In this article we will discuss about Corynebacterium Diphtheriae which causes a disease called Diptheria in humans:- 1. Morphology and Staining of C. Diphtheriae 2. Cultural Characteristics C. Diphtheriae 3. Pathogenicity 4. Clinical Features 5. Laboratory Diagnosis 6. Production of Toxin 7. Treatment and Prophylaxis.
Contents:
- Morphology and Staining of C. Diphtheriae
- Cultural Characteristics of C. Diphtheriae
- Pathogenicity
- Clinical Features of C. Diphtheriae
- Laboratory Diagnosis of C. Diphtheriae
- Production of Toxin by C. Diptheriae
- Treatment and Prophylaxis of C. Diptheriae
1. Morphology and Staining of C. Diphtheriae:
C. diphtheriae are slender, slightly curved bacilli measuring 3µ x 0.3 µ with club or dumbbell-shaped appearance. In culture, involution forms can be seen.
They are Gram-positive bacilli arranged in palisades or Chinese letter; stained with methylene blue, they show beaded or barred appearance, which is characteristic of C. diphtheriae intermedius type, because of the presence of metachromatic or volutin granules. Diphtheroid bacilli which are non-toxigenic, do not have volutin granules.
By Neisser’s method, volutin granules are stained blue black and the cytoplasm is brown; by Albert’s stain, volutin granules are blue black and the cytoplasm is greenish.
2. Cultural Characteristics C. Diphtheriae:
Aerobe, optimum temperature 37°C; On Loeffler’s serum medium, the colonies are small, granular, grey with irregular edges.
Based on their characteristic growth C. diphtheriae are classified into C. diphtheriae, gravis type, intermedius type and mitis type.
Toxin:
C. diphtheriae produces a powerful exotoxin (Protein) which consists of two fragments — A and B. Both fragments are necessary for toxic effect, Fragment A has an enzymatic activity of the toxin. Fragment B is responsible for binding the toxin to the cells.
Toxoid:
A filtrate (toxin) of broth culture of a toxigenic strain is treated with 0.3% formalin and incubated at 37°C until toxigenicity has disappeared. This toxoid is purified and standardised in flocculating units (Lf doses). ,
3. Pathogenicity of C. Diphtheriae:
Diphtheria toxin is absorbed into the faucial mucous membrane and causes the destruction of the epithelium; the necrosed epithelium becomes embedded in exuding fibrin, red and white cells so that greyish “pseudo membrane” is formed.
An effort to remove this false membrane may result into severe bleeding. This toxin has an affinity for cardiac muscle causing endocarditis, cardiac failure, may damage liver, kidneys, adrenals with haemorrhage.
4. Clinical Features of C. Diphtheriae:
In respiratory tract, sore throat, fever usually develops. Prostration and dyspnoea soon follow because of the obstruction which may even cause suffocation. Irregular cardiac rhythm indicates damage to the heart. Later there may be difficulty in vision, speech, swallowing or movement of legs or arms. In general, C. diphtheriae gravis, intermedius types tend to produce more severe disease than mitis type.
5. Laboratory Diagnosis of C. Diphtheriae:
Specific treatment must never be delayed for want of laboratory reports, if the clinical feature is suggestive of diphtheria.
A. Specimens: The nasal, throat swabs must be obtained.
B. Smears: Smears stained with methylene blue show beaded rods in typical arrangement.
C. Culture: Loeffler serum and potassium tellurite chocolate agar media should be inoculated. Colonies on tellurite medium are sufficient to recognise the types of C. diphtheriae.
Any diphtheroid like organisms should be submitted to the toxigenicity test, as only toxigenic strains of C. diphtheriae can cause diphtheria.
Tests for toxigenicity can be done in three ways:
6. Production of Toxin:
The pathogenicity is due to elaboration of powerful exotoxin by lysogenic C. diphtheriae. The toxigenic property depends on the presence of a tox gene determinant controlling toxin production. The tox gene can be transferred from one bacterium to another by lysogenic phages of which beta phage is most important.
The beta phage is transmitted to daughter cells from generation to generation. The pathogenicity is maintained as long as the bacteria is lysogenic when bacteria is freed of its phage, such as culture in medium containing anti-phage serum, the toxigenic strain gets transformed into non-toxic. About 90-95% gravis and intermedius strains are toxigenic and only 80-85% mitis strains are toxin producing.
The structural gene for diphtheria toxin for diphtheria toxin (tox) forms part of the phage genome. It is a temperate phage. In addition to beta phage (p-phage), other phages (a, p, L r) are also known to carry tox genes. Tox phages (P) of C. diphtheriae can lysogenize C. pseudo tuberculosis and C. ulcerans and cause them to secrete toxin.
1. In Vivo Test:
An emulsified culture is inoculated into two guinea pigs, one of which has received 250 units of diphtheria antitoxin intraperitoneally two hours previously. The unprotected animal dies within 2-3 days and the protected one will survive.
2. In vitro (Elek’s agar gel precipitation) Test:
A strip of filter paper soaked with antitoxin is placed on an agar plate containing horse serum. The cultures to be tested for toxigenicity are streaked across the plate at right angles to the filter paper. After 48 hours’ incubation, the antitoxin diffuses from the filter paper and toxin from the toxigenic cultures resulting into the line of precipitation.
3. Tissue Culture Test:
The toxigenicity of C. diphtheriae can be shown by incorporation of culture into an agar overlay of cell culture monolayer. Toxin produced diffuses into the cells and kills them. Schick test is a skin test to diagnose diphtheria.
Diphtheria toxin is very irritating and results into a marked local reaction when injected intradermally unless it is neutralized by circulating antitoxin. One Schick test dose of unheated toxin is injected into the skin of one forearm and an identical dose of heated toxin is injected into another forearm as a control.
The test should be read at 24, 48 hours and 6 days and interpreted as:
1. Positive reaction (susceptibility to diphtheria toxin i.e. absence of adequate amount of antitoxin). Toxin produces rednesss and swelling that increases for several days and then slowly fades leaving a brownish pigmentation. The control site shows no sign.
2. Negative reaction (adequate amount of antitoxin present). Neither injection site shows no reaction.
3. Pseudo-reaction: Schick test may be complicated by hypersensitivity to materials other than the toxin contained in the injection. A pseudo-reaction shows redness and swelling on both arms, these signs disappear simultaneously on the second or third day. It constitutes a negative reaction.
4. Combined reaction begins like a pseudo-reaction with redness and swelling at both injection sites; the toxin later continues to exert its effects; however, whereas the reaction at the control site subsides rapidly. This denotes hypersensitivity and susceptibility to toxin.
7. Treatment and Prophylaxis of C. Diphtheriae :
Diphtheria antitoxin is obtained from animal immunized with repeated injection of concentrate toxoid. The antitoxin (20,000 to 100,000 units) is injected intramuscularly or intravenously after skin test on the day the diphtheriae is diagnosed clinically.
The treatment must be guided by antimicrobial drug susceptibility test. Antimicrobial drugs inhibit the growth of diphtheria bacilli in the throat and, hence, they arrest the toxin production.
Fluid Toxoid:
A filtrate (toxin) of broth culture of a toxigenic strain is treated with 0.3% formalin and incubated at 37°C until toxigenicity has disappeared. This toxin is purified and standardised in flocculating units (L.F. doses). Fluid toxoid is not used as such but it is absorbed on aluminium hydroxide for delayed absorption. It is the better antigen.
Such toxoid is commonly combined with tetanus, toxoid and pertussis vaccine as a single injection of “Triple vaccine” and diphtheria, pertussis and tetanus in immunization of children. Three doses are given intramuscularly at 4-8 weeks intervals from 2 months of age (i.e. 2, 4, 6, 15- 19 months; 4-6 years) as per manufacturer’s directions.
