- Clostridium tetani is a gram-positive, anaerobic bacterium that causes the disease tetanus. It forms spores that are found in soil and can infect wounds. - It produces two toxins - tetanolysin and tetanospasmin (the neurotoxin responsible for tetanus symptoms). - Diagnosis involves culturing samples from infected wounds under anaerobic conditions. Laboratory tests and animal inoculation can confirm toxicity. - Treatment focuses on controlling spasms and maintaining airway/nutrition support. Antitoxin can neutralize unbound toxin and antibiotics are given. Immunization with tetanus toxoid provides the best long-term

1. ClostridiumTetani
By: Moneta Minoy
2nd yr
Microbiology dept.

2. Introduction
•Cl. tetani is the causative agent of tetanus, a
disease, which has been known since very early
times.
• Cl. tetani is widely distributed in soil and intestine
of man and animals.
•It is ubiquitous.

3. Morphology
• It is a Gram positive, slender bacillus (measuring 4-8 µm x 0.5
µm)
• with spherical, terminal spores giving the bacillus the
characteristic drumstick appearance
• It is non-capsulated and motile (except Cl. tetani typeVI) with
peritrichate flagella.
• The young cultures are strongly Gram positive but older cultures
may show variable staining and may even be Gram negative.

5. Culture
• It is an obligate anaerobe which grows on ordinary media.
• Growth is improved by the addition of blood or serum.
• The optimum temperature for growth is 37°C and pH 7.4.
• It can grow well in cooked meat broth (CMB), thioglycollate broth, nutrient
agar and blood agar.
• In CMB, growth occurs as turbidity and there is also some gas formation.
The meat is not digested but becomes black on prolonged incubation
• The bacilli produce a swarming (thin spreading film) growth on blood agar.
• On horse blood agar, they produce ex-haemolytic colonies which
subsequently develop into beta-haemolytic, due to the production of a
haemolysin (tetanolysin)

7. Biochemical Reactions
• Cl. tetani has slight proteolytic, but no saccharolytic property
• Gelatin liquefaction occurs very slowly.
• It does not ferment any sugar.
• It forms indole
• MR andVP negative.
• H2S is not formed.
• Nitrates are not reduced.
• A greenish fluorescence is produced on media containing neutral red (e.g.
MacConkey's medium)

8. Resistance
• The resistance of Cl. tetani spores to heat vary in different strains
• Most of the strains are killed by boiling for 10-15 minutes but some resist
boiling for 3 hours.
• Autoclaving (at 121 °C for 20 minutes) kills the spores of most strains.
• Spores are also killed with Iodine (1 % aqueous solution), hydrogen peroxide
(10 volumes) and glutaraldehyde (2%) within a few hours.
• The spores may survive in soil for years.

9. Classification
• Ten serological types (types I to X) of Cl. tetani have been recognised based
on type specific flagellar (H) antigens by agglutination test.
• TypeVI strains are non-flagellated.
• All the types produce the same toxin, which can be neutralised by antitoxin
produced against any one type.

10. Toxins
• Cl. tetani produces two distinct toxins- tetanolysin (haemolysin) and
tetanospasmin (neurotoxin).
1. Tetanolysin- Tetanolysin is a heat labile, oxygen labile toxin which
causes lysis of erythrocytes of several animal species, especially the rabbit and
the horse.
• It may act as a leucotoxin but its pathogenic role is not clear.
2. Tetanospasmin-Tetanospasmin is a heat labile, oxygen stable,
powerful neurotoxin and rapidly gets destroyed by proteolytic enzymes.
It is protein in nature. It is plasmid coded.This is responsible for clinical
manifestations of tetanus.
Tetanospasmin is a good antigen and is specifically neutralised by the
antitoxin.

11. Pathogenesis
• Cl. tetani has little invasive power.
• Tetanus develops following the contamination of wound with Cl. tetani
spores.
• The source of infection may be soil, dust, faeces etc. Infection strictly
remains localized in the wound.
• Germination of spores and toxin production occur only if favourable
conditions exist, such as reduced 0-R potential, devatilised tissues and
foreign bodies.

12. Pathogenesis
• Pathogenic effects are mainly due to tetanospasmin (neurotoxin) of Cl.
tetani.
• The tetanus toxin specifically blocks synaptic inhibition in the spinal cord.
The toxin acts presynaptically.
• The abolition of spinal inhibition leads to uncontrolled spread of impulses
initiated anywhere in the central nervous system.
• This results in muscle rigidity and spasms due to the simultaneous
contraction of agonists and antagonists.

15. Laboratory Diagnosis
• The diagnosis of tetanus should always be made clinically and laboratory
tests are done only to confirm it.
• Laboratory diagnosis may be made by demonstration of bacilli by
1. Microscopy,
2. Culture
3. By Animal inoculation.
• Specimens generally collected are wound swab, exudate or tissue from the
wound.

16. Microscopy
• Gram staining may show Gram positive
bacilli with drumstick appearance but
these are indistinguishable from
morphologically similar non-pathogenic
bacilli like Cl. tetanomorphum and Cl.
sphenoides.
• Hence microscopy alone is unreliable
but diagnosis by culture is more
dependable.

17. Culture
• Specimen is inoculated on freshly prepared blood agar and incubated at 37°C for
24-48 hours under anaerobic conditions.
• The incorporation of polymyxin B in culture medium makes it more selective as
clostridia are resistant to this antibiotic
• Cl. tetani produces a swarming growth.The specimen is also inoculated in three
tubes of cooked meat broth.

18. Culture
• One of these tubes is heated at 80°C for 15 minutes, the second tube for 5
minutes and the third left unheated. Heating for different periods is to kill
vegetative bacteria, while leaving tetanus spores undamaged.
• These cooked meat broth are incubated at 37°C and subcultured on blood
agar plates daily for upto four days.
• Gram stained smear from culture shows typical Gram positive bacilli with
drumstick appearance.

19. Toxigenicity test / Animal Innoculation
• Pathogenicity of the isolated organism is established with demonstration of
toxin production.
• It is best tested in animals.
• 0.2 ml of 2 to 4 days old cooked meat culture is injected into the root of the
tail of one mouse (test animal), same amount is injected into another animal
(control) that has received tetanus antitoxin (1000 units) an hour earlier.

20. Result ofToxigenicity test
• In positive case, the test animal develops symptoms within 12- 24 hours,
beginning with stiffness of the tail.
• Rigidity proceeds to the leg on the inoculated side, the another leg, trunk
and forelimbs, in that particular order. Death occurs within two days, but
may be killed earlier as the ascending tetanus is very much diagnostic.
• The control animal does not show any change due to neutralisation of toxin
by antitoxin.

21. Prophylaxis
• The available methods are: 1.)Surgical 2.) Antibiotics 3.) Immunisation
• (1) Surgical- It aims at removal of foreign body, blood clots etc., in order
to prevent anaerobic conditions favourable for the bacillus.
• Depending on the type of wound, surgical prophylaxis may vary from simple
cleansing to radical excision.

22. 2. Antibiotics
• Antibiotics destroy or inhibit tetanus bacilli and other pyogenic bacteria in
wounds.
• Thus the production of toxin is prevented.
• Long acting penicillin injection or erythromycin may be given.
• Antibiotic prophylaxis does not replace immunisation but is a useful
adjunct.

23. 3. Immunization - Active
• It is the most effective method of prophylaxis.
• Tetanus toxoid (formol toxoid), which is available either as 'plain toxoid', or
adsorbed on aluminium 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 first two doses.
• 6- 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.

24. • Tetanus toxoid is given along with diphtheria toxoid and pertussis vaccine
(DPT) in children.
• Pertussis 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 five years.

25. 3.Immunization- Passive
• Antitetanus serum (ATS), prepared by immunising horses with toxoid, has
been used for preventing tetanus.
• The dose is 1500 IU by intramuscular route immediately after the person is
wounded.
• The dose is same for adults and children.
• Being a horse serum, it carries the risk of hypersensitivity reaction,
therefore, a skin test is recommended before administersing ATS.
• Homologous serum prepared from humans, human antitetanus
immunoglobulins (HTIG), is now being used without the risk of
hypersensitivity.
• Dose of 250 units is used in prophylaxis.

26. 3. Immunization- Combined
In non-immune person, it is ideal to immunise with:
• First dose of tetanus toxoid in one site along with administration ofATS or
HTIG in another arm,
• followed by the second and third doses of tetanus toxoid at monthly
interval.
• In combined prophylaxis, adsorbed toxoid should be used as the immune
response to plain toxoid may be interfered with antitetanus serum.

27. Treatment
• Tetanus patients are treated in special isolated units.The reason for
isolation is to protect them from noise and light which may provoke
convulsions.
• These patients are not infectious and person to person transmission does
not occur at all.
• Treatment consists of controlling spasms, maintaining airway by
tracheostomy and attention to feeding.
• The antitoxin may be used to neutralise unbound toxin.
• Antibiotic therapy with penicillin or metronidazole should be started and
continued for a week or more.
• Patients recovering from tetanus should be immunised with full course of
toxoid, as an attack of the disease does not confer immunity.

28. Thank you!!!