Corynebacterium diptheriae

Introduction

• Causative agent of Diptheria in humans.• Small, pleomorphic (club-shaped), gram-

positive bacilli that appear in short chains (“V” or “Y” configurations) or in clumps resembling “Chinese letters”.

• Cells contain metachromatic granules (visualize with methylene blue stain)

• Lipid-rich cell wall contains meso-diaminopimelic acid, arabino-galactan polymers, and short-chain mycolic acids.

• Morphology :• Thin, slender, gram positive, non sporing, non-

motile bacilli with average size 3-6umx0.6-0.8um.

• club shaped due to presence of metachromatic granules at one or both ends, also called volutin or babes-Ernst granules.

• Appear in short chains (“V” or “L” configurations) or in clumps resembling “Chinese letters” or cuneiform arrangements.

• Special stains like albert [ malachite green & toludine blue], neisser or polychrome methylene blue are used for staining.

• The bacilli look green and metachromatic granules look bluish black by using albert stain.

• There are 3 biotypes- gravis, intermedius and mitis.

• Cultural characteristics:• Media enriched with blood serum or egg.• Hiss serum water:• Liquid media containing serum- growth seen as

turbidity and pellicle formation.• Loefflers serum slope: • Rapid growth 6-8 hours-small circular, white or

creamy and glistening.• Tellurite blood agar: • Potassium tellurite 0.04%- act as selective media• Black colonies due to reduction of tellurite to

tellurium.• Colonies appears after 48 hrs.

Tellurite blood agar

• Tinsdale agar • contains sheep’s blood, bovine serum, cystine

and potassium tellurite is selective medium.• Brown halo surrounding the colony is

differentiating feature.

• Biochemical reactions:• Ferment glucose and maltose with acid but no gas.• Don’t ferment lactose, mannitol or sucrose• Don’t hydrolyse urea or form phosphatase.• Gelatin is not liquified

Toxins:Exotoxin: diptheria toxin

• Pathogenesis:• Most common in children of 2-10 years.• Incubation period-3-4 days• Mode of transmission- droplet spread• The diphtheria may be following clinical types:1. Faucial 2. Laryngeal3. Nasal4. Conjunctival5. Otitic6. Vulvovaginal7. Cutaneous mainly around mouth and nose.

Faucial diphtheria is the commonest type.

The toxin has both local as well as systemic effects.

• Local effects:• Bacilli remain confined to the site of entry

usually upper respiratory tract• Multiply and produce toxin.• Toxin causes local necrotic changes along with

superficial inflammatory reactions• The necrosed epithelium together with

fibrinous exudates, leucocytes, erythrocytes and bacteria constitute pseudomemebrane.

• Which makes problem in swallowing.

• Systemic effects:

• Toxin diffuses to blood streams and causes toxemia• The toxin has got affinity for cardiac muscle,

adrenals and nerve endings.• It acts systematically on the cells of these tissues• The bacilli themselves do not play any part in the

systemic effects because they neither penetrate in to the tissue nor pass into blood stream producing bacteremia.

• Complications:• Local• The pseudomembrane may extend to the larynx

which may lead to laryngeal obstruction, asphyxia and death.

• Systemic• Diphtheritic myocarditis which may terminate in

heart failure and death.• Polyneuropathy and post diphtheritic paralysis of

palatine and ciliary muscles• Degenerative changes in adrenal, kidney and liver

may occur.

• Laboratory diagnosis:

• Isolation of organism

• Demonstration of its toxicity by virulence tests.

• Collection of specimens:• two Swabs from lesions e.g. throat, nose, larynx,

ear, conjunctiva, or skin.

• Direct microscopy:• Smears are stained with both gram and albert stain.

• Diphtheria bacilli show beaded slender green rods in typical chinese letter pattern.

• Culture:• The swabs are inoculated on the following culture media:• Loefflers serum slope:• Growth appears within6-8 hours.• Subculture is done on tellurite blood agar and plate is

incubated at 37 c for 48 hours.• Tellurite blood agar:• These plates have to be incubated at 37c for at least 48

hours before declaring these as negative, as growth may sometimes be delayed.

• Blood agar:• Useful for differentiating streptococcal or staphylococcal

pharyngitis which may simulate diphtheria.

• Colony morphology and staining:• Loefflers serum slope: small circular, white or

creamy and glistening.• Tellurite blood agar: Black or grey coloured colonies.• Smears are stained with both gram and albert stain.

• Diphtheria bacilli show beaded slender green rods with bluish black metachromatic granules, in typical chinese letter pattern.

• Gram stain is done to identify vincents spirochaetes and fusiform bacilli.

• Biochemical tests:• Hiss serum water is used for testing fermentation of

carbohydrates.

• Ferment glucose and maltose with acid but no gas.

• Don’t ferment lactose, mannitol or sucrose

• Don’t hydrolyse urea or form phosphatase.

• Gelatin is not liquified.

• Virulence tests:• These tests demonstrate the production of

exotoxin by bacteria isolated on culture.• Virulence testing may be done by: - In vivo: Guinea pigs and rabbits- by subcutaneous

or intracutaneous.- In vitro: Eleks gel precipitation test and tissue

culture tests

• Prophylaxis:• Active immunisation- Started at 6 weeks of age by toxoid in combination

with pertusis and tetanus vaccine [DPT, Triple vaccine].

- 3 doses are given by intramuscular route at an interval of 4-6 weeks.

- Booster dose at 18 months and 5 years.

• Passive immunisation- 500-1000 units of antitoxin[Anti diphtheric serum,ADS] is administered subcutaneously.

• Combined immunisation

• Schick test:• Done to demonstrate circulating diptheria antitoxin.

Treatment:

• Erythromycin (orally or by injection) for 14 days (40 mg/kg per day with a maximum of 2 g/d), or

• Procaine penicillin G given intramuscularly for 14 days (300,000 U/d for patients weighing <10 kg and 600,000 U/d for those weighing >10 kg).

• Patients with allergies to penicillin G or erythromycin can use rifampin or clindamycin.