CORYNEBACTERIUM GROUP Lecture Notes

- They are a diverse group of aerobic, non-spore forming Gram positive bacilli.
- Many members of the Corynebacterium group are members of the normal flora of the skin and mucous membranes of humans/ called diphtheroids
- Other Corynebacteria are found in animals and plants.
- Corynebacterium diphtheriae is the most medically important member of this group as it can produce a powerful exotoxin that causes diphtheria disease in humans.

Corynebacterium diphtheriae

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  • Gram positive bacilli with one swollen end and another tapering end (club shaped), non-motile, non-spore forming and non capsulated. They have Chinese letters like arrangement.
  • They contain inclusion granules called metachromatic or volutin granules, that can be demonstrated by methylene blue or Neisser stains. It is the causative organism of diphtheria.

Diphtheria

- It is an acute infectious disease confined to man which has virtually disappeared in developed countries following mass vaccination. It is characterized by local inflammation accompanied by toxemia.
- Human are the only resevoir

Pathogenesis

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- C. diphtheriae  infects the respiratory tract by droplet inhalation , or infect the skin by direct contact.
- Most clinical infections are contracted from carriers rather than symptomatic patients.
- C. diphtheriae carriers usually harbor the organism in their throat or nose.

- The organism doesn’t penetrate deeply into mucosal tissue and bacteremia doesn’t occur.
- The organism starts ,to multiply at the site of entry and produces  small amount of exotoxin to kill any phagocytic cells and then multiplies freely causing inflammation of the throat with local necrosis.
- At the same time the organism secretes exotoxin to the blood causing toxemia.

Diphtheria Exotoxin:

- It is a heat labile polypeptide composed of two fragments; A (active) and B (binding).
- The ability of C. diphtheria to produce toxin is due to presence of prophage in the bacterial chromosome, the gene for toxin production is carried on this prophage. Thus the acquisition of phage leads to toxigenicity, i.e. lysogenic conversion, while invasiveness is under the control of bacterial genes.

Clinical features:

- The incubation period of diphtheria is 2-5 days, the patient present with malaise, sore throat and moderate fever.
- In nasopharyngeal infection a thick, adherent pseudomembrane is present on one or both tonsils or adjacent pharynx. The pseudomembrane may involve nasal mucosa, the pharyngeal wall and soft palate with cervical lymph glands edema.
- Laryngeal involvement leads to laryngeal and lower airway obstruction (suffocation).

-  Cutaneous diphtheria occurs in tropical countries, the lesion is characterized by a non healing ulcer covered by necrotic pseudomembrane.
- Intoxication takes the form of myocarditis ,peripheral neuritis,visual disturbance ,difficulty in swallowing and paralysis of the arms and legs.

Laboratory Diagnosis of Diphtheria:

- The diagnosis is made on clinical backgrounds, supported by a history of contacts, lack of history of immunization, or traveling to endemic areas.

- The clinician should inform the laboratory about the presumptive diagnosis because diphtheria requires special culture media. The diagnosis depends on isolation of C. diptitheria and detection of toxin production by this strain.

- Specific treatment should never be delayed for laboratory diagnosis.

Specimens:

- Swabs from throat, nose, or any other lesion must be taken prior to any treatment.

1. Microscopic examination of a direct smear:

- Gram stained smear is suggestive for diagnosis, but a negative result does not exclude diphtheria.

- Gram stained film shows Gram positive bacilli with one swollen end and another tapering end (club shaped). The organism has Chinese letter like arrangement, non-motile, non capsulated and non spore forming.

- Smears stained with methylene blue and Neisser stains: the organism appears beaded due to volutin granules

2. Cultivation and identification:

•The organism can’t grow on simple media but better on media enriched with serum. Specimen is cultured on:

• Loeffler’s medium: under aerobic condition, for 12-18 h, at 37°C.

• Blood tellurite medium: it is a selective and a differentiating medium. Three biotypes of diphtheria can be identified after 2-3 days.

 Identification:

a. Gravis type.

b. Intermedius type.

c. Mitis type.

• Colonies are identified by film stained with Gram or methylene blue.

3. Toxigenicity Tests:
Such tests are performed only in the reference laboratories.

a. Elek’s test: (in-vitro virulence test)

• It is an example of precipitation test.

• The organism to be tested is streaked on serum agar medium then, a filter paper soaked with antitoxic serum is put perpendicular to the organisms, and the plate is incubated at 37°C for 2 days.

• If the organism is toxigenic the antitoxin diffuses from the filter paper and reacts with the toxin forming lines of precipitation. Absence of precipitation lines indicates that the strain is non toxigenic.

3. Toxigenicity Tests:

b. Tissue culture test:

c. polymerase chain reaction.

D- ELISA TEST.

E- animal pathogenicity test.

Treatment:
1- Diphtheria antitoxic serum

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- should be given without waiting for laboratory results if the clinical picture is strongly suggestive of diphtheria.

- The antitoxin neutralizes the toxin before it causes irreversible damage.

- Dose of 20,000-100,000 units should be given IM or IV.

- Side effects or complications like hypersensitivity reaction e.g. anaphylactic shock or serum sickness may develop to the horse serum (as the antitoxin is prepared by injection of horse with toxoid).

- To avoid this, sensitivity test to horse serum must be done before treatment. In allergic patients hyposensitization is done by injecting slowly increasing doses of antitoxin.

Treatment Diphtheria :
2- Antibiotics:

- Penicillin or erythromycin reduce the number of diphtheria bacilli in the throat and decrease the toxin production.

- Also they help in eliminating coexistence of streptococci with diphtheria.

- Antibiotics must be given with antitoxic serum.

Prophylaxis :

1- Active immunization:

• Diphtheria can be prevented by active immunization with toxoid.(a toxin losses its toxicity but remain antigenic)

•  Several forms of toxoid are available.e.g:

a. Fluid formol toxoid.

b. Alum precipitated toxoid.

They are given in combination with pertussis vaccine and tetanus toxoid (DPT) at age of 2, 4, 6 month I.M. Booster doses are given at 1.5 year, at school age, and every 10 years which contain diphtheria and tetanus toxoids (DT) only.

2. Passive Immunization:

By antitoxic serum to contacts with cases (1000-1500) units.

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