Corynebacterium

            Corynebacteria are Gram positive, non-acid fast, non-motile rods. They frequently show club-shaped swellings and hence the name, ‘coryne = club’. The most important genus is C. diphtheriae, causative agent of diphtheria.

Corynebacterium  diphtheriae

Morphology & Cultural characteristics - The diphtheria bacillus is a slender rod, with a tendency to club at one or both ends. It is pleomorphic. They are nonsporing, noncapsulated, nonmotile. Cells often show septa and branching is frequently observed. Eventhough gram positive, they tend to be decolourise. Polymetaphosphate granules can be seen in the cells. Stained with Loeffler’s methylene blue, the granules take up a bluish purple colour and hence they are called metachromatic granules/volutin granules(Babes- Ernst bodies). They are often situated at the poles of the bacilli and are called polar bodies. The bacilli are arranged in characteristic fashion, ie., they are usually seen in pairs, palisades, small groups, at various angles to each other, resembling letters ‘V’, ‘L’. this has been called the chinese letter or cuneiform arrangement. This is due to the incomplete separation of the daughter cells after binary fission.

            Growth is scanty on ordinary media. Enrichment with blood, serum, egg is necessary for good growth. The optimum temperature is 37⁰C(15-40⁰C), optimum pH is 7.2. It is an aerobe and a faculatatively anaerobe. The media employed for cultivation are Loeffler’s serum slope – very rapid growth, colonies seen in 6-8 hours, first small, circular white opaque discs but enlarge on continued incubation and acquire a distinctive yellow tint and Tellurite Blood Agar – in which 0.4% tellurite inhibits other bacteria, diphtheria bacilli reduce tellurite to metallic tellurium which is incorporated in the colonies giving them a grey or black colour. But delayed growth of 2 days. Based on colony morphology in TBA diphtheria bacilli classified into 3 types : gravis, intermedius and mitis.

●    Gravis – uniformly stained short rods, some degree of polymorphism, few or no granules. Dull greyish black, opaque colonies, in 2-3 days 3-5mm diameter colony with raised dark centre, radially striated periphery. They are weakly hemolytic, causes mild type of disease.

●    Intermedius – long, irregularly barred cigar-shaped rods,highly pleomorphic, less granulation. Small, grey colony with a darker centre and a shining surface. They are nonhemolytic, causes intermediate type disease.

●    Mitis – long, curved, pleomorphic, prominent granulation, grey opaque colonies, with regular margins and glossy smooth surface. They are beta hemolytic, causes severe type of disease.

Diphtheria bacillus ferment glucose, galactose, maltose and produce acid, virulent strains ferment sucrose. No proteolytic activity, no urease.

Susceptibility- C. diphtheriae is readily killed by moist heat at 58⁰Cin 10 minutes, and also by the commonly used disinfectants. But it is relatively resistant to drying and may remain alive for weeks in dust and on fomites when dry and protected from sunlight.

Antigenic Structure- possesses heat labile type specific protein antigens on the surface, ie., K antigen and heat stable polysaccharide O antigen.

Toxin – Virulent strains produce a very powerful exotoxin, which are responsible for pathogenic effects. About 95-99% gravis and intermedius are toxigenic, 80-85% mitis are so. The diphtheria toxin is a protein and has been crystallized, with a molecular weight of 62,000. It is extremely lethal. It consists of 2 fragments A and B, necessary for toxic effect. When released by the bacterium, the toxin is inactive because the active site on fragment A is masked. Activation is accomplished by proteases present in the culture medium and infected tissues. All the enzymatic activity is present in fragment A. Fragment  B is responsible for binding the toxin to the cells. But the fragment B antibody prevents the binding of the toxin to the cells. The toxin is labile, prolonged storage, incubation at 37⁰C for 4-6 weeks treatment with 0.2-0.4% formalin, acid pH converts it to toxoid. The toxigenicity of the diphtheria bacillus depends on the presence in it of corynephages which determines genetically the toxin production. Also toxin production is influenced by the concentration of iron in the medium. The diphtheria toxin acts by inhibiting protein synthesis.

Pathogenicity – Humans are the only natural host for C. diphtheriae. It is usually spread by droplets of respiratory secretions. Infection usually begins in the pharynx 2-4 days after exposure. The organism, damaged epithelial cells, fibrin and blood cells combine to form a pseudomembrane. It is not a true membrane, but it adheres to the underlying tissue. If removed it leaves a raw, bleeding surface, that is soon covered by another pseudomembrane. This will block the airway and cause suffocation. The organisms very rarely invade deeper tissues or spread to other sites, but the extremely potent toxin spreads throughout the body and kills cell by interfering with protein synthesis. The heart, kidney and nervous system are susceptible. Sometimes they invade the nasal cavities, which have relatively few blood vessels. There they cause a milder disease because less toxin enters the blood. They invade the skin in cutaneous diphtheria and cause tissue injury.

Laboratory diagnosis – It consists of isolation of diphtheroid bacillus and demonstration of toxicity. One or two swabs from the lesions are collected using a tongue depressor. Diphtheria bacillus may not always demonstrable in smears from the lesion, also could not be differentiated from some commensal bacteria, normally found in the throat. Other than smear examination, it is important to identify Vincent’s angina. For this a Gram/Leishman stained smear is examined for Vincent’s spirochaetes and fusiform bacilli. Toxigenic diphtheria bacilli identified in smears by immunofluorescence.

For culture the swabs are inoculated on Loeffler’s serum slope, tellurite blood agar and observed characteristic colonies. Smears stained with methylene blue will show the bacilli with metachromatic granules and typical arrangement.

Virulence tests – Any isolate of the diphtheria bacillus should be tested for virulence. There are in vivo and in vitro tests.

In vivo tests – (a) Subcutaneous test : The growth from an overnight culture on Loeffler’s slope is emulsified in 2-4 ml broth and 0.8 ml of the emulsion injected subcutaneously into 2 guinea pigs, one of them treated with antitoxin previously. If the strain is virulent, the unprotected animal will die within 4 days. (b) Intracutaneous test : The broth emulsion of the culture is inoculated intracutaneously into 2 guinea pigs, one should be protected with antitoxin, while the other with less units of antitoxin intraperitoneally, to prevent death. Toxigenicity is indicated by inflammatory reaction at the site of injection, progressing to necrosis in 48-72 hours.

In vitro tests – (a) Elek’s gel precipitation test : A rectangular strip of filter paper impregnated with the diphtheria antitoxin is placed on a surface of a 20% normal horse serum agar in a petridish (medium will be fluid). When the agar has set, the surface is dried and narrow streaks of the strain are made at right angles on the filter paper strip. The plate is incubated at 37⁰C for 48 hours. Toxins produced will diffuse in the agar and produces a line of precipitation where it meets the antitoxin. The presence of arrowhead lines of precipitates indicates that the strain is toxigenic.

(b) Tissue culture test : The toxigenicity of diphtheria bacilli demonstrated by incorporating the strains in the agar overlay of cell culture monolayers. The toxin produced diffuses into the cells below and kills them.

Epidemiology – Diphtheria was formerly an important pediatric disease, by mass immunisation the disease has been eradicated from advanced countries. Asymptomatic carriers in endemic areas are the most important source of infection. Nasal carriers harbour the bacilli for longer periods than pharyngeal carriers. Cutaneous infections stimulate natural immunity. In some cases toys and pencils may act as vehicles of infection. The infection may spread through milk of infected cows.

Prophylaxis – Diphtheria controlled by immunisation. Active immunisation provides herd immunity and lead to eradication of the disease. Diphtheria toxoid given in children as a trivalent preparation containing tetanus toxoid and pertussis vaccine as DPT or triple vaccine. Passive immunisation is an emergency measure when susceptibles are exposed to infection. It is a subcutaneous administration. Combined immunisation consists of administration of the first dose of adsorbed toxoid on one arm, while ADS(antidiphtheritic serum) is given on the other arm, therefore active immunisation.

Treatment – Antitoxin should be given immediately when a case is suspected. The dosage recommended is 20,000-1,00,000 units for serious cases. Antitoxin not recommended in cutaneous diphtheria as the causative strains are non toxigenic. C. diphtheriae is sensitive to Penicillin. Erythromycin is more active than penicillin in the treatment of carriers.

Diphtheroids – Corynebacteria resembling C. diphtheriae occur as normal commensals in the throat, skin, conjunctiva and other areas. They are mistaken for diphtheria bacilli and are called diphtheroids. But they stain more uniformly, possess few or no metachromatic granules and tend to be arranged in parallel rows rather than cuneiform pattern.