In this article we will discuss about Clostridium which is a Anaerobic Sporing, Gram-Positive Bacilli:- 1. Classification of Clostridium Tetani 2. Clostridium Tetani 3. Morphology and Staining 4. Cultural Characteristics 5. Resistance 6. Antigenic Structure 7. Mode of Infection 8. Laboratory Diagnosis 9. Immunization 10. Prevention and Treatment.
Contents:
- Classification of Clostridium Tetani
- Clostridium Tetani
- Morphology and Staining of Clostridium Tetani
- Cultural Characteristics of Clostridium Tetani
- Resistance
- Antigenic Structure of Clostridium Tetani
- Mode of Infection
- Laboratory Diagnosis of Clostridium Tetani
- Immunization
- Prevention and Treatment of Clostridium Tetani
1. Classification of Clostridium Tetani:
The Clostridia can be classified on the basis of diseases produced by them:
2. Clostridium Tetani:
Causes tetanus characterized by hyper-excitability of voluntary musculature and responses to trivial stimuli. When the muscles of jaw are affected, the trismus occurs as an early sign of tetanus in man, ultimately lockjaw occurs.
3. Morphology and Staining of Clostridium Tetani:
Straight, slender rod shaped Gram-positive organisms, 2-3 x 0.4 – 0.5 µ. There are ten types of CI. tetani which are motile except type 6. Spore is terminal and spherical, 2-4 times the diameter of the bacillus producing drum stick appearance (Fig. 43.1)
4. Cultural Characteristics of Clostridium Tetani:
CI. tetani is an obligatory anaerobe, optimum temperature 37°C, grows on nutrient agar medium under anaerobic condition (Fildes or Mcintosh jar,) Agar plates or culture tubes are placed in an airtight jar from which air is removed and replaced by nitrogen with 10% CO2 or oxygen must be replaced by “Gaspack.”
On solid media, smaller colonies that extend (more than 1 mm in diameter) in a meshwork of the fine filaments are formed. CI. tetani forms haemolysis on blood agar medium. Robertson’s cooked meat medium (RCM) shows slight digestion and blackening of meat due to sulphur containing amino acid. Cultures have an unpleasant slightly pungent smell. Biochemically, CI. tetani are inert.
5. Resistance of Clostridium Tetani:
Majority of CI. tetani strains are killed by boiling for 15 minutes but some withstand boiling for 3 hours and dry heat at 160°C for one hour. The spores can survive in soil for years, but they are killed by autoclaving at 121°C; Iodine (1% aqueous) solution) and hydrogen peroxide (10 volumes) kill the spores within few hours.
6. Antigenic Structure of Clostridium Tetani:
All strains of CI. tetani share a common somatic (O) antigen. On the basis of specific flagellar (H) antigens, ten types (I-X) of CI. tetani are recognised by agglutination test. Type VI of CI. tetani is a non-flagellar strain which is only one non-motile strain.
All types produce the same toxin which is pharmacologically and antigenically identical. Toxin production seems to be plasmid mediated, Exotoxin of CI. tetani has two components.
Toxin:
The potent exotoxin consists of:
(a) Tetanospasmin, and
(b) Tetanolysin; tetanospasmin, essential pathogenic constituent, acts in several ways upon central nervous system (CNS).
It inhibits the release of acetylcholine, thus interfering with neuromuscular transmission, which results into generalized muscular spasms, hyper-reflexia and seizures. This toxin has been separated as a pure crystalline protein which is lethal to a mouse in 0.000001 mg dose.
Thus it is a powerful poison second in potency only to the exotoxin of C. botulinum and its production is favoured by:
(1) Necrotic tissue,
(2) Calcium salts,
(3) pyogenic infection.
The toxin culture filtrate is absorbed by motor end plates and is spread up the spaces between the nerve fibres and reach the CNS or via blood stream. When tetanus toxin is injected into guinea pigs or mice, the animals die within a day or two with typical signs of tetanus. Tetanic spasms may start in the muscles related to the site of injection.
Tetanolysin:
It is heat and oxygen labile and causes lysis of red blood cells of many species of animals (horse, rabbit). It may act as leucocidin and its pathogenic role is still unclear.
Antitoxin:
Tetanus antitoxin, called Anti-Tetanus Serum (ATS) can be obtained by immunizing horses with toxoid and is of value in the prophylaxis of tetanus when given as passive immunization.
Clinical Features:
The incubation period is 4-5 days to weeks. The disease is characterised by convulsive tonic contraction of voluntary muscles. Muscular spasms often involve first the area of injury and infection and then the muscles of jaw (trismus, lockjaw) which contract so that the mouth cannot be opened. Gradually, other voluntary muscles become involved— resulting in tonic spasm.
Tetanus:
It is characterised by severe and painful muscle spasms. Masseter muscles are first affected causing “Lockjaw“; when extensor muscles of the body are affected by the progressive spasm the body becomes arched in opisthotonus patients. Usually the wound is infected by CI. tetani and its spores—which ultimately produce toxin responsible for tetanus. Wounds of face, neck and upper extremities are greater risk.
7. Mode of Infection in Clostridium Tetani:
1. Exogenous Infection:
CI. tetani are usually found in the intestine of man and animals and are excreted out along with faeces which contaminate the soil. The spores of CI. tetani in soil and dust may infect the deep penetrating wound or street accident wound.
The growth of tetanus bacilli is promoted by the anaerobic condition and also by the presence of dead tissue and effused blood. The contaminated surgical equipment’s may infect surgical wound and umbilical stump thorn prick may cause tetanus.
2. Endogenous Infection:
Tetanus may result from septic abortion and puerperal sepsis in a person harbouring CI. tetani in intestine. The spores of tetanus bacilli germinate in wound under a favourable condition (low oxygen tension, presence favourable) of necrotic tissue, effused blood and associated pyogenic infection of the wound.
Tetanus neonatorum, postabortal and puerperal tetanus may cause high fatality (15-50%). The warm climate, unhygienic conditions and poor medical services are the factors responsible for higher incidence of tetanus in developing countries.
8. Laboratory Diagnosis:
Diagnosis rests on clinical picture and history of injury. Anaerobic culture of contaminated tissue may yield CI. tetani. However, the preventive and therapeutic use of antitoxin should never be withdrawn pending the cultural test. Proof of isolation of CI. tetani must rest on production of toxin and its neutralisation by specific antitoxin. Wound swab, exudate, in tissue from wound should constitute as specimens.
1. Direct Smear:
Direct smear after Gram’s staining shows Gram-positive bacilli with drum-stick appearance, morphologically similar to other non-pathogenic bacilli. Hence, they should be distinguished by their specific toxigenic property in mice.
2. Culture:
If the tissue is heavily contamined, the specimen should be heated at 80°C for 10 minutes before culture, because it destroys non-sporing organisms. CI. tetani grow in blood agar and aminoglycoside blood agar under anaerobic condition or in Robertson’s cooked meat medium.
The medium is made in one selective by addition of polymyxin B which inhibit all aerobes but not Clostridia. CI. tetani produce swarming growth after 1-2 days of incubation.
3. Animal Inoculation:
It is carried out to demonstrate the toxigenicity of isolated CI. tetani which establishes the pathogenicity.
Two mice were used, in one mouse, 0.2 ml of 2 to 10 days old cooked meat broth culture filtrate is injected subcutaneously into the right side of the base of the tail of the mouse (test animal), same quantity of toxin filtrate is injected into the other animal (control) which has already received 1,000 units of tetanus antitoxin one hour prior to testing.
In positive case, the test animal shows the symptoms of tetanus (stiffness, spasm of the tail, hind limb) within 24 hours and death. The control animal shows no symptoms due to neutralisation of toxin by antitoxin.
9. Immunization:
Tetanus is a fatal disease and can be prevented by immunization:
1. Active Immunization:
It can be done by Tetanus Toxoid (TT) which is prepared by detoxifying the toxin with formal (formal toxin) and by absorbing the toxoid into aluminium hydroxide or phosphate (API).
Dose:
Two doses of toxoid (API) of 0.5 ml each are injected intramuscularly at initial stage at an interval of 6 weeks. After 6 to 12 months a third dose (0.5 ml) of toxoid is administrated. During the first year of life all children are immunized by DPT vaccine which contains diphtheria toxoid, pertussis vaccine and tetanus toxoid in which vaccine acts as an adjuvant.
Thus immunity is conferred for 7 to 10 years. After 5 years, on entry into school, a booster dose of toxoid is given and thereafter booster dose is given every 7 to 10 years.
2. Passive Immunization:
It is carried by anti-tetanus serum (ATS). This ATS is obtained by immunizing horses with toxoid and is used as prophylaxis in a dose of 1,500 International Units (IU) by intramuscular injection immediately after injury for systemic protection against toxin.
ATS is repeated at weekly intervals till the risk of tetanus persists. The dose is same for adults and children. The ATS has a risk of hypersensitivity reaction. Before administrating ATS, a skin test (subcutaneous injection of 0.1 ml ATS) is performed.
The risk of heterologous serum (ATS) can be avoided by using human tetanus immunoglobulin (HTIG) which is a homologous serum prepared from humans and used in advanced countries in prophylaxis and treatment of tetanus. A single dose of 250 units is used in prophylaxis.
3. Combined Immunization:
After an injury, in emergency it is advisable to immunise an individual with tetanus toxoid in one arm along with the administration of 1,500 IU of ATS or 250 units of HTIG in another arm. The other doses of toxoid are given at appropriate intervals. In prophylaxis, penicillin is also recommended.
10. Prevention and Treatment:
Prevention depends upon:
1. Active immunization with toxoid,
2. Proper care of wound contaminated with soil,
3. Prophylactic use of antitoxin,
4. Administration of penicillin.
Antitoxin:
Tetanus antitoxin can neutralize the toxin, but only before it becomes fixed in nervous tissue. Because of hypersensitivity to foreign serum, the administration of human antitoxin is preferable. Patients who develop symptoms of tetanus should receive muscle relaxants, sedation and assisted ventilation.
Surgical removal of necrotic tissue. Penicillin inhibits the growth of CI. tetani and stops further toxin production. Booster shot, when a previously immunized individual sustains a potentially dangerous wound, an additional dose of toxoid should be injected to restimulate the antitoxin production.
Control:
Three injections of alum adsorbed tetanus toxoid (IT) comprise the initial course of immunization followed by another dose of about one year later. Initial immunization should be carried out in all children during the first year of age. A booster dose is given upon the entry into school. The immunization by triple vaccine can also cover tetanus.