the medium only on
cell death and autolysis. It is believed to be synthesized initially as
a non-toxic protoxin or progenitor toxin. Trypsin and other
proteolytic enzymes activate the progenitor toxin to produce active
toxin .
Pathogenicity:
46. The toxin has been isolated as a pure crystalline protein
which is probably the most toxic substance known .It has a
MW of 70,000 and the lethal dose for mice is 0.000,000,033
mg. The lethal dose for human beings is probably 1-2 μg. It is
a neurotoxin and acts slowly ,taking several hours to kill. It is
one of the most potent toxins known to mankind. The
toxin is relatively stable, being inactivated only after 30-40
minutes at 80°C and 10 minutes at 100°C. Food suspected to
be contaminated with the botulinum toxin can be rendered
completely safe by pressure cooking or boiling for 20
minutes. It resists digestion and is absorbed through the
small intestines in active form. It acts by blocking the
production or release of acetylcholine at the synapses and
neuromuscular junctions. Onset is marked by diplopia ,
dysphagia and dysarthria due to cranial nerve involvement. A
symmetric descending paralysis is the characteristic pattern,
ending in death by respiratory paralysis.
47.
48.
49. � Clinical uses of toxins: A small quantity of C.botulinum
type A toxin injected into a muscle selectively weakens it by
blocking the release of acetylcholine at the neuromuscular
junction. Muscles so injected atrophy but recover in 2-4
months as new terminal axon sprouts form and restore
transmission. Intramuscular injection of the toxin , first used
to treat strabismus, is now recognized as a safe and effective
symptomatic therapy for man neuromuscular diseases. The
botulinum toxin can be toxoided. It is specifically neutralized
by its antitoxin and is a good antigen. The toxins produced
by the different types of C.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 .
50. Botulism
Classic botulism is a food poisoning in which a patient first
begins to experience difficulties in focusing vision, in
swallowing, and in other cranial nerve functions, 12 to 36
hours after ingesting toxin-containing food but not
necessarily viable organisms. There is no fever or sign of
sepsis. A progressive paralysis of striated muscle groups
develops, and mortality is about fifteen percent, the patient
usually succumbing to respiratory paralysis. Recovery is
protracted, lasting several weeks.
Clinical manifestations:
51. � Infant botulism: The most common form of
botulism in the United States today is infant
botulism or a cause of floppy baby syndrome.
C. botulinum colonizes the large bowel of
infants, 3 to 24 weeks of age, and the toxin
produced is slowly absorbed. Constipation,
feeding problems, lethargy, and poor muscle
tone are common early signs. Certain formula
supplements, such as honey contaminated with
C. botulinum spores, may transmit the organism.
The condition is possibly a cause of sudden
infant death syndrome, but recovery is the
usual outcome, following symptomatic
treatment that may be prolonged.
Cases occur in infants below six months. Older children and adults are not susceptible. The
manifestations are constipation, poor feeding, lethargy, weakness, pooled oral secretions, weak or
altered cry, floppiness and loss of head control. Patients excrete toxin and spores in their feces.
Toxin is not generally demonstrable in blood. Degrees of severity vary from very mild illness to fatal
disease. Some cases of sudden infant death syndrome have been found to be due to infant
botulism. Honey has been incriminatedas a likely food item through which the bacillus enters the
gut.
Management consists of supportive care and assisted feeding. Antitoxins and antibiotics are
not indicated.
52. � Wound botulism: A rare form of botulism occurs
when a wound becomes contaminated with the
organism, and toxin is absorbed from that site.
53. � Microscopy:
Diagnosis may be confirmed by demonstration of the bacillus or the
toxin in food or feces. Gram-positive sporing bacilli may be
demonstrated in smears made from the food. C.botulinum may be
isolated from the food or the patient's feces .
� Animal inoculation: The food is macerated in sterile saline, and the
filtrate inoculated into mice or guinea pigs intraperitoneally. Control
animals protected by polyvalent antitoxin remain healthy.
� Cultural characteristics:
It is a strict anaerobe. Optimum temperature is 35°C but some strains
may grow even at 1-5°C. Good growth occurs on ordinary media.
Surface colonies are large, irregular and semitransparent, with a
fimbriate border. Biochemical reactions vary in different types. Spores
are produced consistently when grown in alkaline glucose gelatin
media at 20-25°C. They are not usually produced at higher
temperatures .
Laboratory diagnosis:
54. � As most cases of botulism follow consumption of inadequately
canned or preserved food , control can be achieved by proper
canning and preservation. When an outbreak occurs, a pro
phylactic dose of antitoxin should be given intramuscularly
to all who consumed the food article.
� Active immunization has been shown to be effective.
If immunization is needed, as in laboratory workers exposed to
the risk, two injections of aluminium sulphate adsorbed toxoid
may be given at an interval of ten weeks, followed by a booster a
year later Antitoxin may be tried for treatment. Polyvalent
antiserum to types A, B and E may be administered as soon as a
clinical diagnosis is made.
� Supportive therapy with maintenance of respiration is of
equal or greater importance.
Prevention and treatment:
