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Clostridium tetani – Overview

Tetanus is caused by C. tetani, an obligate anaerobic Gram-positive bacillus. Tetanus is an infectious disease characterised by increased muscle tone and spasms caused by ...

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Tetanus is caused by C. tetani, an obligate anaerobic Gram-positive bacillus. Tetanus is an infectious disease characterised by increased muscle tone and spasms caused by the release of tetanospasmin, a neurotoxin produced by C. tetani when it is inoculated into humans.

Morphology of Clostridium tetani

C. tetani demonstrates the following characteristics:

  • 4–8 m in length, C. tetani is a slender, Gram-positive bacillus. Typically, young cultures are Gram-positive, whereas older cultures are Gram-variable or even Gram-negative.
  • This bacillus is straight with parallel sides and rounded ends. The bacillus occurs in singles and occasionally in chains. The bacteria consist of round, terminal, and bulging spores giving drumstick appearance to the bacillus. The spores are rarely seen even in clinical specimens from lesions or in culture media.
  • Except for type VI, all strains of C. tetani are motile due to the presence of flagella. Bacilli of strain type VI lack flagella and are therefore non-motile.
  • Bacteria are encapsulated.

Geographical distribution of Clostridium tetani

  • Tetanus is a widespread disease. The disorder is primarily prevalent in developing nations.
  • The disease is prevalent in agricultural regions, rural areas, warm and humid climates, and the summer months.
  • Tetanus affects individuals of all ages, with the highest incidence among infants and adolescents.
  • Overall, the annual incidence of tetanus is 0.5–1 million cases, predominantly in impoverished nations.
  • In underdeveloped nations, 50% of tetanus-related deaths are caused by neonatal tetanus.

Habitat of Clostridium tetani

  • Organisms of C. tetani are discovered in dirt, animal faeces, and occasionally human excrement, in addition to inanimate things.
  • In some conditions, the spores can persist for years and are resistant to disinfectants and even 20 minutes of boiling water.

Reservoir, source, and transmission of infection of Clostridium tetani

  • Spores of C. tetani are the infectious form of the bacteria.
  • Soil, animal faeces, and rarely human excrement, as well as spore-contaminated inanimate objects, are the most common sources of infection.
  • Risk factors for newborn tetanus include unvaccinated moms, home birth, and improper umbilical cord cutting.
  • Animal excrement, clarified butter, and other substances applied on the umbilical stump are additional risk factors for newborns.
  • The following wound types are more prone to tetanus: (a) severely polluted wounds; (b) wounds exposed to saliva or faeces; (c) stellate, ischemic, or infected wounds; (d) deep (1 cm) wounds; and (e) avulsions, punctures, and crush injuries.

Culture of Clostridium tetani

C. tetani is an anaerobe by necessity. Due to their extreme sensitivity to oxygen, the bacteria can only grow in the absence of oxygen. Bacillus develops optimally at 37 degrees Celsius and a pH of 7.4. C. tetani may grow in both standard and serum- and blood-enriched medium.

RCM medium

  • C. tetani thrives in RCM media.
  • In addition to producing turbidity, the bacteria create some gas in the medium.
  • Although the meat has not been digested, prolonged incubation causes it to become black. 

Blood agar

  • C. tetani causes alpha- hemolysis surrounding colonies on blood agar.
  • Upon prolonged incubation, the bacteria create tetanolysin, a hemolysin that converts alphahemolysis into beta-hemolysis.
  • Surface colonies tend to swarm throughout the entire agar surface. C. tetani creates a very fine, translucent growth film that is difficult to observe except near the colonies’ margins.

Gelatin stab culture

  • C. tetani liquefies gelatin anaerobically.
  • Therefore, the bacillus generates a growth resembling a fir tree in gelatin stab culture during anaerobic incubation.

Nutrient agar slope

  • Inoculation of the bacteria into the condensation water at the foot of the slope of nutritional agar, followed by 24 hours of anaerobic incubation, produces a pure colony of C. tetani at the top of the slope in the tube.
  • This technique, known as the Fildes technique, is routinely used to isolate pure colonies of C. tetani.

Biochemical reactions of Clostridium tetani

C. tetani exhibits the following responses:

  • C. tetani possesses moderate proteolytic activity but no saccharolytic activity.
  • There is no fermentation of sugars. It does not generate H2 S and has no effect on nitrates.
  • It is positive for indole, but negative for MR and VP.
  • It creates a greenish glow on neutral red-containing MacConkey medium.

Susceptibility to the physical and chemical agents

  • Different strains of C. tetani spores exhibit varying heat resistance.
  • Boiling at 100°C for 10–15 minutes and autoclaving at 121°C for 20 minutes kills the majority of spores.
  • A 1% aqueous solution of iodine and 10% hydrogen peroxide will also kill them. The spores are immune to the majority of antiseptics.
  • They are not destroyed by solutions of 5% phenol or 0.1% mercuric chloride. They may persist for years in soil.

Typing

  • C. tetani are categorised into 10 serological kinds (types I to X) based on agglutination.
  • Each strain produces the identical toxin.
  • The antitoxin standard neutralises the toxin.

Pathogenesis and Immunity 

C. tetani is a bacillus that is noninvasive. It only causes disease by producing toxins, which are the most significant virulence factors.

Virulence factors of Clostridium tetani

The following toxins are produced by C. tetani: (a) tetanolysin, (b) tetanospasmin, and (c) neurotoxic or nonspasmogenic toxin. Tetanolysin and tetanospasmin are two significant toxins with different pharmacological and antigenic properties. Recently, nonspasmogenic toxin or neurotoxic has been found.

Tetanospasmin

Tetanospasmin is the toxin that causes the clinical symptoms of tetanus. The toxin is created throughout the stationary phase of development, but it is not released until the bacteria are lysed.

  • Tetanospasmin is a protein composed of a single polypeptide chain with a molecular weight (MW) of 151,000 daltons. Upon release from the bacillus, an endogenous protease splits the peptide into two chains: a light chain (A) of 52,000 MW protein and a heavy chain (B) of 93,000 MW protein; the two chains are held together by the noncovalent forces of a disulfide bond.
  • The refined poison is incredibly strong. The minimum lethal dose (MLD) of a poison is 50–75 10–6 mg for animals and 130 ng for humans.
  • Different animal species have a wide range of vulnerability to tetanospasmin. Birds and reptiles possess exceptional resistance to the poison. Equine species are the most vulnerable to the toxin, followed in descending order by guinea pigs, goats, and rabbits.
  • The toxin blocks synaptic inhibition in the spinal cord by inhibiting the release of neurotransmitters such as gamma-aminobutyric acid (GABA), glycine, and others. This results in uncontrolled spread of impulses throughout the central nervous system (CNS).
  • Toxin binding cannot be reversed.
  • Antigenic, yet harmless, tetanus toxoid. Tetanus toxin is converted into toxoid by formaldehyde treatment.

Virulence factors of Clostridium tetani 

  • Tetanospasmin: Tetanospasmin is a potent heat-labile toxin that inhibits the release of neurotransmitters (e.g., GABA, glycine, etc.) and, as a result, prevents specific synaptic inhibition in the spinal cord. Without any inhibitory regulation, motor neurons undergo a persistent excitatory discharge.
  • Tetanolysin: Tetanolysin is a heat- and oxygen-sensitive hemolysin. It is related to other clostridial hemolysins and streptolysin O antigenically. The toxin’s pathogenicity is questionable, and it appears to have no part in the aetiology of tetanus.
  • Neurotoxic: A peripherally active, nonspasmogenic neurotoxin of uncertain importance. This is the third recently found toxin. It is a peripherally active and nonspasmogenic neurotoxic. Unknown is the involvement of this toxin in the pathophysiology of tetanus.

Pathogenesis of tetanus 

Tetanus is caused by C. tetani spores entering the body. Under favourable conditions of anaerobiosis, the spores germinate into a vegetative state and then create toxins such as tetanospasmin and tetalysin. Wounds with low oxidation–reduction potential, such as those with (a) dead devitalized tissue, (b) a foreign body, or (c) an active infection, promote anaerobiosis in tissues. Locally and peripherally at the myoneural junction of the nervous system, tetanospasmin is absorbed and transported centripetally into CNS neurons.

  • The 100 kDa heavy chain of the toxin is responsible for protein transport and particular binding to brain cells.
  • The light chain inhibits the release of GABA and glycine, two key inhibitory neurotransmitters. Consequently, inhibition of motor reflex reactions to sensory stimuli fails. In the absence of reciprocal inhibition, this results in a widespread contraction of the agonist and antagonist musculature, exhibiting the features of a tetanic spasm.

The shortest peripheral nerves rapidly transport poison to the central nervous system, resulting in facial deformation and back and neck stiffness. Depending on the route of administration, tetanospasmin’s toxicity varies. The method of administration also affects the progression of the disease’s clinical presentation.

  • Injections intraneurally or straight into the CNS are the most deadly.
  • Toxins administered intravenously, intramuscularly, and subcutaneously are effective, but toxin administered orally is ineffective because it is eliminated by digestive enzymes.

Tetanus in experimental animals

  • Experimental tetanus in animals, such as mice, is also affected by the route of toxin administration.

Intramuscular injection of toxin

  • The result is ascending tetanus. Intramuscular injection in one of the hind limbs is connected with the onset of tonic muscle spasm in that limb.
  • This is due to the toxin’s effect on the spinal cord segment.
  • Once the toxins penetrate the synaptic connections, they prevent inhibition or eliminate the nerve impulse by inhibiting or eliminating the nerve impulse, respectively.
  • The nerve continues to transmit impulses, causing spasms or tetani in the afflicted muscles.
  • Muscle stiffness is the earliest indication, and jaw muscles are frequently the first to develop symptoms.
  • This condition is known as lockjaw; as the disease progresses, other muscles spasm.
  • Although brief, the spasm occurs frequently and causes excruciating agony and tiredness. This is referred to as local tetanus.
  • As a result of the poison spreading up the spinal cord, ascending tetanus develops. This syndrome affects the opposite hind limb, trunk, and fore limbs.

Intravenous injection of toxin

  • This results in descended tetanus. In this condition, spasticity develops initially in the head and neck muscles and then travels downhill, similar to how tetanus occurs in humans.

Host immunity 

Protective antibodies against tetanus toxin are created. Antibodies combine specifically with free toxin to inhibit its activity.

  • The prevalence of tetanus-protective immunity is highest among children and those aged 6 to 39.
  • The protective immunity against tetanus declines with age. A poor level of immunity has been determined through serological analysis among the elderly in various nations. Approximately fifty percent of those older than fifty are nonimmune because they were never vaccinated or did not receive booster doses.
  • Clinical tetanus does not induce immunity; therefore, patients who survive the disease must be actively immunised with tetanus toxoid to prevent recurrence.

Clinical Syndromes of Clostridium tetani

The incubation time of tetanus varies from a few days to several weeks, but is typically between 6 and 12 days. The incubation period depends on (a) the distance between the primary wound infection and the central nervous system (CNS), (b) the inoculating dose of bacteria, (c) the toxigenicity of the bacteria, and (d) the immune status of the host. C. tetani can cause the following types of tetanus: (a) generalised tetanus, (b) neonatal tetanus, (c) localised tetanus, and (d) cephalic tetanus.

Generalized tetanus 

  • The most typical kind of tetanus is generalised tetanus. It occurs when the poison released at the wound site travels to many nerve terminals via the lymphatics and blood.
  • This is due to the fact that the blood–brain barrier limits direct toxin passage into the CNS. The severity of the injuries ranges from minor to severe crush injuries.
  • The incubation period ranges from 7 to 21 days, depending on the wound’s proximity to the CNS.
  • Involvement of the masseter muscle causes trismus, the most common and earliest symptom of the condition.
  • Other indicators include difficulty swallowing, impatience, and restlessness.
  • As the condition worsens, patients develop generalised muscle rigidity accompanied by occasional reflex spasms in reaction to stimuli such as noise or touch.
  • Opisthotonus is a condition characterised by flexion and adduction of the arms, clenching of the fists, and extension of the lower extremities that is caused by tonic contractions.
  • During these episodes, individuals experience intense pain and intact sensorium. Fractures, tendon ruptures, and abrupt respiratory failure may result from the spasms.
  • Risus sardonicus, often known as a sardonic smile, is a common trait caused by the continuous tension of facial muscles.
  • Due to the time needed for intra-axonal antitoxin transport, the disease may worsen for up to two weeks despite the administration of antitoxin.
  • The prognosis of tetanus depends on (a) the incubation period, (b) the time from spore injection to the onset of the first symptom, and (c) the time from the onset of the first symptom to the onset of the first tetanic spasm.
  • Tetanus with a brief incubation period is more dangerous than tetanus with a prolonged incubation period. Typically, recovery takes between 2 and 4 months.

Neonatal tetanus 

  • Neonatal tetanus is a broad form of tetanus caused by infection in a newborn. It mostly affects developing nations and is a leading cause of infant mortality.
  • Infection results through the use of a contaminated blade, knife, or other material to cut or dress the umbilical cord in babies, especially those born to unvaccinated mothers.
  • Typically towards the end of the first week of life, infected newborns develop irritability, poor feeding, and severe spasms.
  • The disease has an extremely bad prognosis for survival. The death rate is more than 70%.

Localized tetanus 

  • Tetanus localised is an uncommon form of tetanus. The sickness is localised to an extremity with a contaminated wound and solely affects the nerves that supply the affected muscle.
  • Due to a dysfunction in the interneurons that inhibit the alpha-motor neurons in the afflicted muscles, the condition is characterised by muscular rigidity.
  • Localized tetanus has no CNS involvement and extremely low fatality rates.

Cephalic tetanus 

  • Localized tetanus is a kind of cephalic tetanus.
  • The disorder is typically caused by head trauma or infection of the middle ear.
  • Incubation time is extremely brief (1–2 days).
  • Localized or generalised symptoms may include solo or combined dysfunction of the cranial motor nerves, most commonly the seventh cranial nerve. This condition has an unfavourable prognosis.

Laboratory Diagnosis of Clostridium tetani

The clinical manifestation of tetanus and C. perfringens infection informs the laboratory diagnosis. Laboratory testing is only performed on patients to confirm the clinical diagnosis.

Specimens 

  • The specimens comprise necrotic tissue fragments extracted from the depths of wounds.
  • Wound swabs are not suitable specimens.

Microscopy 

Gram staining of C. tetani smears is beneficial but frequently ineffective and inaccurate.

The presence of drumstick bacilli in wound tissue is not diagnostic for tetanus. This is due to:

  • Some wounds may contain C. tetani without developing tetanus.
  • In addition, C. tetani may not be distinguishable by microscopy from morphologically similar Clostridium species, such as C. tetanomorphum and C. sphenoides.

Culture

  • The specimens are inoculated onto blood agar and incubated for 24–48 hours under anaerobic conditions.
  • C. tetani generates a swarming growth that spreads across the plate. Additionally, the specimens are inoculated into three tubes of RCM medium, one of which is heated at 80°C for 15 minutes, the second for 5 minutes, and the third for 24–48 hours at 37°C.
  • Subcultures are thereafter created everyday on blood agar for up to four days. C. tetani is isolated from the swarming margins of blood agar colonies in pure culture.
  • Because tetanus is caused by a small number of organisms and because many organisms are killed when exposed to air during collection processing, only 30% of tetanus cases result in a positive culture.

Identification of bacteria 

The distinguishing characteristics of C. tetani are their morphology, culture, and toxigenic properties.

Toxigenicity testing 

C. tetani strains are evaluated for toxin production using the following tests:

In vitro neutralization test on blood agar

  • The examination is conducted on a blood agar containing 4% agar. A high concentration of agar is used to prevent C. tetani from swarming.
  • One-half of the medium is inoculated with tetanus antitoxin (1500 units per mL), whereas the other half is antitoxin-free.
  • Each side of the plate is stab-inoculated with C. tetani strains and incubated anaerobically for 48 hours.
  • The colonies of C. tetani exhibit hemolysis on the portion of the blood agar lacking antitoxins, but not on the portion of the agar containing antitoxins.
  • This is due to the antitoxin in the agar inhibiting the hemolytic activity of the toxin. This test is beneficial for proving that the colony is C. tetani, however it is not a reliable test.

In vivo neutralization test in mice

  • In this test, 0.2 mL of a 2–4-day-old cooked meat culture of C. tetani is implanted into the root of the tail of two mice each; one of the mice is given 1,000 units of tetanus antitoxin 1 hour before to the test (control animal).
  • The other mouse lacks any antitoxin protection (test animal). Test animals acquire symptoms of ascending tetanus due to tetanospasmin generated by C. tetani 12–24 hours after bacterial inoculation.
  • The symptoms begin with stiffness in the tail and swiftly spread to the leg on the inoculated side, the opposite leg, the trunk, and the forelimbs.
  • Typically, the animal dies within 48 hours. The vaccinated control animal displays no symptoms.
  • Testing for toxicity in mice is a valid way for identifying the colony as C. tetani.

Serodiagnosis

  • Patients’ serum contains neither antibodies to tetanus toxin nor tetanus toxin itself, so serological tests are not performed.

Other tests

The spatula test

  • This is an extremely helpful and straightforward bedside diagnostic test for tetanus. The oropharynx is touched with a spatula or tongue blade for this examination.
  • Typically, this induces a gag reaction and the patient attempts to evacuate the spatula (negative test).
  • If tetanus is present, patients suffer a reflex spasm of the masseters and bite the spatula (positive test).
  • The test is 100% sensitive and 100% specific.

Identifying features of Clostridium tetani

  • Extremely thin, transparent layer of growth that tends to cover the entire agar surface.
  • Produce alpha-hemolytic colonies initially on blood agar, which turn beta-hemolytic after prolonged incubation due to the synthesis of tetanolysin.
  • In the Gram-stained smear of the colony, Gram-positive bacilli with conspicuous terminal spores (drumstick look) are seen.
  • Bacteria that are mobile (excluding type VI) and encapsulated.
  • Avoid fermenting any sugars.
  • Testing for toxicity in mice is a valid way for identifying the colony as C. tetani.

Treatment of Clostridium tetani

Treatment for tetanus includes (a) early supportive care, (b) wound debridement and treatment, (c) cessation of toxin generation, (d) neutralisation of unbound toxin, (e) control of clinical symptoms, and (f) management of sequelae. It consists of antibiotic therapy and immunoglobulin therapy.

Antibiotics therapy

  • Antibiotics are used to inhibit C. tetani from multiplying in the wound, hence stopping the generation and release of toxins.
  • The current antimicrobial medicine of choice is metronidazole, with penicillin serving as an option.
  • Tetracycline is an alternate medication for patients with penicillin or metronidazole allergies.
  • Other antimicrobials used to treat tetanus include clindamycin, erythromycin, and vancomycin.

Human immunoglobulin therapy

  • Human tetanus immunoglobulin (TIG) is administered to neutralise unbound tetanus toxins and prevent circulating tetanus toxin from reaching the central nervous system (CNS).
  • At the time of diagnosis, a single total dose of 3000–6000 IU is administered intramuscularly to children and adults.
  • The baby dose of 500 IU for neonatal tetanus has proven beneficial.
  • Equine tetanus antitoxin (ATS) is utilised in regions where TIG is unavailable.
  • Tetanus antitoxin is provided intramuscularly as a single dosage of 50,000–100,000 IU following sensitivity and desensitisation testing, as required. Always, a portion of this dose (20,000 IU) is administered intravenously.

Prevention and Control of Clostridium tetani

Active immunization

  • Tetanus is entirely prevented by vaccination.
  • Active immunisation with tetanus toxoid vaccine is the key to preventing tetanus.
  • Toxoids are used for vaccination, and they are either simple toxoid or adsorbed on aluminium hydroxide or phosphate.
  • The toxoid may be administered alone or in combination with diphtheria toxoid and acellular pertussis (whooping cough) vaccination (DTaP or triple).

Immunization for prevention of neonatal tetanus

  • Increasing immunisation among women of childbearing age, particularly pregnant women, and enhancing maternity care prevent neonatal tetanus.
  • Tetanus toxoid is administered to previously unimmunized pregnant women twice throughout pregnancy, 4–6 weeks apart, especially in the last two trimesters and again at least 4 weeks prior to delivery.
  • Antitetanus antibodies from the mother are transmitted to the foetus, and this passive immunity is effective for several months after birth.

Passive immunization Human

  • TIG is used to immunise passively. In nations where TIG is unavailable, ATS is also utilised for the same purpose.
  • Passive vaccination is administered intramuscularly with 250–500 IU of TIG or subcutaneously or intramuscularly with 1500 units of ATS.
  • When a wound is contaminated or likely to contain necrotic tissue, passive immunisation is indicated for: (a) unvaccinated individuals and (b) those whose immunisation status is unknown.

Combined immunization

  • After exposure, secondary tetanus protection is achieved through wound washing, debridement, and combined immunisation.
  • Combined immunisation is achieved by administering (a) human TIG or the ATS and (b) tetanus toxoid simultaneously.
  • If the patient has not been previously immunised with at least three doses of toxoid, it is indicated.

Vaccines

Tetanus toxoid and the triple vaccine containing tetanus toxoid are highly effective and safe. The toxoid induces the development of protective antibodies in nearly all immunocompetent individuals. Up to 90 percent of inoculated individuals 15 years after vaccination had protective blood antibody levels, according to numerous studies. Extremely modest and occasional adverse effects include mild fever and injection-site pain, redness, or edoema. The vaccine cannot transmit tetanus to patients. The primary prevention of tetanus is achieved with vaccination at ages 2, 4, 6, 12–18 months, and 4–6 years with the triple vaccine. A booster dose of tetanus-diphtheria (dT) is provided between the ages of 11 and 12 and then every 10 years. Tetanus toxoid is provided in two doses 4–6 weeks apart, followed by a third dose 6–12 months later, for the primary immunisation of unimmunized children aged 7 or older. Booster doses are delivered every ten years or at the time of a serious accident if it happens more than five years after the initial vaccination. Tetanus toxoid vaccination is advised for all people who have not received a booster dose during the past ten years.

  • Adults recovering from tetanus infection.
  • Adults who have never gotten a tetanus vaccination.

References

  • George EK, De Jesus O, Vivekanandan R. Clostridium Tetani. [Updated 2022 May 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482484/
  • Hodowanec, A., & Bleck, T. P. (2015). Tetanus (Clostridium tetani). Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 2757–2762.e2. doi:10.1016/b978-1-4557-4801-3.00246-0 
  • Hodowanec, A., & Bleck, T. P. (2015). Tetanus (Clostridium tetani). Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 2757–2762.e2. doi:10.1016/b978-1-4557-4801-3.00246-0
  • Fishman, P. S. (2009). Tetanus Toxin. Botulinum Toxin, 406–425. doi:10.1016/b978-1-4160-4928-9.00034-2 

Citation

APA

MN Editors. (November 29, 2022).Clostridium tetani – Overview. Retrieved from https://microbiologynote.com/clostridium-tetani-overview/

MLA

MN Editors. "Clostridium tetani – Overview." Microbiology Note, Microbiologynote.com, November 29, 2022.

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